2012 EAS Abstracts - Eastern Analytical Symposium

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2012 EAS Abstracts
November 2012
2012 EAS Abstracts
This volume contains the final abstracts for the oral and poster presentations which take place
on Monday, November 12, through Thursday, November 15, 2012. Additional abstracts received
after this volume was finalized are provided in the Addendum to the Final Program. If an abstract
is not provided in this volume or the Addendum, then the presenting author did not supply an
abstract. For each abstract provided, a complete mailing address for the presenting author is
shown. Additional authors are indicated, however, their mailing addresses are not provided.
Schedule and meeting room information for the technical sessions, as well as information concerning short courses, exhibitor workshops, and the exposition, are contained in the Final Program Book.
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1
2012 EAS Abstracts
November 2012
ABSTRACTS OF TECHNICAL PAPERS
1
Food for Chemometrics
Ron Wehrens, Fondazione Edmund Mach, Via E. Mach 1, San Michele
all’Adige, I-38010 Italy
Food science forms a rich source of applications for chemometrical methods, ranging from authentication to profiling and biomarker selection. Samples are often measured in a high-throughput manner, putting constraints on the types of data analysis,
in the sense that manual tuning is to be avoided as much as possible. At the same
time, the large number of samples allows for more powerful methods to be employed. In this paper, an overview is presented of some applications at Fondazione
Edmund Mach, focusing on the analysis of fruits that are of large economic importance locally, such as apples and grapes, and of food products such as cheese and
wine. As an example of methodological research fed by the richness of available
data, recent results are presented on biomarker selection, addressing the need for
more objective cutoff values and more robust selection schemes.
of the individual variables. As an example problem we use metabolomics datasets
(GC-MS and NMR) of the cerebrospinal fluid and plasma in a Multiple Sclerosis
study. [4]
[1] S. Yu et al., Kernel-based Data Fusion for Machine Learning. Methods and applications in Bioinformatics and Text mining, Springer: Berlin 2011.
[2] P. Krooshof et al., Analytical Chemistry 82 (2010) 7000-7007.
[3] Postma et al. Analytica Chimica Acta 705 (2011) 123-134.
[4] Smolenska et al. PLoS ONE (2012).
5
Novel Approaches to Increasing Selectivity, Peak Capacity, and
Resolution in Chromatographic and Electrophoretic Separations
Joe P. Foley, Drexel University, Department of Chemistry, 3141 Chestnut
St., Philadelphia, PA 19104, Adam Socia, Donna Blackney Beckett
Two unconventional approaches to liquid-phase separations, sequential elution liquid chromatography (SE-LC) and dual-opposite-injection capillary electrophoresis
(DOI-CE) are described. In SE-LC, a sample comprised of different classes of compounds is introduced into a liquid chromatographic system that is highly retentive
for all compounds because of a weak mobile phase that is initially employed. The
highly-retained compounds are then subjected to a sequence of selective mobile
phases, each of which is designed to elute one class of compounds. The result is
the sequential elution and separation of the sample components by class and also
within class, i.e., each compound within a category is also separated from other
compounds in the same category. The peak capacity in SE-LC is usually 2-3X larger
than conventional high-pressure liquid chromatography (HPLC) or ultra pressure liquid chromatography (UPLC), resulting in a much greater probability of resolving the
compounds of interest. In DOI-CE, the electro-osmotic flow is suppressed by one of
several methods reported in the literature, and the same sample is introduced into
both ends of the capillary in order to allow for the unbiased, simultaneous analysis
of cationic and anionic species. Cationic analytes migrate from the anodic end of the
capillary toward a centrally-located detector (e.g., absorbance or C4D), and anionic
analytes likewise migrate from the cathodic end toward the detector. Voltage polarity, a small amount of hydrodynamic pressure, and the relative migration distance of
the anions and cations can be varied to optimize selectivity and resolution. Representative applications are described.
2
Data Analysis Strategies for Comprehensive Two-Dimensional
Liquid Chromatography
Sarah C. Rutan, Virginia Commonwealth University, Box 842006,
Department of Chemistry, Richmond, VA 23231, Robert C. Allen, Hope
P. Bailey
Comprehensive two-dimensional liquid chromatography shows great promise for
the analysis of complex metabolomic samples, but due to the higher complexity of
this type of data, new chemometric approaches must be developed to be able to
maximize the information yield from these data. Both multivariate curve resolution –
alternating least squares (MCR-ALS) and parallel factor analysis (PARAFAC) have
been shown to be quite useful in this regard. Some examples of useful strategies for
data analysis, including combining the use of MCR-ALS and PARAFAC, correction
for retention time shifts and background contributions, and a new unimodality constraint for MCR-ALS and PARAFAC are discussed in this presentation. Applications
of these methods to designed calibration experiments are illustrated.
3
Chemometrics for Process Understanding
Anna de Juan, University of Barcelona, Faculty of Chemistry, Diagonal,
647 Barcelona 08028 Spain, Joaquim Jaumot, Sílvia Mas, Sara
Piqueras, Ludovic Duponchel, Benoit Igne, C. A. Andersson, Romà
Tauler
Process understanding is essential in industry in order to optimize the efficiency
of operational procedures and ensure the quality of end products. Knowing a process implies getting a clear description of the evolution of the events taking place
(mechanism, side phenomena, etc.) and of the structural identity of the compounds/
signal contributions involved. Process monitoring is often carried out by spectroscopic methods. In this context, multivariate curve resolution (MCR) methods are
well suited to interpret this kind of data since they describe the original process/
batch measurement through a simple bilinear model formed by chemically meaningful process and spectral profiles. Relevant assets of MCR methodologies as applied
to process analysis are: a) the possibility to analyze multi-technique or multi-experiment (multi-batch) datasets of very diverse nature and data structure, b) the use
of hybrid methodologies, combining soft-modeling with hard-modeling constraints,
which include mechanistic information on the process, c) the adaptation to deal
with processes monitored by complex instrumental measurements, such as hyperspectral images, and d) the inclusion of internal calibration procedures, which allows
the use of MCR for end-point process detection and for the description of the heterogeneity evolution from a global point of view or in a compound-wise fashion. All
the advantages mentioned above are shown with real examples of pharmaceutical
and bio-analytical interest.
6
High-Throughput Electrophoresis and Application to Screening,
Protein Analysis, and In-Vivo Sensing
Robert Kennedy, University of Michigan, 930 N. University Ave., Ann
Arbor, MI 48109
Advances in microfluidic and capillary electrophoresis have allowed separations to
be obtained on the seconds scale and better. Microfluidics further allows integration
of functions so that entire assays may be performed at high-throughput including
the sample preparation steps. In this talk we review advances of these ideas for
protein analysis, screening, and in-vivo sensing. Perhaps the most common protein analysis method is Western blot. While powerful, this method has not been
successfully migrated from conventional gel electrophoresis and requires over four
hours to complete. Although rapid protein separations can be achieved on chip, little
work has been done to incorporate the immunoassay needed for Westerns. We are
developing a microfluidic system that allows rapid protein separation, blotting, and
immunoassay. Projected throughput is 90 seconds per assay for 96 samples. We
have also developed electrophoretic separations of protein-protein complexes that
have interest for drug discovery. To enable the electrophoresis separations to be
used in screening campaigns, we have interfaced fast separations to a segmented
flow system that allows rapid introduction of nanoliter samples on a microfluidic device. Finally, we have used in-vivo sampling interfaced to segmented flow capillary
electrophoresis to perform high resolution monitoring in-vivo. The instrumentation
and methods are presented in this talk with emphasis on integration of steps for
high-throughput analysis.
4
Strategies for Data Fusion
Lutgarde Buydens, Radboud University Nijmegen IMM, Heyendaalse
weg 135, Nijmegen 6525 AJ The Netherlands
Due to the ever-increasing production of complex data by a large variety of analytical technologies, chemometric data analysis and data mining have become crucial
tools in modern analytical laboratories. The omics field is a clear example of this. In
the last decade data fusion has become widespread in the omics field. It is common
to perform linear data fusion. However, many biological or chemical data display
complex, e.g., non-linear, parameter dependences. The linear methods are bound
to fail in such situations. In this lecture two alternative approaches are be presented.
The first one is based on the hierarchical fusion of mid-level fusion models. The
second method is a non-linear kernel fusion model in order to cope specifically with
nonlinearities.[1] We use our pseudo-sample approach [2,3] to reveal the contribution
7
Distinguishing Retention Mechanisms in Liquid Chromatography
Apryll M. Stalcup, Dublin City University, Irish Separation Science
Cluster, Glasnevin, Dublin 9 Ireland
Traditional chemical separation methods rely primarily on exploiting a single facet
of all the potential ways in which molecules can interact. However, molecules are
seldom so well-behaved with each other in the liquid or gas state so as to interact
through only one interaction mode. The same intermolecular interactions responsible for the phase behavior of pure substances (e.g., dispersion, hydrogen bonding,
dipole-dipole, dipole-induced dipole, electrostatic, etc.) can be creatively exploited
2
2012 EAS Abstracts
November 2012
11
to accomplish the desired separations. In some ways, the separation system reflects the global interactions between the solute and the separation system, which
acts as a molecular amplifier of these interactions. Further, separation techniques
are also incredibly sensitive tools for investigating and amplifying subtle differences
in intermolecular interactions. Current research is directed toward understanding
those interactions and how they may be extended to other applications. Linear solvation energy relationships are being used to understand interactions between solutes and surface-confined ionic liquids under a variety of chromatographic modes.
Examples from current research are used to illustrate these points.
Revisit Bioanalytical Assay Development Strategy Through a
Clinical Study with Pilot DBS Sample Collection
Qin Ji, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543
In last several years, dried blood spot (DBS) sampling reemerged and has attracted great interest in the pharmaceutical industry as a micro-sampling technology
for drug discovery and development studies, especially as a viable alternative to
plasma/serum sample collection for toxicokinetic (TK) and pharmacokinetic (PK)
studies. Along with identifying the challenges and obstacles that must be overcome
prior to applying this technology routinely, industry has gained a better understanding of bioanalytical assay development for DBS samples. One critical parameter
that needs to be addressed during assay development and validation is “elution
efficiency” of the analyte as it transitions from the DBS card to solution phase during
sample preparation process. In this presentation, an assay development strategy
that emphasizes the “elution efficiency” is discussed. The factors affecting “elution
efficiency” are explored. Practical solutions for achieving good assay “elution efficiency” to ensure the accurate sample analysis of clinical study samples are suggested. Other related discussions include advantages and disadvantage of using
“center” and “whole” spot punch for DBS sample analysis.
8
Tailored Nanocrystalline Silicon as a Gas Chromatography
Detector Platform
Frank V. Bright, University of Buffalo, SUNY, Department of Chemistry,
Materials Science and Engineering, Natural Sciences Complex, Buffalo,
NY 14260, Justin M. Reynard, Nathan S. VanGorder, Caley A. Caras,
Randi E. Deuro
Porous silicon (pSi) can be produced from crystalline silicon (cSi) wafers using a
variety of methods. Quantum confinement effects within the pSi nanocrystals produce a material that exhibits visible photoluminescence (PL). The as prepared pSi
(pSi-ap) nanocrystallite surfaces are rich in SiHx (x=1-3) species and one can readily create oxidized pSi (pSi-ox) with new Si-O-Si, OySiH (y=1-2) and Si-OH surface species. Researchers have previously exploited the pSi surface chemistries
in areas ranging from solid-state lighting to chemical sensing. This presentation
focuses on the analytical performance of pSi-ap, pSi-ox, and surface modified pSi
as a detection platform for gas chromatography.
12
The Lessons Learned for Dried Blood Spot (DBS) Method
Development and Validation: The Difference between Regular
Plasma Bioanalysis Methods versus Dried Blood Spot
Naidong Weng, Janssen Research and Development, 1000 US Highway
202, Raritan, NJ 08869, Shefali R. Patel
DBS has garnered attention within the pharmaceutical industry. Compared to plasma methods, DBS need more time for development. DBS presents some recognized technical challenges and additional challenges are also gradually uncovered.
Analyte extractability from DBS cards is one of the pivotal parameters. The recovery
is dependent upon the type of cards, storage conditions, extraction solvent/technology, and age of the spots. Here, we discuss our investigation on the identification
and mitigation of an extractability problem in a DBS assay. The method validation
for fluoxetine met acceptance criteria, but when storage stability experiments were
attempted after 26 days, low accuracy values (70% -80%) were observed for the
quality control samples. Thorough trouble-shooting was conducted, and many parameters that could affect the analyte extractability and stability were investigated.
In fact, a well thought out strategy must be used to differentiate reduced extractability vs. poor stability. When validating a DBS method, one should pay attention to
analyte extractability from DBS cards. Implied from the above results, one can no
longer validate a DBS method while choosing to perform longer-term extractability
and stability experiments at a later date. The initiation of method development and
validation needs to be moved ahead – they must be conducted well prior to initiation
of the in-life studies. We discuss DBS method validation strategy in this presentation
with the focus on the extractability.
9
Carbonaceous Nanomaterials as Stationary Phases for Liquid
Chromatography
Luis A. Colon, SUNY Buffalo, Department of Chemistry, MS 578 NSC,
Buffalo, NY 14260, John C. Vinci, Lisandra Santiago-Capeles, Zuqin
Xue
Column technology continues to be a central area of research in liquid chromatography. The development of new separation media is essential to achieve a high
degree of selectivity, high efficiency and fast separations to embrace the increasing
demands imposed by sample complexity in chemical analysis. Research efforts in
our laboratory have focused on the development and study of new chromatographic
media that can be implemented in different formats (i.e., micro-particulates, monoliths, and thin films). Recently, we are investigating the use of nanometer size particulates as adsorbent media for chromatography. We are producing carbon-based
nanoparticulates (C-NP) in the regime of 10 nm to be used as stationary phase
once they have been immobilized on the surface of chromatographic supports. In
an initial approach, the nanoparticles have been attached on allyl-silica via radical
initiation after nanoparticle hydrogenation. Other attachment strategies are also being used. Silica monoliths and particulates have been modified with C-NP. In this
presentation, we discuss our synthetic/processing approach to produce the C-NP
modified silica materials, physical characterization, and our first observations of the
chromatographic behavior of these materials.
13
Ultramicro Extraction, Nanospray, and Dried Blood Spots: Towards
Digitization of a High Sensitivity MS Workflow
Gary Valaskovic, New Objective, 2 Constitution Way, Woburn, MA 01801
Dried blood spots (DBS) have rapidly emerged in the pharmaceutical industry as
a highly cost effective method for low-volume sampling, storage, and retrieval of
specimens prior to assay by liquid chromatography tandem mass spectrometry (LCMS/MS). Prior to analysis, a punch (typically 3 mm) from the spot (typically 15 μL) is
subjected to solvent extraction and subsequent analysis by conventional (mm bore)
LC-MS/MS. Inefficiencies in extraction and volume handling limit assay sensitivity.
Insufficient limits of quantification often preclude the use of DBS for high potency
and/or inhaled compounds. Here we present segmented-flow sample handling as a
means to combine low-flow electrospray with high-throughput analysis. Segmented
flow sample handling enables the further manipulation, concentration, separation,
and storage of sample on the picoliter to nanoliter scale. The results of a preliminary
study combining segmented flow with micro DBS punches (< 0.4 mm) and a microfluidic flow-through extraction cell are presented. Microfluidic extraction improves
extraction efficiency by approximately thirteen-fold over conventional methods.
Simultaneous LC-MS/MS Determination of Acetaminophen,
Acetaminophen-Glucuronide and Acetaminophen-Sulfate in
Human Dried Blood Spot Samples in Support of a Pilot Study to
Assess the Feasibility of Remote PK Blood Sampling Methodology
Wenkui Li, Novartis Institutes for BioMedical Research, One Health
Plaza, MS: 435/3123, East Hanover, NJ 07936
Clinical research can be revolutionized by collecting pharmacokinetic (PK) samples
onto bar-coded dried blood spot (DBS) cards and utilizing a smart phone based barcode scanning application to mark the time of PK sample collection. The feasibility
of this combination was tested in a small, single dose PK study with acetaminophen.
Eligible, healthy volunteers were trained on DBS sample collection by finger stick
and use of the barcode application. Subjects were then given a single 500 mg oral
dose of acetaminophen, which was followed by collecting and time stamping the
scheduled DBS samples without supervision at 30 min, 1, 4, 6 and 8 h post-dose.
A sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method
was developed and validated for the simultaneous determination of acetaminophen
and its major metabolites, acetaminophen glucuronide and sulfate, in the collected
DBS samples. The assay dynamic range was from 50.0 to 5000 ng/mL for each
compound using a 1/8 inch (3-mm) disc punched from a DBS sample. The validated
assay was successfully implemented in the study with the obtained PK parameters
in agreement with the values reported in literature. The combination of DBS sampling and smart phone sample collection time recording was demonstrated to be
suitable for remote PK sample collection for clinical research.
10
14
Perspectives on Column Equivalency: Technical Challenges and
User Perspective
Brian A. Bidlingmeyer, Agilent Technologies, 2850 Centerville Rd.,
Wilmington, DE 19808, William L. Champion
This presentation is divided into two sections – technical challenges and user perspective. The first question to be answered is what does the term “equivalent column” imply? Clearly equivalency can have a number of meanings; and the several
technical issues are discussed. The key is that the column provides performance
that meets the intended use of the separation. This requires some understanding by
the user of the purpose of the separation in order to establish an equivalent separation. The second half of the presentation addresses the user’s perspective. For
knowledgeable users this may be trivial; however, for less experienced users this is
often more difficult to assess. In establishing guidelines, there should be provision of
how similar the separation needs to be to be considered “equivalent.”
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2012 EAS Abstracts
November 2012
15
United States Pharmacopeia Website on Column Equivalency
Margareth Marques, United States Pharmacopeia, 12601 Twinbrook
Pkwy, Rockville, MD 20852
There are more than 300 brand names for C18 columns and more than 75 brand
names for C8 columns available worldwide and not all of them are equivalent. This
diversity makes it very laborious and difficult to find possible high-pressure liquid
chromatography column alternatives. The US Pharmacopeia is managing a web
site that offers two databases that allow the search of possible alternative columns.
One database classifies the columns based on evaluation using the NIST SRM 870
(www.NIST.gov) and in the second database the column classification is based on
research published by Snyder LR et.al. in J. Chromatogr. A 2004; 1060:77-116 and
J. Chromatogr.A 2004; 1057:49-57. Besides allowing the search for possible alternative columns, columns for orthogonal methods can also be found.
on the microbial activity, redox potential, pH, temperature, presence of metals and
nature of the protein sequences. Different pathways of degradation are observed
where the protein chains undergo changes, from hydrolysis to cross-linking and
residues side-chain modifications. Similarly, specific amino acids are affected by
photo-oxidation, resulting in the formation of photoproducts that tend to change the
color of the fibers (yellowing, discoloration, fading), as well as the physical properties such as strength. This paper describes the pathways of amino acid oxidation
and formation of specific biomarkers in both wool and silk.
19
Using Protein Mass Fingerprinting to Further our Understanding of
Cultural Heritage
Dan Kirby, Straus Center for Conservation and Technical Studies,
Harvard / Art Museums, 32 Quincy St., Cambridge, MA 02138
Proteins are present in artworks in many forms, such as paint binders, adhesives
and coatings, and in our museum laboratory we have implemented protein mass
fingerprinting (PMF) of tryptic peptides for the routine identification of proteinaceous
materials in artworks. PMF provides a simple, rapid, sensitive, and specific method
for protein analysis and is an attractive alternative to methods currently used. With
PMF and an empirically derived set of marker ions, we have successfully identified
protein components in a wide variety of artworks including 14th Century altarpieces,
modern paintings and fashion fabrics from Mariano Fortuny. PMF can be used not
only to identity proteins, but also to determine their species of origin. This feature
is the result of the fact that, whereas all proteins of a given kind are very similar,
for example collagen in mammals, they are not identical, but contain mutations that
have occurred over millennia giving rise to species-specific peptide markers. Taking
advantage of this capability, we have analyzed parchment-containing materials from
many objects of cultural heritage. Using either published marker ions or in-house
generated reference PMF’s, we have successfully identified the species origin of
parchment in, for example, 16th and 17th C legal documents, a 7th C Coptic Codex,
Duchamp’s Etant donnés, historic Alaskan kayaks, and a purse from 1800’s Pennsylvania. In this presentation we describe our method in detail, show examples of
current analytical results, and discuss plans to extend PMF and species identification to new, important areas of cultural heritage.
16
The Hydrophobic Subtraction Model of Reversed-Phase Selectivity
– Application to the Problem of Column Equivalency
Dwight R. Stoll, Gustavus Adolphus College, 800 W. College Ave., Saint
Peter, MN 56082, Eric Talus, Ian Gibbs-Hall
Since its inception over a decade ago, the hydrophobic model of reversed-phase
high-pressure liquid chromatography (HPLC) column selectivity has and continues
to play an important role the HPLC method development. In this presentation we
discuss the application of the model to the particular problem of choosing columns
with a high degree of similarity. Two of the greatest strengths of the model are its
comprehensiveness, with over 570 columns characterized to date, and its availability, wherein these data are freely and publicly available via the United States Pharmacopeia website (http://www.usp.org/app/USPNF/columns.html). The model and
the associated database are a rich source of information that can complement other,
more focused approaches to assessing the similarity of two phases for a particular
application. In this presentation we review the fundamental basis of the model, including a description of the physico-chemical stationary phase/solute interactions
that are accounted for by the model, and briefly summarize the data collected to
date. We then describe various approaches to finding similar columns, including
fast and simple pair wise column comparisons, and visualization tools that allow
several columns to be compared at once. Finally, we share some thoughts about
the future of the model and its implementation, with an invitation for suggestions as
the number and diversity of available columns continues to grow.
20
A Simple Protocol for MALDI-TOF-MS Analysis of Lipids and
Proteins in Single Microsamples of Paintings
Cosima D. Calvano, Dipartimento di Chimica, Università degli Studi di
Bari “Aldo Moro,” via Orabona 4, Bari, 70126 Brazil, Inez Dorothé van
der Werf, Francesco Palmisano, Luigia Sabbatini
A simple protocol, based on Bligh-Dyer (BD) extraction followed by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, for fast identification of paint binders in single microsamples is proposed. We
demonstrated that the BD method is effective for the simultaneous extraction of lipids and proteins from complex, and atypical matrices, such as pigmented paint layers. Detection of lipid classes, e.g., triacylglycerols (TAGs) and phospholipids (PLs),
and their oxidation by-products was accomplished, whereas proteins could be identified by peptide mass fingerprinting. The effect of some pigments on ageing of lipids
and proteins was also investigated. Finally, the proposed protocol was applied to
the study of a late-15th century Italian panel painting and a 12-13th century painted
crucifix, allowing the identification of various proteinaceous and lipid substances in
organic binders, such as egg white, egg yolk, casein, animal glue, and drying oil.
17
Complementing USP Column Equivalency Databases with HPLC
Selectivity Measurements in Your Lab
Richard Henry, Pennsylvania State University, 983 Greenbriar Dr., State
College, PA 16801
High-pressure liquid chromatography (HPLC) column retention and selectivity is determined by different chemical interactions that occur between sample components,
stationary phases and mobile phases. Once promising HPLC conditions have been
established by screening, analysts may wish to identify several equivalent columns
for method development to insure success and provide back-up if required. This can
be easily done using the United States Pharmacopeia (USP) website (www.usp.org/
app/USPNF/columns.html) where databases have been created for many commercial HPLC columns using National Institute of Standards and Technology (SRM 670)
and PQRI (hydrophobic-subtraction) test mixes. This talk describes simple HPLC
experiments that can be done in your lab to complement equivalency results from
the USP database. Excel plots and Neue S values are shown to work well under
gradient conditions. The ability to rapidly conduct local selectivity experiments allows the following important enhancements to using on-line databases: 1) estimate
equivalency for columns that are not part of the online database; 2) measure equivalency with your own test mix to confirm USP results and investigate how changes in
mobile phase solvent, pH and temperature affect selectivity values; 3) compare different lots of the selected column phase for equivalency; and 4) investigate column
life and determine how well selectivity is maintained under use conditions.
21
Biological Clocks: High-Throughput Identification of Deterioration
Markers and Dating of Museums’ Proteinaceous Specimens
Mehdi Moini, Smithsonian Institution, 4210 Silver Hill Rd., Suitland, MD
20746
Cultural heritage contains a large number of precious proteinaceous specimens
such as silk and wool textiles, collagen-containing materials, leather objects, paper,
paint, coatings, binders (and associated adhesives), etc. This presentation discusses the fundamental factors that affect change and degradation of these specimens
at the molecular level using modern technologies. The fundamental factors that are
discussed include: amino acid racemization, and protein deamidation. Our goal is
to develop rapid and reliable methods for finding deterioration markers, and for dating of proteinaceous specimens that consume minimal amount of specimens. To
achieve this goal, several mass spectrometric techniques has been utilized including
capillary electrophoresis mass spectrometry, matrix assisted laser desorption ionization, and nano ultra high-pressure liquid chromatography in conjunction with high
resolution mass spectrometry. In addition, for higher throughput characterization
of deterioration markers and dating of museums’ proteinaceous specimens using
minimal amount of these precious samples, we have developed a rapid and highly
sensitive miniaturized front end based on ultrafast capillary electrophoresis. The
new techniques are alternative to 14th century dating and stress-strain measurement
approaches, both of which require several mg of samples; quantities in this range
are almost impossible to obtain for the precious items such as historic silk samples.
Moreover, the new techniques are useful for dating more recent specimens (less
18
Protein Diagenesis in Ancient Wool and Silk Textiles
Caroline Solazzo, BioArCh, University of York, Department of Chemistry,
York, YO10 5YW United Kingdom
In the past couple of years, amino acid racemization, isotopic, DNA and proteomics
analysis of protein-based fibers have been developed in order to identify, date and
provenance textiles. While these techniques have been extensively applied to other
materials such as bones and teeth, the particular complexity and degradation of
animal fibers has rendered these approaches more challenging. A solid knowledge
of the nature of the materials and their patterns of degradation is an essential step
to undertake further biomolecular studies. For this, proteomics combined with amino
acid analysis is an efficient approach to identify the products of protein degradation.
This paper presents a series of studies on biodegradation and photodegradation of
wool and silk. Protein fibers are constructed from core proteins (alpha-helical keratins for wool and beta-pleated sheets of fibroin for silk) surrounded by smaller amorphous proteins. Preservation of textiles in archaeological environments depends
4
2012 EAS Abstracts
November 2012
than 400 years), where radiocarbon dating yields very poor time resolution. These
techniques are complementary to gas chromatography–mass spectrometry already
routinely used in museums for analysis of volatile organic compounds.
phoric phase of stimulant use. This presentation focuses on the forensic pharmacology of alprazolam, the published studies on the effects of alprazolam and other benzodiazepines on driving abilities and driving under the influence cases. Troubling
issues faced by forensic toxicologists in impaired driver cases such as tolerance,
“therapeutic level” and drug per se laws are covered with selected integration of
published papers, selected case investigations and personal experiences. This presentation highlights the potential adversity of trying to offer interpretative opinions in
a case from a single blood concentration.
22
Using Time-of-Flight and Tandem Mass Spectrometry to Solve
Analytical Challenges for the New Designer Stimulant Drugs
Matthew M. McMullin, NMS Labs, 3701 Welsh Rd., Willow Grove, PA
19090
A collection of synthetic stimulates and hallucinogens are sold under the misleading labels of “bath salts” and “plant food.” These chemicals have been involved
in an increasing number of violent and high publicity cases. Chemically they fall
into four major categories: synthetic cathinones, phenethylamines, piperazines
and tryptamines. Toxicologically these are dangerous chemicals that come in pill
or powder form and are swallowed, snorted, injected or smoked by users. This
new wave of drugs of abuse means that the standard drug screening scope is no
longer adequate, so analytical approaches must be updated. The challenges for
a toxicology laboratory includes the large and ever expanding number of drugs,
lack of absorption, distribution, metabolism, and excretion (ADME) data in man,
their structural simplicity, and their structural similarities including multiple isobaric
pairs to be resolved. In forensic toxicology there is a need to access the impact and
prevalence of driving under the influence of these new drugs, plus testing in support
of post mortem case work. Clinically, detection of these drugs supports physicians
treating patients with adverse events. To address these challenges and to meet the
forensic toxicology standard of practice, which requires two analytically different
tests on two independent aliquots, multiple analytical tools are needed. Our approach is to screen for a defined and expandable menu of these drugs using liquid
chromatography-mass spectrometry time-of-flight (LC-MS/TOF), and confirm positive findings by LC-MS/MS or gas chromatography mass spectrometry (GC-MS).
Our experiences to date are presented from testing for roughly 67 “bath salt” drugs
in human blood, and urine.
25
Update on Novel Synthetic Cannabinoids: Detection, Impairment
and Adverse Effects
Jillian K. Yeakel, Center for Forensic Science Research and Education,
2300 Stratford Ave., Willow Grove, PA 19090, Barry Logan
This presentation considers the impact of the designer drug market on analytical
and interpretive toxicology, focusing on the emerging literature describing adverse
effects of synthetic cannabinoid drugs and their forensic significance. The evolution
of the diverse range of compounds being sold to users are considered, and the issue of designating compounds as scheduled drugs according to state and federal
law are reviewed. The drugs have marijuana-like activity, but are distinguished from
marijuana by more frequent life-threatening adverse effects including cardiovascular events, lowered seizure threshold risk, and greater psychological effects including anxiety and panic. This category of drugs is not detected by traditional immunoassay or gas chromatography mass spectrometry (GC-MS) screening approaches
and the appropriate analytical approach to their detection are considered, including the relative risks and benefits of immunoassay versus liquid chromatography
tandem mass spectrometry (LC-MS/MS) screening approaches. The presentation
considers several case studies involving police investigations, fatalities and driving
under the influence (DUI) arrests, and emphasizes the need for appropriate toxicology testing to support opinions about the cause of the impairment.
26
Adapting Quality-by-Design (QbD) Tools to Analytical ICH Methods
Validation Activities
Elizabeth Hewitt, Millennium Pharmaceuticals, 40 Landsdowne St.,
Cambridge, MA 02144
The International Conference on Harmonization (ICH) method validation using a
QbD approach can be leveraged to give robust methods that help pharmaceutical commercial organizations release materials in a timely fashion and minimize
investigations due to method failures. A systematic process using various QbD
tools are discussed and demonstrated to help achieve understanding of the method
parameters’ impact on test method clinical quality assurances. Examples of QbD
approaches applied to pharmaceutical analytical methods optimization and validation are presented along with strategies to control the quality output of analytical
methods.
Cannabinoid Disposition in Oral Fluid: Quantification,
Pharmacokinetics and Interpretation
Dayong Lee, National Institute on Drug Abuse, 251 Bayview Blvd, Ste.
200, Baltimore, MD 21224, Marilyn A. Huestis
Oral fluid is a new biological matrix for clinical and forensic drug testing. Oral fluid
testing offers non-invasive and directly observable sample collection, reducing the
potential for adulteration and eliminating the need for a special collection facility.
Appropriate interpretation of oral fluid test results requires a comprehensive understanding of drug pharmacokinetics and metabolic patterns in this matrix. Oral
fluid cannabinoid disposition is especially important as cannabis is often the most
frequently detected illicit drug in many programs. To build the scientific database for
cannabinoid oral fluid testing, the Chemistry and Drug Metabolism Section at the
National Institute on Drug Abuse conducted multiple clinical studies, analyzing oral
fluid specimens from cannabis smokers under various conditions of cannabis/THC
administration and discontinuation. In these Institutional Review Board-approved
studies, oral fluid specimens were collected with the Quantisal™ collection device, and ∆9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC, cannabidiol and
cannabinol were quantified by two-dimensional gas chromatography mass spectrometry. This presentation delineates oral fluid pharmacokinetics following oral,
sublingual, and smoked cannabinoids and characterization oral fluid cannabinoid
elimination profiles during extended abstinence. Windows of detection at different
analytical cutoffs, disposition after various routes of administration, and possible
approaches to identifying recent cannabis exposure and minimizing passive contamination are presented. Analyzing multiple oral fluid cannabinoids at low limits
of quantification allows data evaluation tailored to individual purposes of oral fluid
analysis in pain management, dependence treatment, workplace, and driving under
the influence of drug settings. Lastly, additional considerations for oral fluid drug
quantification and interpretation, including analyte stability, inter-subject variability,
and sample collection procedure are discussed.
23
27
Approaches Towards QbD and Risk Analysis in Mid- to Late-Phase
Analytical Method Development
Peter Tattersall, Bristol-Myers Squibb, One Squibb Dr., MS: 92-1-150,
New Brunswick, NJ 08903, Mario Hubert, David Lloyd, Ruben Lozano,
Liya Tang
Quality-by-design (QbD) can be used in a variety of ways to assist in the efficient
development of robust analytical methods. In order to guide analytical scientists
at Bristol-Myers Squibb, a QbD team developed a training guide to capture the
thoughts, tools and pathways that can assist in consistently developing appropriate
methods depending on the technique and analytical concern. During pharmaceutical development it becomes apparent what methods are required to monitor the
critical quality attributes of the product. The methods chosen to monitor these attributes contain their own variability. With this in mind an assessment of the method
requirements can be made. The subsequent method evaluation and development
along with the risk/reward of QbD to enable/guide this can vary from a significant
amount (such as a selective impurity method) to very little (such as a residue on
ignition or an ID test). QbD-developed methods add significant value generating
better understanding of the method operating space, increased method flexibility
(allowance of changes without requiring post-approval process), and greater ruggedness and robustness. This should lead to more efficient method transfer, and
consistent qualities in analytical methods. In this presentation we discuss the training guide, including the risk assessment tools used in method scouting and performance monitoring, followed by the justification and implementation of modeling or
design of experiments at the later stages of development, highlighting their value
for certain methods.
24
Driving Under the Influence of Alprazolam and Other Drugs: What
Might Explain the Explosion of Abuse Across the Nation?
H. Chip Walls, Forensic Analytical & Clinical Toxicology, Miami Police
Training Center, 400 NW 2nd Ave., 3rd Floor, Miami, FL 33128
Alprazolam, a high-potency benzodiazepine, is Food and Drug Administration approved for the treatment of anxiety and panic disorder and is one of the most prescribed medications and abuse drugs worldwide. Daytime sedation and impairment
of cognitive functioning and psychomotor performance is one of the main problems
associated with the use of benzodiazepines as anxiolytics. Patients using alprazolam commonly report adverse events, such as drowsiness, dizziness and reduced
alertness especially in the first month of use. Alprazolam is commonly used with
other recreational drugs presumably to achieve increased intoxication and manage
undesirable drug withdrawal symptoms, to alleviate the panic or paranoia caused
from using high potency cannabis and is frequently used in the downside or dys-
5
2012 EAS Abstracts
November 2012
31
28
Implementation of Quality-by-Design for Analytical Methods during
Development Through Technology Transfer
Rosario LoBrutto, Teva Pharmaceuticals, 223 Quaker Rd., Pomona, NY
10970
Implementation of quality-by-design (QbD) in development allows for improved
understanding of the analytical method focusing on robustness and ruggedness
designed with end user in mind thereby facilitating methods transfer and provides
opportunities for continual improvement. This allows for reduced chance of method
failures during release/stability testing, aids out-of-specification (OOS) investigations, and ultimately increases quality and reduces costs. By integration of elements
of analytical QbD, into the development strategy the critical sources of analytical
variability (continuous and discontinuous) are identified, measured and understood
so that they can be controlled with the appropriate control strategy. QbD implementation is aligned with a risk and science based approach to methods development
and methods validation. The outcome of the risk analysis allows for the identification of the potential critical analytical method factors that could be evaluated using
a statistical design of experiments. Design of experiments (DOE) is used to provide
the most efficient and statistically sound approach to evaluate multiple method factors and their responses (critical method attributes). This provides an excellent opportunity for determination of optimum conditions generated from a limited number
of experiments used in the DOE study. Also, statistical design facilitates an in-depth
understanding of the method and justifies the choice of ranges for method factors
and finds a robust (optimum) region for the final method. Finally an analytical method design space could be developed encompassing the operable ranges for the
potential critical analytical method factors and their interactions and suitable control
strategies can be implemented.
Am I Seeing Things? Data Visualization and Analysis
Katherine A. Bakeev, Camo Software, One Woodbridge Center, Ste.
319, Woodbridge, NJ 07095, Miryeong Sohn
We are often faced with large amounts of data from which we are expected to extract information and develop qualitative or quantitative models that can be used
for monitoring processes. There are many approaches one can take to the analysis
and data modeling, but they must all start with visualizing the data and exploring
the patterns that exist in it. In this presentation, spectral data (NIR and Raman) collected on the same samples with the objective of quantifying the concentration of
components are presented. Aspects of visualization and analysis to help us to see
the information are discussed.
32
Cutting
Edge
Vibrational
Spectroscopy
for
Structural
Characterization of Chiral Drugs and Biologics
Rina Dukor, BioTools, 17546 Bee Line Hwy, Jupiter, FL 33458
No abstract submitted by author.
33
Raman Five Component Identification
Edita Botonjic-Sehic, Morpho Detection, 205 Lowell St., Wilmington, MA
01887
No abstract submitted by author.
34
Selective Comprehensive Two-Dimensional Separation for
Resolution Enhancement in Liquid Chromatography
Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., St.
Peter, MN 56082, Steve Groskreutz, Elliot Larson, Eric Talus, Sabrina
Trudo
An approach to enhancing the resolution of select portions of conventional onedimensional high-pressure liquid chromatography (HPLC) separations was developed, which we refer to as selective comprehensive two-dimensional HPLC (sLC x
LC). This approach breaks the long-standing link in on-line multi-dimensional chromatography between the timescales of sampling the first dimension separation and
the separation of fractions of first dimension effluent in the second dimension. This
allows rapid, high-efficiency separations to be used in the first dimension, while
still adequately sampling first dimension peaks. Transfer, transient storage, and
subsequent second dimension separations of multiple fractions of a particular first
dimension peak produces a two-dimensional chromatogram that reveals the coordinates of the peak in both dimensions of the chromatographic space. Preliminary
calculations suggest that the sLC x LC approach can be used to significantly (e.g.,
by a factor of five) reduce analysis time compared to conventional one-dimensional
separations, for moderately complex samples. Using existing valve technology we
find that the approach is very repeatable (percentage of relative standard deviation
of peak area < 1.5%), even at very short first dimension sampling times - as low
as one second. Example applications of the sLC x LC approach in environmental,
biological, and pharmaceutical analysis are presented. We anticipate that future
advances in the valve and control technology used here will significantly widen the
scope of possible applications of the sLC x LC approach.
29
Research and Development (R&D) Activities in Support of Enabling
Real-Time Release (RTR) in Initial Filing
Yang Liu, Pfizer, Eastern Point Rd., Groton, CT 06340
Most of the current RTR applications are established on a relatively large number
of batch experiences for commercialized products. The batch history demonstrates
the process capability; maps out the normal operating space and exhibits the validity
of the RTR methods. In the QbD paradigm, the process and product understanding obtained during the R&D stage provides valuable information to lay a sound
scientific foundation for RTR applications. For process analytical technology (PAT)
methods, e.g., near- infrared (NIR) methods, the R&D knowledge allows the models
to be established on the product and process design space. This approach can
potentially eliminate or reduce the method development and validation effort at the
commercial stage. In this presentation, the challenges of enabling RTR for new
drug products and how these challenges can be addressed in the research and
development stage are discussed using a recent filing example of NIR for content
uniformity and assay.
30
Accentuate the Positive: Building on Spectroscopy’s Strengths to
Solve Industrial Analytical Chemistry Problems
Nancy Jestel, SABIC, New Technology, 1 Noryl Ave., Selkirk, NY 12158
Vibrational spectroscopy, particularly infrared spectroscopy, is a workhorse technique in any industrial laboratory, from quality assurance to research and development. It provides qualitative and quantitative chemical composition information on
a wide variety of sample forms quickly and easily. However, many industrial problems require information that is beyond the technique’s abilities, such as quantifying
functional groups present in low concentrations (ppm), understanding complex mixtures, or analyzing samples smaller than its spatial resolution limit (~ 10 µm). These
limitations could be enough to prevent vibrational spectroscopy from playing a key
role in innovative research and development projects, but hyphenating or combining techniques and utilizing chemometrics can mitigate many of them. Since our
business› charter is to develop innovative new materials, we have a never-ending
supply of new, widely varied projects that need creative analytical solutions. We
might accomplish this by developing new methods on homemade systems built from
existing equipment or by identifying, evaluating, and implementing new commercial
technology. A variety of examples of this strategic approach are highlighted, including hot cell infrared spectroscopy to examine thermally induced polymer reactions,
the development of liquid chromatography-infrared spectroscopy (LC-IR) methods
to analyze complex polymer mixtures, atomic force microscopy-infrared spectroscopy (AFM-IR) to characterize very small inclusions in plastic parts, and combining process instruments to monitor polymerization reactions in-situ. By leveraging
spectroscopy’s strengths to address its weaknesses, we ensure both that it provides
valuable information to the business and that practicing industrial spectroscopists
have a stimulating work environment.
35
Fast, Direct and Acetonitrile-Free High-Performance Liquid
Chromatographic Enantioseparation of Chiral Benzyl Alcohols
under Reversed-Phase Mode
Ruoqiu Wu, Seton Hall University, Department of Chemistry, 400 South
Orange Ave., South Orange, NJ 07058, John R. Sowa, Jr.
Chiral benzyl alcohols are a series of important organic molecules with broad application in the fields of biocides, agricultural, material, and pharmaceutical industries. Herein, we report the first direct and acetonitrile free high-performance liquid
chromatographic enantioseparation of chiral benzyl alcohols under reversed-phase
mode. Enantioseparation of all of the seven selected chiral benzyl alcohols on the
six polysaccharide-based chiral columns are presented, and six out of seven analytes can achieved base-line separation (Rs > 2.0) under optimized conditions. A green
chemistry concept is introduced by using economical and environmentally friendly
solvents such as methanol, ethanol and water instead of acetonitrile or THF. The
optimized methods are validated and applicable for chemical and pharmaceutical
industries. The effect of temperature and eluting strength on the enantioseparation
is investigated and discussed. Thermodynamic studies indicate transitions of conformation and/or higher order structure of the chiral stationary phases under specific
changes in mobile phases. Entropy-enthalpy compensation under reversed-phase
mode results in relatively constant in enantioselectivity while changing the solvent
strength. Linear entropy-enthalpy compensation curve is obtained in two chiral columns across all of the chiral benzyl alcohols.
[1] T. O’Brien, L. Crocker, R. Thompson, K. Thompson, P. H. Toma, D. A. Conlon,
B. Feibush, C. Moeder, G. Bicker, N. Grinberg, Anal. Chem., 1997, 69, 1999.
[2] G. Venkatasami, J. R. Sowa Jr., Anal. Chim. Acta, 2010, 665, 227.
[3] T. D. Booth, I. W. Wainer, J. Chromatogr. A, 1996, 741, 205.
6
2012 EAS Abstracts
November 2012
39
[4] Y.Okamoto, E. Yashima, Angew. Chem., Int. Ed., 1998, 37, 1021.
Novel Developments in Separation Modeling Technologies
Imre
Molnár,
Institute
for
Applied
Chromatography,
Schneegloeckchenstrasse 47, Berlin 10407 Germany
Since regulatory authorities are moving strongly towards separation methods, which
are developed according to quality-by-design (QbD) principles, there is an increased
need to plan and model separation processes based on solid science. There is an
increased request also for continual improvement of ultra- pressure liquid chromatography (UPLC) / ultra high-pressure LC methods in regular time intervals. Another
issue is to ensure a higher degree of method robustness, which allows the trouble
free use of a method under more complex circumstances in a global economy. QbD,
continual improvement and method robustness in HPLC can be achieved by separation modelling. This presentation compares different approaches of control strategies in the rapidly changing analytical arena, which is moving towards technologies
for better drug safety and faster new drug development. It is shown how modelling
creates a platform for design space, helping also to visualize robustness in a unique
way. Separation modelling provides a deep chromatographic understanding and
helps to create the necessary QbD framework for UPLC/UHPLC, achieving optimal
methods in a fast and transparent way.
[5] S. P. Simeonov, A. P. Simeonov, A. R. Todorov, V. B. Kurteva, Amer. J. Anal.
Chem., 2010, 1, 1.
36
Troubleshooting
a
Highly
Challenging
Size
Exclusion
Chromatography Method for a Pegylated Protein Formulation
Joseph J. Valente, Bristol-Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08903, Kathleen A. Kelly, Mark S. Bolgar
Adsorption of high molecular weight species to column resins is a common issue
associated with size exclusion chromatography (SEC) of protein pharmaceuticals.
Systematic screening of column chemistries and mobile phase conditions during
method development is therefore necessary to optimize recoveries. Unfortunately,
samples available during method development may not fully represent the entire
spectrum of samples encountered during product development and stability programs, and while method development is an iterative process, practical and technical challenges can limit redevelopment opportunities. This discussion summarizes
SEC method troubleshooting strategies that were designed in response to a particularly adsorption-prone species observed in a stressed pegylated protein formulation.
Adsorption of this species to column and instrument surfaces complicated quantitative analysis and reduced column lifetime. An unstressed drug product solution
was used to monitor accumulation and carryover effects of the adsorbed species.
Additionally, multiple co-solvents including arginine, Brij-35 and diethylene glycol
were investigated for utility in mitigating adsorption and carryover. Brij-35 altered the
SEC profile making it unsuitable as a mobile phase additive but it was found to be
uniquely effective for removing material that accumulated on the column and in the
HPLC system. Implementation of a periodic Brij-based system cleaning procedure
ultimately improved data quality and extended column lifetime without requiring any
method redevelopment. Further investigations indicated that the adsorption-prone
species was specific to stressed product stored in prefilled syringes. Insights into
the delivery device specificity of this species and the observed effects of Brij-35 are
discussed.
40
Novel, Universal Approach for the Measurement of Natural
Products in a Variety of Botanicals and Supplements
Ian N. Acworth, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Mark Netsch, Marc Plante, Mark Roman
Botanicals contain a great diversity of compounds that can show extreme variation
in their physicochemical properties. Analysis of potentially active components can
be challenging as not all contain a chromophore or can be ionized, thereby limiting
the use of UV absorbance and mass spectrometry, respectively. High-pressure liquid chromatography (HPLC) with charged aerosol detection is a sensitive, universal
(nonselective) approach that can measure any nonvolatile and many semi-volatile
compounds. A number of isocratic and gradient HPLC with the Thermo Scientific
Dionex Corona Charged Aerosol Detector (CAD™) methods were developed and
evaluated for the measurement of phytochemicals extracted from a variety of botanicals including: triterpene glycosides from black cohosh (Cimicifuga racemosa);
ginkgolides and bilobalides from ginkgo (Ginkgo biloba); ginsenosides from ginseng
(Panax ginseng); silibinins in milk thistle (Silybum marianum); ursane and oleanane
triterpenes from gotu kola (Centella asiatica); and diterpene glycosides from stevia
(Stevia rebaudiana). Analytes showed consistent response independent of chemical structure (typically < 10% variability between compounds corrected for gradient
elution). All methods had a wide dynamic range (~four orders of magnitude), good
sensitivity (typically low ng levels of detection), and excellent reproducibility (relative
standard deviations typically < 2%) even at low detection levels. Comparative data
from evaporative light scattering and ultraviolet detection are also discussed.
37
Impact of Pressure on Secondary Structure of Proteins under
UHPLC Conditions,
Alexey Makarov, Novartis, One Health Plaza, East Hanover, NJ 07936,
Rosario LoBrutto, Paul Karpinski
There are several spectroscopy techniques that allow for analyzing protein secondary structure in a solution. However a majority of these techniques require using
purified protein concentrated enough in the solution to produce a relevant spectrum.
Fundamental principles for the usage of ultra high-pressure liquid chromatography
(UHPLC) as an alternative technique to study protein secondary structures in a solution were investigated. Several “model” proteins, as well as several small ionizable
and neutral molecules were used for these studies. The studies were conducted
with UHPLC in isocratic mode using premixed mobile phases at constant flow rate
and temperature. The pressure was modified by a backpressure regulator in a range
of about 6000 psi to about 12000 psi. It was found that when using a mobile phase
composition which facilitates altering of protein secondary structure (alpha-helix denaturation), the retention time factors of the proteins increased linearly upon pressure increase. On the other hand, when using a mobile phase composition which is
less restrictive in terms of protein denaturation, it was observed that the retentiontime factors of the proteins displayed a much steeper increase upon pressure increase. The impact of different mobile phase compositions on the denaturation of
the proteins was also studied using circular dichroism. Moreover, the behavior of
the retention factors of the proteins under different mobile phase compositions and
pressures were studied with different pore size silica’s.
41
Measurement of Essential Nutrients for Algae Growth and Biofuels
Production Using a Discrete Analyzer
Elizabeth A. Badgett, OI Analytical, 151 Graham Rd., College Station,
TX 77845, W. Gary Engelhart, William C. Lipps, Phil Griffiths
The United States Department of Energy issued the National Algal Biofuels Technology Roadmap in May, 2010 which identifies a range of technologies being investigated for production of algae-based renewable fuels and chemicals. The photosynthetic efficiency of algae is significantly higher than terrestrial crops and algae can
be cultivated in saline, brackish or wastewaters in locations lacking arable land.
These favorable characteristics vis-à-vis other biomass feed stocks are a major
factor driving government and private sector research and development projects
to commercialize algae-based fuels. This poster describes the use of a discrete
analyzer for measuring essential nutrients required to optimize propagation of photosynthetic algae that supply feed stock materials for biofuels production. Analytical
results obtained with saline and freshwater growth media samples are presented.
38
Analyzing Phenolic Compounds in New Hampshire Maple Sap
Using HPLC
Elizabeth Brady, University of New Hampshire, Department of Chemistry,
Durham, NH 03824, Sterling Tomellini, Walter Shortle, Barrett N. Rock,
Martha Carlson
Recently, many studies have focused on phenolic compounds in plants due to
their antioxidant, anti-mutagenic and antiradical capabilities. We are interested in
investigating these compounds as possible indicators of environmental stress in
sugar maple trees. Our objective is to develop high-pressure liquid chromatography
(HPLC) methods for the direct analysis of phenolic compounds in maple sap in
an effort to monitor qualitative and quantitative changes throughout the maple sap
tapping season. The analysis, however, poses several challenges including achieving the required sensitivity, working with a biological matrix and the effects of high
concentrations of sugar in the sap. We are also concerned with safety and ease of
use as this method is being developed for future use by high school students. Data
obtained from samples provided by volunteer producers around the state of New
Hampshire are presented and the 2010 and 2011 tapping seasons are compared.
Initial observations suggest trends that could provide potentially valuable information. The development and results of confirmatory liquid chromatography mass
spectrometry studies are also discussed.
42
Protein Structure Analysis of Gonococcal Cell Surface Protein
Antibody Binding Domains Using a Molecular Model Approach
Yu-Shu Ting, Zeus Scientific, 200 Evans Way, Somerville, NJ 08876
Pilin is the major surface protein of bacteria N. gonorrhoeae which is responsible
for the sexually transmitted infections gonorrhea. In the 1980s, after the pilin protein
from a variant of strain MS11 was isolated, clinical laboratories sought to identify
the best peptide sequence antigen to produce antibodies with high specificity and
less cross-activity. Three synthetic peptide sequences (69-84, 48-60 and 135-151)
were found to produce antibodies with high titer levels. The first two sequences are
from the protein’s common region while the third (135-151) is from variable region.
Recently in 2006, the structure of the pilin protein was determined using electron microscopy and X-ray protein crystallography. In this presentation, a protein structure
analysis using research collaboratory for structural bioinformatics protein database
(PDB) (code: 2HI2 and 1IGY) and Swiss PDB viewer molecular model software
was conducted for the analysis of three pilin epitopes (69-84, 48-60 and 135-151)
binding domain against the immunoglobulin. The molecular level analysis provides
structural rationale on why the epitopes exhibits high antibody affinity properties
7
2012 EAS Abstracts
November 2012
found in previous immunoassay techniques. The pilin epitope loop bends due to the
position of proline and provides a “key and lock” fitting to the IgG Fab clam shell size
binding pocket. The pilin epitope at residue 48-60 shows the best fit into the hole
between hyper variable loops from both IgG heavy chains and light chains, which attach to a beta-sheet of immunoglobulin domain. Additionally, the pilin protein structure model elucidated its multiple function site behavior such as glycosylation site,
assembly/disassembly interaction, and protein aggregate.
quickly determine the concentration of prostanoids and sex hormones and their metabolites from urine with high-pressure liquid chromatography (HPLC) and ultraviolet detector. In our study, exercise training was applied to postmenopausal AfricanAmerican women with hypertension. Urine samples were collected before and after
training period. Our study explores the effects of physical training on the treatment
of hypertension and the correlation between inflammation and hormone protection.
46
Analysis of Single Amino Acid Mutations in Intact Proteins
Rob Freeman, SGE, 2007 Kramer Ln., Austin, TX 78758
With the increasing availability of high resolution mass spectrometers the analysis
of intact proteins becomes a feasible alternative to the more commonly employed
shot-gun techniques in proteomics. High resolution separations of proteins are hindered by the size of the analyte molecules and the resulting low diffusion rates
especially inside the pore system of the stationary phase. The stationary phase of
the columns employed in these studies is a 3-μm C8 silica with 1000 Å pore size.
By using a dedicated protein separation column (ProteCol™ C8 HQ1003) we demonstrate that it is possible to separate ribosomal proteins derived from a rat liver
with excellent resolution and identify single amino acid mutants in a 33 kDa protein.
43
Analysis of Polypeptide Glucagon and its Analogs in Biological
Matrices Using Liquid Chromatography Coupled with Mass
Spectrometry
Hang Zeng, Hoffmann-La Roche, 340 Kingsland St., Nutley, NJ 07110,
Zhenmin Liang, David Moore, Catherine Bentzley
Polypeptide drug glucagon and its analogs are developed for treatment of metabolic
diseases such as hypoglycemia, diabetes and obesity. Their high potency normally
results in low levels in biological samples in pharmacokinetic (PK)/ pharmacodynamic (PD) studies. Therefore a sensitive bioanalytical method is required to monitor low ng/mL levels in blood plasma and urine samples at therapeutic doses. This
work describes a highly sensitive, fast liquid chromatographic method with protein
precipitation and mass spectrometric (MS) detections including tandem MS/MS, liner ion-trap and high-resolution time-of-flight or Orbitrap FTMS functions. Signal-tonoise ratio of analyte was enhanced, and endogenous interferences were reduced
by monitoring the selective MS fragment ions or accurate mass measurements.
Sample preparation consisted of simple protein precipitation with mixed solvent containing acetonitrile, 0.5 % formic acid and an internal standard. The analytes were
separated on a Supelco Ascentis Express Peptide C18 fused-core column (particle
pore size 160Å) using a linear gradient elution from the mobile phases consisting of
water and acetonitrile, each containing 0.1 % formic acid. Analytes were detected
with MS in positive ion selected reaction monitoring mode using 4+ charged state
as the precursor ion. A bioanalytical assay with a linear dynamic range from 1~1000
ng/mL in plasma and urine was developed with good accuracy and precision. The
pros and cons of using different MS detection systems and ionization techniques
are discussed in this presentation. This method offers excellent sensitivity with low
sample volume consumption, low cost, fast throughout and high robustness for routine analysis of pharmacokinetic studies.
47
Determination of the Thermodynamics of Retention Using an Octyl
HPLC Stationary Phase Under Varying Mobile Phase and
Temperature Conditions
Caitlin Galvin, Ursinus College, 145 Plymouth Rd., Springfield, PA
19064, Eric J. Williamsen
High-pressure liquid chromatography (HPLC) is a widely used technique that utilizes various physical interactions between an analyte and stationary phase, analyte and mobile phase, and the mobile phase with the stationary phase, to perform
separations. Because the combinations of these physical interactions are complex,
the molecular processes underlying HPLC are not completely understood. Using
an octyl stationary phase, 10% methanol, 90% water mobile phase, and a sodium
nitrite dead time marker, retention of approximately 40 analytes with multiple sites
of functionality was measured at temperatures ranging from 5.0 to 65.0 ˚C. Analytes
containing phenyl, alcohol, cyano, halogen, non-polar, and other functional groups
were studied. Van’t Hoff plot were generally linear with a few exceptions such as anisole and 1-methyl-2-pyrrolidinone. Selectivity plots (the natural log of the selectivity
factors (α) of two analytes versus the inverse temperature in Kelvin) were made to
determine whether the volume phase ratios were constant and to observe differential changes in entropy. Principal component and cluster analysis were applied to
emphasize retention patterns. Trends in retention, and in the enthalpy and entropy
of retention are presented and compared to results obtained while using other stationary phases (octadecyl and fluorinated octyl), other mobile phase conditions (with
organic/water ratios of 20:80, 30:70, and 50:50) and substituting acetonitrile instead
of methanol as the organic component.
44
Determination of Atorvastatin and its Metabolites in Human Plasma
Using Polymeric Solid-Phase Extraction Sorbents, Superficially
Porous HPLC Columns, and Tandem Mass Spectrometry
Ritu Arora, Agilent Technologies, 25200 Commerce Centre Dr., Lake
Forest, CA 92630, David Jones, William Hudson, Joan Stevens, William
Long
Due to the expiration of blockbuster drug patents, there is a tremendous opportunity for generic drug manufacturers to not only manufacture these drugs, but also
develop fast, clean, and sensitive methods for their analysis from biofluids. Lipitor
(the brand name for atorvastatin) is the world’s best-selling drug and is designed
to lower both cholesterol and triglycerides. It is in the drug class of lipid-lowering
agents called statins. It is metabolized to two hydroxylated metabolites, o-hydroxyatorvastatin and p-hydroxyatorvastatin. About 70% of the total plasma activity is
accounted for by its active metabolites. Previous studies conducted on atorvastatin
and its metabolites in human plasma samples involve little or no sample preparation, protein precipitation or liquid-liquid extraction being the methods of choice.
Long analysis times (in the order of 50 min.) are common for these separations. In
this study, a fast and sensitive analytical method are presented using Bond Elut®
Plexa polymeric solid-phase extraction cartridges, Poroshell high-pressure liquid
chromatography (HPLC) columns with tandem mass spectrometry detection. The
benefits of solid phase extraction are compared against alternative sample preparation work flows such as protein precipitation and filtration. This approach offers a
complete, automatable solutions approach for the determination of atorvastatin, and
its metabolites, which can be easily implemented in studies involving pharmacokinetics, drug metabolism, and clinical pharmacology/toxicology.
48
LC Method Comparison for Ultra-Low Concentration of Residual
Acrylamides in Personal Care Formulations
Maria Diss, Ashland Specialty Ingredients, Wayne, NJ 07470, Larry
Feeley, Paul Suszczynski, Qian Wei
Personal care products are quite complex in their formulations. Determining residual
quantities of unreacted synthesis materials such as monomers and solvents is crucial in the safety and product specification of these materials. Developing a method
to analyze and evaluate these residual species within these complex matrices is
most important. In this poster, examination of three liquid chromatography methods
for determination of ultra low concentration of residual acrylamide within a complex
sample matrix is discussed. These methods are compared in terms of precision,
accuracy, efficiency and reproducibility.
49
Detection of Protein Heterogeneity by HPLC
Atis Chakrabarti, Tosoh Bioscience, 3604 Horizon Dr., Ste 100, King of
Prussia, PA 19406
Protein heterogeneity is generated by posit-translational modifications, decomposition or chemical modification. Antibodies and recombinant proteins are now widely
used for the therapeutic treatment. So the evaluation of the heterogeneity of the
therapeutic antibody is essential for the development, stability study and quality
control of the final product. The heterogenic moieties increase the risk of anaphylaxis or immuno reaction if not separated. In this presentation we have shown the
use of high resolution nonporous resin columns for ion exchange chromatography
(IEX) and hydrophobic interaction chromatography (HIC) to detect even one residue
difference of the proteins. Size exclusion chromatography (SEC) is suitably used to
separate the monomer (150 kDa) and the dimer (300kDa) of a human monoclonal
antibody to very good baseline resolution. Papain and pepsin digested antibodies
could be resolved to separate Fc, Fab and intact protein to the baseline resolution
by SEC. Analysis of aggregates, fragments and proteins treated with PEG and the
analysis of hapten conjugated protein using SEC are also reported in this presentation. A new reversed-phase chromatography (RPC) column with large pore size of
45
Chromatographic Analysis of Female Sex Hormones and
Prostanoids in Hypertension Subjects
Guoxiu Wei, Susan Jansen-Varnum, Temple University, Chemistry
Department, 130 Beury Hall, 1901 N. 13th St., Philadelphia, PA 19122
Hypertension is primary contributor to cardiovascular disease. In general, men have
a higher risk of hypertension than women at the same age, and women have an
increasing risk of hypertension after menopause. Several factors are assumed to
contribute to the development of hypertension such as body weight, diet, gene, life
style and gender. Studies have indicated that female sex hormones may play a role
of protection of women from hypertension. Inflammation mediators are also involved
in pathogenesis of hypertension. Studying the level change of inflammatory lipids
and sex hormones are helpful to understand the balance between inflammation and
self-protection in hypertension patients. We have developed a simple method to
8
2012 EAS Abstracts
November 2012
300 Å is shown to be applicable for the protein analysis. Overall this presentation is
focused on the detection and separation of protein heterogeneity by high-pressure
liquid chromatography using four different modes of chromatography such as SEC,
IEX, RPC and HIC.
derstood that two columns should be preferably from the same lot. Using a sunscreen sample, two sets of experiments were performed, each with 20 injections:
for one set, two new columns were used, and for the other set two previously-used
columns were used. The results clearly confirm the significant productivity increase
that was expected. They also show that both the chromatographic profile and response precision of the two columns in each set were not different from one another.
Furthermore, the results show that there was no significant difference between the
older and newer columns.
50
Reduction of Matrix Interference in Bioanalytical LC-MS Analyses
by Simultaneous Protein Precipitation and Phospholipid Removal
in One Step
Xiaoning Lu, Supelco/Sigma-Aldrich, 595 North Harrison Rd., Bellefonte,
PA 16823, Craig R. Aurand, Roy Eksteen, David S. Bell
Endogenous proteins and phospholipids are two of the major causes of matrix effects in liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses.
The concentrations of these proteins and phospholipids are often high and variable. Liquid-liquid extraction and solid phase extraction are two commonly used
procedures for cleanup of such proteins and phospholipids during the preparation
of biological samples. Both of the procedures, however, involve multiple steps and
tend to be time consuming and labor-intensive. While the use of protein precipitation affords a faster method for sample preparation, this technique is not effective in
removing phospholipids. The presence of phospholipids in the sample extract can
significantly impact the quality of the data derived from using this technique. We
have developed a one-step procedure for simultaneous protein precipitation and
phospholipid removal from biological matrices. The procedure involves the use of
zirconia-modified silica sorbents to selectively remove phospholipids from biological
samples. And the proteins are precipitated with organic solvents. The protein precipitation and phospholipid removal are realized in a single step. Our results indicate
that phospholipids in various biological fluids such as plasma and serum were effectively eliminated and ion suppression of the MS signals was greatly reduced. We
demonstrate with several application examples that our procedure leads to higher
sensitivity and better data quality.
54
LC-MS Methods Development for Retention of Polar Analytes
Using a Mixed Mode Approach
Van Truong, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Roy Helmy,
David Waterhouse, David Schenk
The objective of this study was to develop a general open-access liquid chromatography mass spectrometry (LC-MS) method that is able to retain and separate polar
analytes. The separation of highly polar analytes using traditional reversed-phase
chromatography can be problematic due to insufficient retention. One alternative
that has been utilized to deal with this issue is separation under hydrophilic interaction liquid chromatography (HILIC) conditions. Nevertheless, the mobile phases
used in HILIC methods are not always MS friendly. In this study, the stationary
phase evaluated was a mixed mode, Imtakt Scherzo SW-C18. This stationary
phase is a C18 loaded phase containing a low amount of strong ionic ligands, and
the analytes are retained through a combination of ion exchange, reversed-phase,
and normal phase interactions. The developed method was successfully applied in
the analysis of various radio-labeled and non-radio labeled compounds synthesized
within Merck, as well as catalog products, such as Phenylephrine and Metformin.
55
www.hplcsimulator.org - a Free, Open-Source, Sophisticated HPLC
Simulator for use in all Levels of HPLC Education and Training
Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., St.
Peter, MN 56082, Paul Boswell, Peter Carr, Mark Vitha, Gary Mabbott,
Megan Nagle
A free, open-source high-pressure liquid chromatography (HPLC) simulator has
been developed at www.hplcsimulator.org to fill a present need for an HPLC simulation resource that is user-friendly, inexpensive, and mimics the sophistication of current instrumentation. The intuitive controls offered by this site allow users to easily
adjust a wide number of experimental parameters, including temperature, flow rate,
and column properties, and immediately observe the impact of any changes on the
provided chromatogram. The simulator is simple enough for a beginner, yet offers
a depth of complexity to challenge more advanced users. A detailed help menu,
tutorials, and a number of sample classroom activities are freely available for use on
the site. Some initial results of implementing the HPLC simulator in undergraduate
quantitative analysis courses are discussed.
Structure Elucidation of a Process Impurity in a P38 Map Kinase
Inhibitor Drug Substance Using LC-MS and NMR
Scot J. Campbell, Boehringer Ingelheim, 900 Ridgebury Rd., PO
Box 368, Ridgefield, CT 06877, Fenghe Qiu, Alice Granger, Daniel L.
Norwood, Zhulin Tan, Jeff Song
Pharmaceutical impurity identification is not only a regulatory requirement, but also
a critical problem solving tool during chemical process development and pharmaceutical product development. Elucidation of the chemical structure of an unknown
by-product or a degradation product is the first step toward understanding of the
formation chemistry of the impurity and subsequent process and formulation optimization. This presentation gives a case study in which an unknown process impurity
was observed in a P38 MAP kinase inhibitor drug substance. The level of the impurity was at approximately 0.2% at release, which is above the International Conference on Harmonization (ICH) identification threshold for impurities in a new drug
substance. After a multidisciplinary analytical investigation, the chemical structure
of the impurity was fully characterized, as shown below. This presentation describes
the details of the structure elucidation process using liquid chromatography mass
spectrometry (LC-MS), isolation, and nuclear magnetic resonance (NMR) characterization, and discusses the formation mechanism of the impurity.
52
56
51
Requirements, Recommendations and Considerations for Optimal
UHPLC
Wilhad M. Reuter, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484
With the significant rise in interest and implementation of ultra high-pressure liquid chromatography (UHPLC) systems, one must also consider the necessary demands of operating such a system, especially for routine operation. These demands
are typically greater than can be expected for a conventional LC system. As such,
there are many that are still somewhat unfamiliar with the key requirements and
concerns. This poster focuses on these fundamental requirements/considerations,
including instrumental bandwidth, tubing concerns, loop/sample volumes and provides key recommendations. Where relevant, these are highlighted by specific
before-and-after examples.
Dissolution Experiments at Varying pH in Presence of Lipid Layer
to Mimic Absorption and Using Polymers to Prevent Drug
Precipitation
Jon Mole, Sirius Analytical, 100 Cummings Center, Suite 231C, Beverly,
MA 01915, John Comer, Karl J. Box
A suspension of drug (~10mg) and PVP polymer (5 - 30mg) is introduced into a
stirred chamber containing 15mL of phosphate/acetate buffer plus hydrochloric acid
at pH2 to represent stomach conditions. An aliquot of lipophilic solvent is introduced
above the aqueous layer. A fiber-optic probe monitors the UV spectra of the solution at regular time intervals. The pH is varied with time by addition of an alkaline
solution to represent passage of the compound from the stomach to the intestine.
The concentration in solution vs. time at each pH is calculated from the spectra.
The method was evaluated with papaverine, a compound with a basic pKa at 6.37.
Under aqueous conditions and without lipid present, the papaverine had dissolved
after 30 minutes at pH 2, and it remained in solution at pH 3.8 and pH 5.4. However,
concentration in the aqueous phase dropped rapidly at pH 7.3 and heavy precipitation was observed as the solubility is exceeded. In the presence of lipid, the concentration in the aqueous phase decreased at pH 5.4 as the papaverine was absorbed
into the lipid layer. However, precipitate was still observed in the aqueous layer at
pH 7.3. A similar pattern was observed when 30mg of PVP polymer grade K29-32
was included. However, with 30mg of PVP copolymer grade S630 drug precipitation
was prevented, allowing for more drug absorption into the lipid. These experiments
could be used to help select suitable formulations for enhanced solubility and oral
absorption.
53
Concomitant Use of Two LC Columns to Reduce the Equilibration
Time in the Analysis of Sunscreen Agents
Njies Pedjie, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484
The advent of the ultra high-pressure liquid chromatography (UHPLC), with pumps
capable of generating pressure up to 18000 PSI, has brightened the horizon of the
liquid chromatography technology. A typical twenty minute separation on a traditional HPLC is now often achieved in less than three minutes. A faster separation
also means a reduction in solvent usage and waste, less environmental impact and
substantial savings. However, there is still room for improvement, especially regarding gradient methods, as the re-equilibration time can be as long as the run time.
The technology presented in this application note eliminates the equilibration time
between injections, by the concomitant use of two columns and two pumps. While
the sample is being injected in one column, the other column is allowed to equilibrate, and vice versa. For the effective use of this strategy, it must be established
that both columns exhibit similar chromatographic profiles and responses. It is un-
9
2012 EAS Abstracts
November 2012
57
Stability Testing of Mercapto Compounds Using High-Performance
Liquid Chromatography and Argentometric Titration
Peter P. Yeh, MacDermid, 245 Freight St., Waterbury, CT 06702
The stability of 2-mercaptobenzoic acid (2-MBA) in a sodium hydroxide solution
under constant reflux has been investigated. Samples of the refluxing solution were
taken periodically and submitted for analysis using the high-performance liquid
chromatography (HPLC) method to determine the 2-MBA concentration. In this analytical procedure, 2-MBA was eluted off a reversed-phase column and was detected
using a spectrophotometric detector. Our investigation has shown that 2-MBA was
unstable in the caustic medium and was converted completely to the disulfide compound via oxidation within 5 or 6 hours. In comparison, 2-MBA in de-ionized water
was found to be relatively stable at room temperature, loosing approximately 20% of
its initial concentration after three days. The stability of mercaptopropyltrimethoxysilane (MPTMS) in dipropylene glycol methyl ether (DPGME) has also been investigated. Targeting its thiol group, MPTMS was determined via argentometric titration
using a silver nitrate volumetric solution as titrant and a silver electrode for potentiometric measurement. A significant change in the electrode potential during titration
signaled the total consumption of the analyte by the titrant and thus the end of the
titration. The stability of MPTMS in a DPGME solution containing 4% water was
found to be only slightly lower than that of the same solution without added water.
In this paper, details of the stability testing protocols and the analytical procedures
used for determining the analyte concentration are presented. The test results on
several mercapto compounds and the significance of their solution stability on their
intended use in catalysis are also discussed.
N-protected compounds are being followed by HPLC and the products are being
characterized using nuclear magnetic resonance and infrared. Microwave assisted
syntheses of the N- protected PMPA using the above groups are also being investigated to prepare the compounds on a small scale.
60
Orthogonality Studies to Accelerate Preparative Chromatographic
Method Development
Amanda Marie Makarewicz, Merck, 126 East Lincoln Ave., Rahway, NJ
07933, William Long, Derek Von Langen
One goal of the Merck Center of Excellence for Separations and Purifications is to
provide preparative chromatographic support for drug discovery. Towards that end,
we are driven to maximize throughput with a minimum of resources. In an effort to
achieve these goals, we implemented an orthogonal column screening technique.
This technique affords for a rapid identification of suitable preparative chromatographic conditions. To illustrate the application of this technique, we screened (in
collaboration with Agilent Technologies) a representative set of small molecule pharmaceutical compounds on five unique stationary phases under mobile phase conditions suitable for preparative scale chromatography. The poster to be presented
illustrates the results of this study and demonstrates the utility and application of
this technique.
61
Determination of Temperature-Dependent Degradents of Chemical
Vapor Deposition (CVD) Precursors using Pyrolysis-GC/MS
Anna L. Johnson, Arkema, 900 First Ave., King of Prussia, PA 19406
Fully understanding the thermal decomposition products of CVD are invaluable in
optimizing the atmospheric pressure CVD (APCVD) coating process used to coat
substrates for optoelectronic applications. Several deposition parameters affect the
quality of the resulting film, the most notable being temperature and precursor reactivity with an oxygen source. During deposition, the production of excess by-products or an inefficient reaction pathway may affect multiple performance parameters.
The precursors used to produce APCVD films are often pyrophoric alkyl metal complexes, complicating conventional analytical techniques. The solution presented is a
closed system that simulates the APCVD coater environment by coupling the volatile precursor streams to a pyrolysis-GC/MS. Utilizing the pyrolysis instrument, the
high temperatures used to simulate the heated substrate are easily obtained in an
airtight, water free system. The mass spectrometer coupled to the pyrolysis unit, is
used to elucidate the thermal decomposition products.
58
Application of LC-NMR and Solid-State NMR to the Problem of
Container Closure Extractables
James O. Mullis, Boehringer Ingelhiem, PO Box 368, Ridgefield, CT
06877, Mark Davis, Thomas Egert, Nina C. Gonnella, Shirley Rodriguez,
Daniel L. Norwood
In the development of drug product container closure systems and medical devices, regulatory approval may require identification of extractables from plastic
and elastomeric materials. These packaging materials may come into contact with
the drug product formulation and have the potential to leach chemical additives
and other substances into the drug product. Hyphenated techniques such as liquid
chromatography mass spectrometry (LC-MS) are typically utilized to elucidate the
molecular structures of unknown extractables/leachables, however mass spectral
information alone can be insufficient for full structure elucidation of many unknown
chemical entities. Nuclear magnetic resonance (NMR) spectroscopy, in concert with
mass spectral data, is capable of structure elucidation of many organic chemical
entities which elude structural elucidation by mass spectrometry. However, NMR
techniques are extremely insensitive relative to mass spectrometry. This poster
presents the application of automated solid-phase extraction (SPE) LC-NMR for
the structural elucidation of extractables from container closure system construction materials. The authors show that SPE isolation methods were optimized with
model compounds over a wide range of Log P using a reverse-phase high-pressure
liquid chromatography (HPLC) platform. Unambiguous structure elucidation of individual extractables was accomplished using on-line SPE LC-NMR, followed by
chromatographic peak isolation and further analysis by high-field cryo-probe NMR.
In addition, data are shown of a solid-state NMR (ssNMR) analysis of a cryo-ground
packaging component to demonstrate the direct analysis of potential extractables.
62
Rapid Measurement of Olive Oil Adulteration with Soy Based
Vegetable Oil Using DSA-TOF MS with Minimal Sample Preparation
Avinash Dalmia, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484,
Blas Cerda, Craig Whitehouse
Among edible oils, olive oil shows important and outstanding characteristics due to
its differentiated sensorial qualities (taste and flavor) and higher nutritional value.
Olive oil is one of the most adulterated food products of the world, due to its relatively low production and higher prices as compared to vegetable and seed oils, to
increase economic profits. The addition of vegetable oils, of low commercial and nutritional value compared to olive oil, causes frequent problems for regulatory agencies, oil suppliers and consumers. Extensive scientific effort has been expended to
develop a rapid, reliable and cost effective analytical approach for measurement of
adulteration of olive oils with other oils. In the past, methods employing gas chromatography mass spectrometry (GC-MS) and high-pressure liquid chromatography
(HPLC) hyphenated to MS (HPLC-MS) have been implemented for this purpose.
These methods are time consuming, expensive and require extensive sample
preparation. Recently, we have developed direct sample analysis (DSA) ionization
source which utilizes a field free reagent ion generator. Olive oil samples were rapidly analyzed using this DSA ion source with minimal or no sample preparation. In
this study, the high-throughput DSA ion source was interfaced to a PerkinElmer
Time-Of-Flight Mass Spectrometer (TOF-MS) operating with high resolution and accurate mass analytical capability. Olive Oil, adulterated with soy based vegetable
oil, was analyzed in both positive ion and negative ion mode. Addition of soy based
vegetable oil to olive oil was measured down to levels of 2.5 % with an analysis time
less than 30 seconds.
59
Exocyclic N-Protecting Groups for 9-[(R)-2(phosphonomethoxy)
propyl]adenine (Tenofovir) a Precursor for Tenofovir Disoproxyl
Fumarate (TDF) an HIV Drug
Saraswathi Narayan, Stevenson University, 1525 Greenspring Valley
Rd., Stevenson, MD 21153, Janine Ladzinski, Jahnavi Patel, Amber
Woodruff, Hira Munir, Joseph M. Fortunak
Tenofovir disoproxyl fumarate (TDF) is a nucleotide reverse transcriptase inhibitor (NtRTI). TDF is a challenging drug to synthesize for several reasons. Tenofovir 9-[(R)-2(phosphonomethoxy)propyl]adenine (PMPA) is usually condensed with
chloromethyl isopropyl carbonate followed by the salt formation with fumaric acid.
One of the most common challenges faced during this process is the formation of
unwanted side products that results in low product yield. Many of the reagents used
during the synthesis of TDF are expensive. Additionally, lengthy extraction times results in the decomposition of the product in solution. The present experiment focuses on the protection of the primary amine group of PMPA to reduce the occurrence
of side products and possibly to prevent decomposition of products during extraction
process. The protected PMPA will then be condensed with chloromethyl isopropyl
carbonate followed by the removal of the protective groups by hydrogenation. Addition of chloromethyl isopropyl carbonate (CMIC) to the N,N-dibenzylformamidine
protected PMPA resulted in 54.64% yield (based on high-pressure liquid chromatography (HPLC)) of the exocyclic N-protected Tenofovirdisoproxy compound. Several
other protective groups like benzoyl, p-toluyl, Fluorenylmethoxycarbonyl (Fmoc)
and 2, 2, 2-Trichloroethyloxycarbonyl (Troc) were used. The syntheses of these
63
Tovacian Chemistry: A Quaternion Based Approach to ICP-MS
Design
Willie Johnson, Jr., GFT, 687 Walnut, Apt. 1, Easton, PA 18042
The Gyroscopic Force Theory introduces Tovacian Chemistry; a new field of chemistry wherein chemical or tovacian bonds are formed via the orthogonal interaction
of quaternions and their conjugates. Two identical moving charges, orthogonally
interacted, may not form an electrostatic repulsion. Indeed they may attract. Interacting the E’s of two moving charges along the x axis evinces an electrostatic
repulsion. Changing the angle of interaction of one charge to 30º diminishes that E’s
x component, diminishing the repulsion. At 60º diminution further increases until at
90º there is no E x component for that particular charge and therefore no electro-
10
2012 EAS Abstracts
November 2012
static repulsion. Charges are quaternions where ab = -cos q +e cos q. Quaternions
do not commute; ab does not equal ba. Rather ab =-ba. Cosine and sine pairs, respectively, about the unit circle represent quaternions. At 60º we find (1/2, (sqrt3)/2)
or ab. Its 90º counterpart is (-(sqrt3)/2, 1/2) which is -ba. Thus quaternionically (1/2,
(sqrt3)/2) = (-(sqrt3)/2, 1/2), therefore the formation of conjugates. The quaternion
(-(sqrt3)/2, 1/2) has a polar opposite counterpart, ((sqrt3)/2, -1/2). This must also
be equal to its 90º counterpart (1/2, (sqrt3)/2). At every 90º a conjugate is formed
being both equal to and opposite of its 90º counterpart. Therefore quaternions, as
conjugate charges, may attract. Conjugate interaction implies the obviation of the
strong force. In terms of inductively coupled plasma mass spectrometry engineering
and design, we interact ions in such a way as to minimize space charge repulsive
affects.
bottom and fly ash (as incineration by-products) using GC-MS/MS. During this study
instrumental limit of detections using GC-MS/MS were calculated in the low fg/ml
concentration ranges. This and further analytical performance is discussed alongside GC-HRMS; especially degree of agreement between the techniques.
67
A New Syringe Line for Highly Sensitive Applications
Dan R. DiFeo Jr., SGE, 2007 Kramer Ln., Austin, TX 78758, Rob
Freeman
As instrumentation becomes more sensitive and able to detect ever-smaller concentrations of sample, cleanliness and reactivity of the analysis system becomes
increasingly important. This issue has been addressed in most part by the instrumentation manufacturers using shorter, smaller diameter transfer lines and materials with very low reactivity. There have been no significant gains in the sample
introduction phase of the analysis system, namely the syringe, for some time. A
new range of syringes has been specifically designed, drawing on over 50 years of
knowledge in the analytical chemistry field, to address this issue. With increasing
sensitivity of detectors any sample carryover or any interaction of the syringe materials with the sample can negate the analysis. This new range of syringes is customizable; the user can select the most suitable syringe components for the analysis and
be assured they will have minimal effect on the analysis. These syringes also take
advantage of technology that dramatically improves syringe lifetime and stability.
This poster demonstrates the improved results seen when using new Diamond MS
syringes designed for higher sensitivity analyses.
64
An Economical Air-Based DART Ionizer-Quadrupole-MS for Rapid
Synthesis Monitoring and Quality Control Applications
Brian Musselman, IonSense, 999 Broadway, Ste. 404, Saugus, MA
01906, Joseph Lapointe, Michael Festa
A direct analysis in real-time (DART) is an open air ionization source has been coupled to a compact, fume hood-designed single quadrupole mass spectrometer. The
DART-expression™ MS utilizes a desorption chemical ionization sampling card, the
OpenSpot® (OS) Sample Card, to which liquid transferred from the reactor can be
applied directly minimizing sample clean-up. Determination of mass is completed
in seconds. The OS card uses a resistively heated metal screen to vaporize both
high and low vapor pressure molecules in under 20 seconds per sample. Samples
presented for characterization using a direct analysis in real time open air ionization
source (DART) are normally vaporized by using either pressurized gas chromatography grade helium, or nitrogen supplied from a bulky gas cylinder. Pressurized
gases require constant monitoring, physical movement of cylinders, and with the
cost of these gases increasing is a major component of the operating budget for
liquid chromatography mass spectrometry (LC/MS) systems. In order to reduce the
cost and complexity of the instrumentation we have investigated the use of air as
the DART carrier gas. A comparison of the relative ionization capability of the ambient ionization system using air vs. nitrogen and air vs. helium are discussed with
emphasis on practical quality control applications for chemical and food samples.
68
Alkyl Mesylate Formation Studies Using Pharmaceutically Relevant
Conditions
Amy F. Birch, Johanna Ubben, Fenghe Qiu, Boehringer Ingelheim,
Danbury, CT 06810
Methanesulfonic acid (MSA) is a common salt form of active pharmaceutical ingredients (APIs). There has been an ongoing concern that genotoxic alkyl sulfonates
such as methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and isopropyl methanesulfonate (IPMS) potentially can form during the manufacturing of
the API or the formulated product in the presence of alcohols. There is a regulatory
expectation that the pharmaceutical industry assess and control the risk of alkyl and
aryl esters of sulfonic acid in the API and drug product, if an API is a mesylate salt.
Analytical procedures were developed to support mechanistic studies on the formation of these alkyl mesylates. Our GC laboratory conducted experiments to study a
variety of conditions to see if they promoted mesylate formation. Three pathways
that can potentially lead to mesylate formation were looked at: 1) MSA plus an alcohol, 2) MSA plus an ester, and 3) transesterification. Our analyses found that a
mesylate will not form from a mesylate salt and an alcohol. The type of solvent used
affects the potential formation of mesylates. Mesylates will form from MSA and an
alcohol much faster than from MSA and an ester. Transesterification does not occur
easily in the conditions used for these studies. Once the mesylate API is made, if an
API or product is not acidic, it is an unfavorable matrix for the formation of genotoxic
esters of mesylate under pharmaceutically relevant conditions.
65
Minimizing False Negatives and False Positives in High-Resolution
Mass Spectrometry Through Valid Signal Extraction Widths and
Processing Techniques
Kevin Siek, LECO Corporation, 3000 Lakeview Ave., Saint Joseph, MI
49085, Joe Binkley, Jeffrey S. Patrick
High resolution mass spectrometry, like other growing technologies, can expand
the scientist’s horizons of research, while simultaneously offering faster avenues
to incorrect conclusions. Discussions with users and some examples published in
literature reveal conclusions based on raw extracted ion chromatograms (XIC’s) of
statistically invalid widths. Typically presented raw XIC widths are often too narrow
for the claimed resolving power of the instrument and represent an unacceptable
likelihood of false negatives due to the possible presence of larger partially resolved
isobars. High resolution time-of-flight mass spectrometry with comprehensive collision induced dissociation (CID) and advanced software tools for data interrogation
were successfully used in different applications to extract signals of drug metabolites and pesticides from close isobaric interference. Signal amplification strategies
based on comprehensive CID data allow valid XIC widths to be used without appreciably increasing the likelihood of false positives. Exploitation of parametric statistics
for spectral deconvolution of high-resolution data is also illustrated.
69
Sensitive Analysis of Underivatized Amino Acids and Peptides
Using UHPLC with Charged Aerosol Detection
Christopher A. Crafts, Thermo Fisher Scientific, 22 Alpha Rd.,
Chelmsford, MA 01863, Marc Plante, Mark Netsch, Bruce Bailey
The quantification of derivatized amino acids by ultra high-pressure liquid chromatography (UHPLC) with UV detection is one of the most widely used methods to
date. Unfortunately, separation and detection of underivatized amino acids is difficult as many of them are similar in structure, and few possess adequate chromophores. Derivatizing agents, either pre- or post-column, may overcome some of
these issues, but can add time and complexity to the method. Presented here is a
simple UHPLC gradient method using a new RSLC 2.2-µm 2.1 x 250 mm column
with charged aerosol detection (CAD) for the measurement of 22 underivatized
amino acids. Retention of hydrophilic amino acids was achieved by the addition
of a volatile ion-pairing agent heptafluorobutyric acid (HFBA) to the mobile phase.
All analytes were resolved in less than 10 minutes with a limit of detection of low
nanograms on column, making this approach ideal for the analysis of amino acids
standards and starting materials in quality control. The work was expanded with a
comparison of CAD with inverse gradient and UV detection for monitoring digestion of bovine serum albumin by Trypsin. This technique offers a complementary
approach to the traditional UV at 214 nm with potential for more uniform response
across both large and small peptide chains and the ability to measure free amino acids which may be missed because of insufficient chromophores. These fast UHPLC
methods provide a simple alternative to the more time consuming or less sensitive
amino acid methods typically used to measure analytes in a simple matrix.
66
Polychlorinated Dibenzo-p-Dioxins and Furans (PCDD/Fs) in
Environmental Samples and Incinerator Ash Using High Sensitivity
GC-MS/MS
Robert Biniakewitz, Thermo Fisher Scientific, Austin, TX 78728, Paul J.
Silcock, Inge de Deobbeleer, David Steiniger, Eric Phillips, Chris Hunter,
David Gardner, John Fardon
The measurement of backgrounds levels of PCDD/Fs in the environment is a widespread activity carried out by many regulatory agencies globally. The chronic toxicity
of these compounds to humans and wildlife at extremely low concentrations requires that the techniques used in determination must be both sensitive and selective enough to allow high confidence results. This is especially true when measuring background levels in environmental matrices, such as soil and sediment or by
products from waste incineration processes. Traditionally high resolution magnetic
sector gas chromatography mass spectrometry (GC-HRMS) instrumentation has
delivered the required analytical performance and has become the gold standard
technique. In recent years there has been more interest in GC triple quadrupole
instrumentation for this purpose, especially in the area of food safety control. For
this and environmental analysis, it is necessary to deliver data that correlates very
closely with HRMS systems which means especially sensitive triple quadrupole systems are required. Presented is the determination of PCDD/Fs in sediments, soils,
11
2012 EAS Abstracts
November 2012
73
70
Carbohydrate Analysis Using HPLC with PAD, FLD, Charged
Aerosol and MS Detectors
Bruce Bailey, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Paul Ullucci, Marc Plante, Christopher A. Crafts, Ian N. Acworth
Carbohydrates are a structurally diverse group of compounds that can be categorized as monosaccharides, disaccharides, oligosaccharides, glycoproteins and others. Carbohydrates are difficult to analyze because they have similar physical and
chemical characteristics, and do not have a suitable chromophore for UV detection.
Several different high-pressure liquid chromatography (HPLC) methods using various detector strategies (pulsed amperometric electrochemical, fluorescence following derivatization, charged aerosol detection and mass spectrometry were developed to help study carbohydrates and examples for each approach are presented.
Although fluorescent tags improve the chromatographic resolution and detector
sensitivity, they can lead to increased assay variability. Various HPLC modes can
be used for carbohydrate separations, with ion exchange, hydrophilic interaction
liquid chromatography (HILIC) and reversed-phase (RP) on porous graphite column
(PGC) being the most common. HPLC provides for simple chromatographic methods when direct detection using PAD or mass detectors such as evaporative light
scattering detector (ELSD), mass spectrometry (MS), and charged aerosol detection (CAD) are employed. The CAD is an ideal detector when combined with HILIC
or RP/PGC for measuring different carbohydrates. It is a mass-sensitive detector
which can measure any non-volatile, and many semi-volatile compounds, typically
with low ng sensitivity. Unlike ELSD, it shows high sensitivity, wide dynamic range,
high precision, and more consistent inter-analyte response independent of chemical
structure. For the analysis of glycans liberated from glycoproteins the utility of the
LC-MS-CAD platform is presented where the CAD is used for quantitative analyses
while the MS provides structural verification. The advantage of this approach over
methods using fluorescent tags is discussed.
The Efficient Method Development for the Analysis of Proteins,
Peptides, and Synthetic Oligonucleotides with a Novel Hybrid
Reversed-Phase Column
Takashi Sato, YMC Co., 284 Daigo-cho, Karasuma Nishiiru Gojo-dori,
Shimogyo-ku, Kotyo, 600-8106 Japan, Ernest J. Sobkow, Naohiro
Kuriyama, Noriko Shoji, Chie Yokoyama, Takatomo Takai, Akiko Matsui
The recent development of bio-pharmaceutical industry has been remarkable and an
effective analytical method with higher sensitivity, superior selectivity, and increased
speed has been required in the characterization of peptides, proteins and oligonucleotides by high-pressure liquid chromatography (HPLC). The method development of reversed phase HPLC requires optimization of several conditions, such as
bonded-phase, column efficiency, solvent type, pH and temperature. pH and buffer
type is the most important parameter to control retention of biomolecules which
have multiple ionic functional groups. Furthermore, temperature often becomes a
key tool to achieve better peak shapes and resolution for larger molecular-weight
compounds. Although silica based reversed phase columns have been widely used
for separation of biomolecules, they have low stability under alkaline conditions and
a limited usable pH range. To improve the chemical stability at an expanded pH
and temperature range, we have developed a new type of organic/inorganic hybrid
reversed phase column, YMC®-Triart C18 and YMC®-Triart C8. The novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety
of mobile phase conditions. In this poster, we show characteristics of this new hybrid
column, and some example cases of efficient method development in separation of
the biomolecules such as peptides, proteins and synthetic oligonucleotides.
74
Improvement in Productivity of the Method Development for the
Drug Analyses by Using a Novel Hybrid Reversed-Phase Column
Takashi Sato, YMC Co.¸ 284 Daigo-cho, Karasuma Nishiiru Gojo-dori,
Shimogyo-ku, Kotyo, 600-8106 Japan, Ernest J. Sobkow, Naohiro
Kuriyama, Noriko Shoji, Chie Yokoyama, Takatomo Takai, Akiko Matsui
Silica based reversed phase columns have been widely used for analytical and preparative chromatographic field. The silica based packing materials have low stability
under alkaline conditions, and have a limited usable pH range. Recently, there has
been much attention given to hybrid materials that have two aspects of inorganic
and organic character to improve the chemical stability. We have developed a new
type of hybrid reversed phase, YMC®-Triart C18 and YMC®-Triart C8. YMC®-Triart
columns are based on multi-layered organic/inorganic hybrid particles with 5 mm,
3 mm and a novel 1.9 mm diameter which are produced with a combination of
our existing technologies for silica manufacturing and flow micro-reactor. We also
have applied the optimized technology of surface modification for hybrid silica of
YMC®-Triart columns to all particle sizes for improving durability, scalability, selectivity and peak shapes for various types of compounds. In this poster, we evaluate the
pH stability and chromatographic performance of YMC®-Triart columns comparing
commercially available columns, and show some application data utilizing characteristics of this material.
71
Improve the Chromatographic Performance of your Assay without
Changing Method or HPLC Equipment
Luisa Pereira, Thermo Fisher Scientific, Tudor Rd., Runcorn WA7 1TA
United Kingdom, Anthony Edge, Stephen Luke, Robert Wiedemer,
The use of sub-2-µm particle packed columns is now well established in the analytical laboratory. The small particle diameters improve the separation kinetics and
therefore efficiency, but have some practical issues. Conventional high-pressure
liquid chromatography (HPLC) systems cannot operate at the flow rates that facilitate the optimum performance on such columns due to the backpressure generated. Partially porous particles, with a diameter between 2- and 3-µm, have been
more recently introduced into the analytical laboratory and these provide similar
performance to sub-2-µm particles at significantly lower column backpressures. The
very tight particle size distribution of solid-core materials results in columns with
high permeability, and therefore for the same nominal pressure they give better
separations than fully porous materials. Often however, solid-core particle packed
columns are narrow-bore and short which has implications in terms of the system
set-up. System dead volume and operating parameters have to be optimized in
order to get the best possible performance out of the column. The work presented
in this poster demonstrates that it is possible to significantly improve assay performance without the need to make changes to the operating parameters or system
set-up. With the correct choice of HPLC column it is possible to dramatically improve
separation efficiency, resolution, sensitivity and peak capacity over those obtained
with conventional fully porous 5- and 3-µm particle packed columns, with less than
50% increase in backpressure. Improvements are demonstrated for both isocratic
and gradient methods. Some reduction in analysis time is also possible.
75
Direct Analysis of Surfactants Using HPLC with Charged Aerosol
Detection
Marc Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Bruce Bailey, Ian N. Acworth, Christopher A. Crafts, Daniel
Kutscher
Surfactants are a diverse group of chemicals whose structures vary widely but typically contain an oil-soluble hydrocarbon chain and a hydrophilic group. Surfactants
can be categorized based upon their structure and include nonionic, anionic, and
cationic classes. They have widespread use as detergents in shampoos and cleaning products, ion pairing agents used in chromatography, and complex dispersants
used to treat oil spills. Many of these commercial surfactants are mixtures of members of a homologous series, and such mixtures can be defined using liquid chromatography. Chromatographic approaches can separate the molecules on the basis of
carbon chain length, chain branching, or positional isomer distribution. Surfactants
typically do not contain a UV chromophore so are usually measured using reversed
phase high-pressure liquid chromatography (RP-HPLC) with non-suppressed or
suppressed mode conductivity or indirectly using photometric detection. Charged
aerosol detection can measure any non-volatile, and many semi-volatile compounds, typically to low nanogram sensitivity. Furthermore, as response is similar
for all compounds and independent of chemical structure, charged aerosol detection is ideal for measurement of surfactant species. Generally, the reproducibility
for methods using charged aerosol detection is better than 2% relative standard
deviation. Sensitive methods are described herein, using the new Thermo Scientific
Acclaim Surfactant Plus HPLC column, for the analysis of various surfactant classes
including anionic alkyl sulfonates (lauryl sulfate), cationic quaternary amines (laurylmethylbenzyl-amine), non-ionic block copolymer (Pluronic F-68), and complex
mixtures of oil dispersants (Span 80).
72
Utilizing a HILIC Column for Improved Separation and MS
Sensitivity of Polar Compounds
Jared Benedict, AkzoNobel/Kromasil, 281 Fields Ln., Brewster, NY
10509
Traditionally, polar compounds like organic acids, nucleobases, and water soluble
vitamins have been a challenge to separate on a standard reversed phase column
like C18. Kromasil HILIC-D is designed for the best selectivity of polar compounds
and true orthogonal selectivity when compared to a C18 column, normally giving
an opposite elution order. The diol derivatized Kromasil HILIC-D provides excellent reproducibility when compared to hydrophilic interaction liquid chromatography (HILIC) columns based on standard bare silica. This paper demonstrates mass
spectrometry (MS) compatibility since the phase is low bleed and the solvents used
when running in HILIC mode are optimal for MS. Therefore the MS sensitivity can
be 100 times better when using Kromasil® HILIC-D over traditional reversed phase
columns. Taken together, utilizing a HILIC column can provide a solution when analyzing polar compounds.
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2012 EAS Abstracts
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76
liquid chromatography (HPLC). Superficially porous (also known as Fused-Core®)
silica particles can be obtained with a variety of bonded phases, pore sizes, particle sizes, and shell thicknesses. Columns packed with 2.7-µm diameter FusedCore particles have been shown to exhibit surprising efficiency. These columns
can demonstrate performance superior to equivalently sized porous particles.
In addition, these columns show comparable performance to columns packed
with sub-2-µm particles, but at less than half the back pressure. High column
efficiencies have been observed for the use of bare silica Fused-Core particle
columns operated in hydrophilic interaction liquid chromatography (HILIC) mode.
The objective of this study has been to develop highly polar bonded-phase surface
modifications of Fused-Core silica particles for HILIC separations that maintain the
high efficiencies previously observed with bare Fused-Core silica, but that also
exhibit the advantages of covalently-modified HILIC packing materials. To this end,
a variety of highly polar covalent bonded phases were applied to Fused-Core silica
particles. Novel hydroxylated bonded phases are observed to be highly hydrophilic,
exhibiting typical HILIC retention properties. A selected material, commercialized as
Halo® Penta-HILIC , contains five hydroxyl groups on the bonded ligand and shows
high efficiency and reduced ionic interactions with ionizable compounds, including
bases, acids, and zwitterions. We demonstrate applications of such highly polar
bonded phases for a variety of HILIC separations of small molecules, including antibiotics, antidepressants, drugs of abuse, and nucleosides.
The Importance of Column Selection in Reversed-Phase Method
Development
Kenneth D. Berthelette, Waters, 34 Maple St., Milford, MA 01757, Mia
Summers
Chromatographic columns compatible with ultra high-pressure liquid chromatography (UHPLC) instrumentation are offered in a wide variety of particle sizes, column
dimensions and column chemistries. Column choice can significantly impact the
effectiveness of a separation and affect the extent of downstream method development required. Here, we compare samples on a variety of column chemistries with
different base particles and ligands. Three different types of samples were analyzed
using six different column chemistries to show the effect of stationary phase selection on the initial separation. The result of varying mobile phase pH using high pH
compatible columns is also demonstrated. Finally, the effect of changing particle
size on the speed of separation is examined. All samples were run on an ACQUITY
UPLC H-Class system equipped with a column manager. Knowledgeable selection
of the appropriate column stationary phase, based on the analytes present in the
sample, can lead to early generation of more effective separations and reduce the
need for extensive method development.
77
Three Novel Silica Based SEC Columns Designed for the Separation
of Antibody Monomer from its Dimer, Higher Aggregates and
Antibody Fragments
Atis Chakrabarti, Tosoh Bioscience, 3604 Horizon Dr., Ste 100, King of
Prussia, PA 19406
Monoclonal antibody is widely being used in the field of biotherapeutics. Separation
of the pure antibody monomer needs to be very well resolved from its dimer and
higher aggregates. Similarly for quality control and regulatory purpose the separation of antibody fragments is also very much essential. The species other than
the monomer might induce toxic side effects to the body if not removed. We have
designed three novel silica based size exclusion chromatography (SEC) columns
specially designed for these purposes. TSKgel UltraSW Aggregate is especially
designed for the separation of aggregates. It is packed with 3-micron particle with
30nm pore size with a dimension of 7.8mm ID x 30cm. TSKgel UltraSW Aggregate
has a wider separation window and higher resolving power for oligomers and aggregates with high molecular weight, compared to the conventional column. TSKgel
SuperSW mAb HTP is a semi-micro short column packed with 4 micron particles
and 25 nm pore size, with a dimension of 4.6mm ID X 15 cm; for the separation of
dimer, monomer and fragments at the same resolving power but in half the analysis
time compared to the conventional column. TSKgel SuperSW mAb HR is a standard 7.8mm ID X 30cm long column packed with the same 4 micron particles as
SuperSW mAb HTP but for high resolution. Here we report the efficient separation of aggregates induced by heat denaturation and acid denaturation using these
columns. Also we report the separation of fragments generated by the digestion of
the antibody by papain. This study shows that these three novel SEC columns are
suitable for the analysis and purification of antibodies.
80
Effects of Particle Size and Shell Thickness on Fused-Core®
Column Performance
Stephanie A. Schuster, Advanced Materials Technology, 3521 Silverside
Rd., Suite 1-K, Quillen Bldg., Wilmington, DE 19810, Joseph J.
DeStefano, William L. Johnson, Jason M. Lawhorn, Joseph J. Kirkland
Fused-core (superficially porous or porous shell) particles have been shown to have
distinct advantages over comparable totally porous particles for separating small
molecules. Columns of fused-core particles show higher efficiency than totally porous particles of similar size because of superior eddy diffusion properties (smaller
van Deemter A term) resulting from the homogeneous packed beds made possible
by the exceptionally narrow particle size distributions of the fused-core particles.
The efficiency for columns of 2.7-µm fused-core particles actually rivals that for sub2-µm totally porous particles with only about one-half the back pressure. Wider-pore
fused-core particles now are commercially available for separating larger molecules
such as peptides and proteins. These particles show special advantages with these
larger molecules for fast separations at high mobile phase velocities because of
superior mass transfer (kinetic) properties (smaller van Deemter C term) in the thin
porous shells. Fused-core particles with a wide range of particle sizes and porous
shell thicknesses have been synthesized to allow the study of the effects of these
physical characteristics on chromatographic performance. This report describes the
effect of these particle characteristics on several factors of separation importance,
including reduced plate height, separation efficiency, and sample loading. Chromatograms demonstrating the advantages of using fused-core particles over totally
porous particles are shown for a variety of applications.
78
Looking Beyond ODS - Unique Stationary Phases for 2-µ HPLC
Columns
Matthew Przybyciel, ES Industries, 701 South Rt 73, West Berlin, NJ
08055
Reversed-phase high-pressure liquid chromatography (HPLC) is widely used for
separation of many pharmaceutical compounds. A majority of these separations
are based on ODS type columns. However, retention and separation of various
compounds have proven to be a challenge. Many of these types of compounds are
not retained, poorly retained or not separated on most conventional ODS reversedphase columns, even when these ODS column are packed with highly efficient sub2-µm particles. Fortunately, to deal with these types of analytes an alternative mode
of chromatography can be utilized using polar, fluorinated and other non-hydrocarbon based stationary phases. These columns can to be used in the traditional reverse phase mode as well as hydrophilic interaction liquid chromatography (HILIC).
HILIC uses mobile phases containing between 5 - 20 % water for the retention of
polar compounds. These new HPLC columns packed with 1.8 um particles have
been engineered to specifically for high pressures. We provide examples and applications on the chromatographer can benefit from these types of stationary phases
using the 1.8 um particle size format. We demonstrate how these HPLC columns
can provide for the high resolution separations over a wide variety flow rate conditions and mobile compositions.
81
Column Lifetime Study of a Silica-Based Diol-Bonded SizeExclusion Chromatography Column for Protein Separations
Bruce Kempf, Tosoh Bioscience, 3604 Horizon Dr., Ste 100, King of
Prussia, PA 19406, Atis Chakrabarti
Development of a reliable analytical high-pressure liquid chromatography (HPLC)
method requires a stable column yielding a high degree of reproducibility of retention
time, peak symmetry and column efficiency over a large number of injections, while
such quality characteristic are expected to be independent of the lot of base silica as
well as the bonding and packing procedures. Size exclusion chromatography (SEC)
performed under aqueous conditions, also known as gel filtration chromatography
(GFC), is popular among biochemists for isolation and quality control of monoclonal
antibodies and other proteins and peptides. Column lifetime is an important issue,
not just for the analyst but also from a financial perspective. We report the results of
a column lifetime study of silica-based TSKgel G3000SWxl 5-μm, SEC columns with
dimension of 7.8mm ID x 30cm; a column type that is widely used for the separation
of monoclonal antibodies. We evaluated the performance of three columns each
from three different commercial lots using a mobile phase consisting of 100mmol/L
KH2PO4/K2HPO4, pH 6.7, 100mmol/L Na2SO4 and 0.05% NaN3, UV detection
at 280nm, a flow-rate of 1.0mL/min, at ambient temperature. A standard protein
test-mixture, containing thyroglobulin, µ-globulin, ovalbumin, ribonuclease A, and
p-amino benzoic acid as pore volume marker, was used in this study. Monoclonal
antibody, human sera was also used in this study. To provide extra stress on the
column, we chose not to use guard columns in this study. Results show excellent
reproducibility in retention time with a very low percent relative standard deviation
(%RSD) of <1% (n=10) within the same lot and between the lots.
79
Novel Superficially Porous Bonded Phase for High Speed HILIC
Separations
Stephanie A. Schuster, Advanced Materials Technology, 3521 Silverside
Rd., Suite 1-K, Quillen Bldg., Wilmington, DE 19810, Barry E. Boyes,
Joseph J. DeStefano, William L. Johnson, Joseph J. Kirkland
During the past few years superficially porous silica particles have emerged as preferred materials for high efficiency and high speed separations in high-pressure
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2012 EAS Abstracts
82
November 2012
86
Withdrawn by the author.
Application of a Mixed-Mode Column in Pharmaceutical Analysis:
Two Case Studies
Elvin S. Lee, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ
08904, Hui (Viviane) Zhao, Yan Zha, Xin (Amy) Bu
About fifty percent of active pharmaceutical ingredients (API) are developed as salt
forms to enhance solubility, bioavailability and form stability. In addition, the APIs
often contain impurities derived from their manufacturing process and degradants
which arise upon storage. Typically, the API, counter ion and impurities/degradants
possess very different chemical and physical properties; therefore, requiring separate testing methods using different analytical techniques to assure the quality control of the drug substance. Method development, validation, and routine analysis
can be very challenging and consume considerable time and resources. Analytical methods that enable the simultaneous quantitation of compounds with different properties would greatly enhance the efficiency of the pharmaceutical analytical
laboratory. In one case, the API under development is an amine with p-toluenesulfonic acid (PTSA) as the counter ion and the other, an acid with diispropyl amine
(DIPA). The major impurities/degradants include amine, acid, neutral and amino
acid (zwitter ionic) compounds. The critical impurities of interest are very polar,
therefore difficult to retain using traditional reversed-phase columns. To overcome
these challenges, we investigated various mixed-mode columns that possess multiple separation mechanisms, including hydrophobic interaction, anion exchange,
and/or cation exchange. As a result, the same column was shown to be able to
simultaneously analyze the API, the counter ion and impurities/degrandants in one
case and API and impurities/degrandants in the other in single HPLC methods using
a mixed-mode column with mobile phases containing sulfuric acid modifier. Validation demonstrated that the established methods are specific, accurate sensitive and
precise. This application of mixed-mode column significantly improves analytical
efficiency, ease of sample analysis and technology transfer.
83
High-Throughput LC-MS Application for Bioanalysis of Pain
Management Drugs
Craig R. Aurand, Supelco/Sigma-Adlrich, 595 North Harrison Rd.,
Bellefonte, PA 16823, David S. Bell, Jennifer Claus
For efficient therapeutic drug monitoring, it is important for clinicians to have access
to fast/robust analytical methods for accurate assessment of drug efficacy. Industrial
trends toward highly specific liquid chromatography mass spectrometry (LC-MS)
applications over traditional enzyme-linked immunosorbent assay (ELISA) type immunoassay has resulted in the need for high-speed chromatographic assays along
with simplified sample preparation methods. Often the limitation of a bioanalytical
technique is based upon the effectiveness of the sample preparation technique.
Plasma and serum samples are often suspect to assay irregularities due to matrix
induced interferences. In this study a combination of fast chromatographic separation along with a novel sample preparation platform is utilized for the analysis of
pain management drug tapentadol and associated metabolites. Here a bioanalytical
assay for tapentadol and associated metabolites is demonstrated using a combination of hydrophilic interaction liquid chromatographic (HILIC) separation along
with a novel phospholipid depletion device for efficient analysis of plasma samples.
Targeted depletion of phospholipid matrix interference was achieved using novel
HybridSPE®-Phospholipid 96well plate. Plasma samples spiked with tapentadol and
associated metabolites were prepared using the HybridSPE®-Phospholipid sample
prep technique and assayed using the Ascentis Express HILIC phase, resulting in a
fast and accurate bioanalytical assay method.
84
The Development and Application of a New Fused-Core HPLC
Particle
Gaurang Parmar, Supelco/Sigma-Adlrich, 595 North Harrison Rd.,
Bellefonte, PA 16823, Wayne K. Way, Richard Henry, Carmen T.
Santasania
Fused-core columns have enjoyed great acceptance in the last few years. These
columns have provided a new choice for improving the performance of traditional
high-pressure liquid chromatography (HPLC) systems. The fused-core particle technology has provided the benefits of high speed and high efficiency without the concerns of smaller particle columns. Due to the high efficiencies at low backpressures,
these particles have benefited conventional HPLC users with little drawback. In this
talk, we discuss a new fused core particle. The new fused-core particle consists of
the familiar solid core and porous shell. A major benefit of the Fused-Core particle is
the small diffusion path compared to conventional fully porous particles. This short
diffusion path reduces axial dispersion of solutes and minimizes peak broadening.
This new column can be interchanged with a standard 5-µm particle and provide
greater efficiency, a hallmark of the fused-core phases. There is no need to change
column dimensions, flow rates, mobile phase conditions, or sample prep. The new
particle excels under high flow rates and high-throughput demands of bioanalytical liquid chromatography mass spectrometry methods. Furthermore, the improved
particle format provides an extremely rugged HPLC column and offers seamless
transfer of methods from traditional porous phases. We discuss small molecule applications in such diverse areas as pharmaceutical analysis, bioanalytical applications and food applications and compare the separations to classic sized materials
to show advantages of this fused-core particle.
87
Evaluation of Low-Temperature Evaporative Light Scattering
Detector (LT-ELSD) for Pharmaceutical Analysis
Chanda L. Rogers, Merck, 126 East Lincoln Ave., Rahway, NJ 07065,
Rong Xiang, Bing Mao
Detection and quantitation for low to no UV chromophore compounds has been a
challenge in the pharmaceutical industry. With the development of aerosol based
detectors, such as charged aerosol detector (CAD) and ELSD, the task of analysis for these types of compounds has become achievable, but there are still some
drawbacks to using these types of detectors for analysis in a regulated industry. In
the evaluation of the LT-ELSD, different types of pharmaceutical compounds, such
as small and large molecules, were used to determine its performance across various factors, such as baseline noise, linearity, sensitivity (limit of detection and limit
of quantitation), reproducibility, and robustness. This evaluation also focused on
mobile phase compatibility of the LT-ELSD compared to the CAD.
88
Metabolomics of Polar Compounds Using LC-MS
Robert Kennedy, University of Michigan, 930 N. University Ave., Ann
Arbor, MI 48109-1055
Metabolomics has emerged as a powerful method to study the function and pharmacology of cells and organisms. The wide polarity range of metabolites presents a
substantial challenge to their separation and detection. Polar compounds are often
difficult to resolve by reversed-phase liquid chromatography (LC). Hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) works well for resolving many highly polar metabolites. Even polar lipids can be well separated on some
columns allowing metabolites from broad classes to be analyzed. In some cases
however, the resolution and sensitivity for detection is inadequate. For example,
many polar neurotransmitters cannot be detected in neuronally derived samples
(e.g., in-vivo dialysate) by HILIC-MS. We have found that benzoylation provides
a versatile and effective derivatization approach for metabolite detection. Benzoyl
chloride reacts with a variety of polar function groups (alcohols and amines) to produce relatively non-polar analytes that can be separated by reversed phase LC.
The electrospray ionization MS sensitivity is enhanced up to 1500 fold. Finally, 13C
labeling allows quantification by this approach. We present application of HILIC and
benzoylation-reversed phase LC for studies of polar metabolites involved in diabetes and drug effects in the brain.
85
Characterization of Carbon-Modified Silicas for use in Analytical
Liquid Chromatography
Ian Gibbs-Hall, Gustavus Adolphus College, 800 West College Ave., St.
Peter, MN 56082, Tuan Tran, Paul Young, Dwight Stoll, Jon Thompson,
Doug Fryer, Conor Smith
We have recently developed a series of novel carbon-modified porous silica materials for use in analytical and preparative separations, and as solid-phase extraction
media. The analytical materials exhibit unique characteristics compared to other
commercially available carbon phases as they are substantially more stable at high
pressures and exhibit acceptable mass transfer characteristics. Users of carbonbased phases are aware that some compounds are very difficult to elute from existing commercial carbon-based materials. The new materials described here significantly address this problem through both the ability to adjust the carbon loading on
the underlying substrate, and the use of a relatively inert substrate. We report on the
basic characterization of a suite of materials that cover a wide range of carbon loading, including: surface area and pore volume measurements, retention of non-polar
and polar compounds, and selectivity. We also show the potential of these materials
for use in two-dimensional separations.
89
Next Generation Glycomics Using Capillary Separations and Mass
Spectrometry
Milos V. Novotny, Indiana University, Department of Chemistry, 800 East
Kirkwood Ave., Bloomington, IN 47405
The multilateral importance of glycans as the selectivity determinants of many biological processes necessitates a deeper insight into the structural richness of both
prokaryotic and eukaryotic glycomes. Oligosaccharides’ propensity to branch and
form numerous isomers presents enormous challenges to analytical methodologies
and instrumental measurements. Much current emphasis in the field of analytical
glycobiology has been to determine structural attributes of trace glycoproteins in
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2012 EAS Abstracts
November 2012
93
biological fluids and tissues. Importance of this direction has been underscored by
numerous connections of aberrant glycosylation to human diseases such as cancer,
cardiovascular problems, and diabetes. Highly sensitive glycomic measurements
are pertinent to the search for disease biomarkers, early disease detection and
prognostic evaluations. In our laboratory, glycomic profiling through matrix-assisted
laser desporption/ionization time-of-flight mass spectrometry has routinely been
established, but the determination of minor profile components necessitates the
development of suitable pre-concentration methodologies based on immunoaffinity
of lectin affinity principles. Brief examples of delving deeper into the human serum
glycomes and glycoproteomes are given in this presentation pertaining to cancer
research. Micro-column liquid chromatography procedures and capillary electrophoresis are demonstrated as complementary approaches to mass spectrometry.
Immobilization of Gold Nanorods onto Polycaprolactone
Nanofibers via Polyelectrolyte Decoration - A SERS Substrate for
Environmental Sensing
John Rabolt, University of Delaware, Department of Materials Science
and Engineering, DuPont Hall 201,
Newark, DE 19716
With their intriguing fine-tunable optoelectronic properties, gold nanorods (AuNRs)
have become a promising building block in the fabrication of surface enhanced Raman scattering (SERS) substrates. In this work, immobilization of AuNRs on electrospun polycaprolactone (PCL) fibers was accomplished by using polyelectrolyte
(PE) decoration. Appropriate charges were imposed on the PCL fibers during the
electrospinning process and the AuNRs were adhered to these fibers by PE multilayer deposition. This multistage process was investigated by Fourier transform
infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). In addition, AuNR/PCL substrates with varied AuNR densities were fabricated and characterized by scanning electron microscopy (SEM). The SERS enhancement response
of these substrates was measured using 4-mercaptopyridine as the probe molecule.
All the substrates showed acceptable Raman signal enhancement which increased
with the AuNR density (detection limit is 10-7M 4-mercaptopyridine for the substrate
with medium AuNR density). The high sensitivity makes the substrate a promising
candidate for the detection of trace amounts of toxic substances in the environment.
Engineering Analytical Separation Media with Nanoscale Features
for Size Separation of Biomacromolecules and Bioparticles
Milton Lee, Brigham Young University, Department of Chemistry and
Biochemistry, MS: C267 BNSN, Provo, UT 84602, Jie Xuan, Pankaj
Aggarwal, Kun Liu, Suresh Kumar, Aaron R. Hawkins, Adam T. Woolley,
John S. Lawson, H. Dennis Tolley
There is considerable interest in developing improved analytical methods for separation of bio-macromolecules (e.g., proteins) and other bioparticles (e.g., viruses)
which range in size from approximately 5 to 200 nm. This effort is challenging
because resolution is more difficult to achieve as analytes increase in size and
mass. To address this challenge, we are developing monolithic separation media
and planar nanofluidic devices that can be used for sieving of these large bioparticles. Monoliths engineered for size exclusion chromatography (SEC) must have
appropriate pore size distribution and exhibit little to no non-specific adsorption of
proteins. We have recently synthesized poly(ethylene glycol) diacrylate monoliths
that demonstrate SEC behavior and resist adsorption of proteins. Correlation of
chromatographic performance to qualitative and quantitative descriptors of monolith
morphology is being provided by capillary flow porometry and three-dimensional microscopy. Fluidic devices containing arrays of parallel planar nanochannels containing segments with varying inner heights were fabricated for size fractionation of bioparticles. These chips were successfully demonstrated for trapping and separating
polymeric nanobeads and virus capsids. A major challenge has been to minimize
nonspecific protein adsorption because of the extremely high surface-to-volume
ratio of the nanochannels. To systematically study a variety of coatings, we investigated protein adsorption in bare and coated silica capillaries with inner diameters as
small as 25-μm. Methods that were effective in fused-silica capillaries were further
evaluated for sieving applications in our nanochannel arrays.
90
94
The Relationship between the Physicochemical Properties of
Phosphoinositides and the Spatiotemporal Control of Membrane
Signaling Events
Arne Gericke, Worcester Polytechnic Institute, Department of Chemistry,
Worcester, MA 10609, Edgar E. Kooijman, Zachary E. Graber, Zhiping
Jiang
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor component in the cell
membrane, but has very important roles for many cell signaling events. While hydrogen bond formation plays a pivotal role in PI(4,5)P2/protein interactions, electrostatic interactions strongly contribute to any protein/PI(4,5)P2 binding event. We
have investigated the ionization state of PI(4,5)P2 in complex lipid mixtures that
contain lipids capable to donate hydrogen for bond formation with PI(4,5)P2 (e.g.,
PE, PI and PS). We find that the ionization state of PI(4,5)P2 is governed by intraand intermolecular hydrogen bond formation, which has also a strong effect on the
lateral distribution of PI(4,5)P2 in the lipid bilayer. For example, in the presence of
phosphatidylinositol two opposing effects lead to a net charge that is similar to the
charge found for PI(4,5)P2 in the absence of PI. The enhanced negative charge in
the membrane due to the presence of PI leads to an increased PI(4,5)P2 protonation (reduced charge). This effect is opposed by PI/PI(4,5)P2 hydrogen bond formation which results in increased deprotonation of the phosphomonoester groups.
As a result, PI appears to have a minor effect on PI(4,5)P2 ionization, however, fluorescence microscopy measurements of PC/PI/PI(4,5)P2 GUVs show a pronounced
effect on PI(4,5)P2 morphology (PI and PI(4,5)P2 form mixed domains). A similar
PI(4,5)P2 de-mixing is observed in the presence of cholesterol, which can presumably be attributed to hydrogen bond formation between the cholesterol hydroxyl
group and the PI(4,5)P2 head group.
91
Tagging Strategies for Capillary LC-MS Metabolomics
James Edwards, Saint Louis University, Department of Chemistry, St.
Louis, MO 63103
The detection, identification and quantification of the collection of small organic molecules in a biological sample are broad and deep analytical challenges. Given the
vast number of metabolites and the wide ranges of their concentrations, current
methods of untargeted analyses cannot hope to analyze the entire metabolome.
Rather our approach is to extract specific classes of molecules from the remaining metabolic milieu. This is expected to enhance the sensitivity of those classes
of molecules believed to be relevant to the biological system. Specific classes of
molecules are tagged based on functionality for analysis by mass spectrometry
(MS). Multiple tags have been synthesized and utilized to both extract these specific
classes of metabolites as well as enhance ionization efficiency. Coupling high efficiency capillary liquid chromatography to MS analyses proves critical to resolving
structural isomers and differentiating subclasses of extracted metabolites. Isotope
and isobaric tagging in addition to extraction tags are discussed. These methods
determine metabolic effects of high glucose stimuli in endothelial cells as a model
of diabetic complications.
95
Vibrational Spectroscopy, Microscopy, and Imaging: Applications
to Skin Biophysics and Pharmacology
Richard Mendelsohn, Rutgers University, Department of Chemistry, 73
Warren St., Newark, NJ 07102, Carol R. Flach
Vibrational microscopy and imaging are poised to address important biomedical
and pharmacological issues, including the diagnosis of pathological states. A major advantage of these technologies is the availability of direct molecular structure
information to complement the spatial images. The current presentation illustrates
these approaches in diverse areas of skin science, as follows: 1) Some technical
advantages of the methods are presented, including identification of lipid packing
and phase transitions in intact stratum corneum, the generation of quantitative IR
images of exogenous agents, and the evaluation of molecular structure in particular
skin regions from confocal Raman microscopy. 2) Two biochemical applications of
these technologies are presented. (i) Pro-drugs are used to enhance the delivery of
therapeutic agents into skin, where they undergo biochemical modifications and are
converted to their active forms. We have demonstrated the feasibility of tracking and
spatially imaging, in intact skin, via confocal Raman microscopy, the pro drug-todrug interconversion of a derivative of the anti-cancer drug 5-fluorouracil. (ii) Wound
healing consists of a complex series of spatially and temporally organized events
to prevent infection and to restore the skin barrier over the wound bed, followed by
remodeling of connective tissue and re-establishment of stratified epidermis. We
investigated the re-epithelialization of skin excisional wounds over a six day period.
We distinguished the spatial distribution of different keratin types during the healing
process. In addition, we detected for the first time a class of conformational disordered lipids in the vicinity of the migrating epithelial tongue.
92
Molecular Spectroscopy of Lipid Organization in Biological
Membranes
David Moore, TRI Princeton, 601 Prospect Ave., Princeton, NJ 08540
A persistent theme in Professor Richard Mendelsohn’s research over the last 30
years is the translation and application of spectra-structure correlations developed
in simple lipid systems to complex systems such as live cells and intact tissues. I
had the pleasure of being involved in some of this work. This presentation highlights
some of the key developments from Professor Mendelsohn’s research focusing
on the use of molecular spectroscopy to characterize lipid organization within the
plasma membrane of living cells. Among the topics covered are the translation of
quantitative molecular spectroscopy methods from simple hydrocarbons to the plasma membrane of live cells and the insights provided by this approach. Examples
include the characterization of lipid domains in erythrocyte cell membranes and their
correlations with cell morphology, and the link between lipid conformational order
and cell viability in mycoplamsa.
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2012 EAS Abstracts
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96
Sensing Shifts in Chemical Parameters in Marine Ecosystems via
Spectroscopic Signatures and Physical Appearance of Microalgae
Cells
Frank Vogt, University of Tennessee, Department of Chemistry, 552
Buehler Hall, Knoxville, TN 37996, Morgan McConico-Lewis, Kendhl
Witt
In industrialized areas, human activities result in a multitude of chemical impacts
on the environment which need to be assessed in order to understand ecological
implications of chemical shifts. While some parameters such as biochemical oxygen
demand or pH can be measured directly, other chemical parameters might be a
better tell-tale but cannot be accessed in such a straightforward manner. We propose an analysis methodology which is based on relating the chemical signatures
of microalgal biomass to the ambient conditions they had been exposed to. It are
shown that based on infrared spectroscopic data a quantitative but highly nonlinear
calibration model can be derived which however requires novel chemometric approaches for calibration and quantitative analyses. As an example, the availability
and sources of nutrient are quantified. However, the discrimination of different cell
types is challenging and would benefit from additional information. We present results demonstrating that the cell size and their shape are discriminators which reflect
selected figures of merit such as cells species and nutrient situations. Both physical
cell parameters can be quantified by means of microscope image analyses applied
to cell cultures, i.e., to a large numbers of cells. We aim at combining spectroscopic
and image analyses for measuring microalgal biodiversity and chemical composition of biomass both of which are hypothesized to be measures for the chemical
stage of an ecosystem.
netic modeling demonstrated that simultaneous measurements of slurries with UV/
Vis ATR and NIR reflectance is an effective means of characterizing the dissolved
fraction and the onset of nucleation and crystallization. Work with research partner
DuPont Crop Protection Science included development of a 4 L glass reactor with
a recirculation loop and sampling system for off-line high-pressure liquid chromatography analysis.
100
Mass Spectrometry of Cultural Heritage Materials: The MaSC
Database
Christopher A. Maines, National Gallery of Art, Sixth & Constitutions Ave.
NW, Washington DC, 20565, David Peggie, Joseph Padfield, Klaas Jan
van den Berg, Ken Sutherland, Catherine Higgitt, Ester S.B. Ferreira
The Users’ Group for Mass Spectrometry and Chromatography (MaSC) is an international forum for interaction, discussion, and exchange of information among
scientists using chromatographic and mass spectrometric techniques for conservation and preservation studies. Among the Group’s activities is the organization of
regular conferences comprising of discussion meetings and specialized practical
workshops on the application of these analytical techniques to the study of art and
historic artifacts. MaSC is working to develop a shared database of mass spectra
relevant to the study of artistic and cultural objects, a prototype version of which
has been distributed to members for evaluation and feedback. The database is also
intended to establish a method by which unknown compounds of common interest is more easily found and collaboratively identified. Successfully characterized
compounds are then submitted to well-referenced and vetted databases, such as
National Institute of Standard and Technology’s, as many of the compounds of interest to MaSC are not found there. Work is ongoing to provide a streamlined interface
for the submission of spectra to the database and its distribution to contributors.
97
Geospatial Pattern Recognition: Can Geographical Information be
deduced from Surface Water Data?
Steven Brown, University of Delaware, Dept. of Chemistry and
Biochemistry, 163 The Green, Newark, DE 19716, Liyuan Chen
Identification of classes of chemical data is an area that has a long history in chemometrics and some well-established methods. Extending assignments of classes
to a geographical location of a sample on the basis of the chemical signature of that
sample is far less common. This presentation reports some results of our focus on
assigning water samples to a specific watershed, based only on the results of trace
metal and isotopic analyses. Some of the challenges inherent in environmental data
are considered. Results of classification and clustering studies are presented.
101
Analytical Measurement Errors: Estimation and Implications for
Figures of Merit
Peter Wentzell, Dalhousie University, Department of Chemistry, PO Box
15000, Halifax, NS B3H4R2 Canada
Measurement errors are a critical part in the characterization of any analytical procedure. Developing models to describe the noise characteristics of instruments is
an important component for understanding the limitations of an analytical method.
However, this process is often complicated by the absence of replicate measurements. While the use of replicate experiments is preferred, this presentation describes a procedure that can be used to circumvent the requirement for replicates
to develop models for noise. The procedure is useful for multichannel instruments
where multiple (non-replicate) samples have been obtained. If time permits, the
implications of this in characterizing limits of detection and other figures of merit
are described.
A Comparative Evaluation of Off-Gassing from Museum Materials
by GCMS Techniques
Adriana Rizzo, Metropolitan Museum of Art, 1000 Fifth Ave., New York,
NY 10028, Tsukada Masahiko
Pollutants held in atmosphere are one of the factors which can cause deterioration
of artwork. The source of pollutants can be the general outside air pollution and/
or off-gassing from materials used inside museum buildings. Especially in an enclosed space such as display cases, avoiding materials for its construction that emit
harmful chemical gaseous substances is crucial. For this purpose, the accelerated
corrosion test is often used for the evaluation of materials for their safe usage in
display and storage of artworks. Sample material is enclosed in a test vessel with
metal coupons under elevated temperature and humidity conditions, and the test
evaluates the effect of off-gassing from the material on the metal coupons. This
test shows the overall impact of off-gassing on metal coupons, but the nature and
extent of off-gassing that affects the metal coupons cannot be understood. In order
to explore this aspect, air from test vessels was analyzed with solid-phase micro
extraction gas chromatography mass spectrometry (SPME-GCMS). Test materials
enclosed in these vessels were also analyzed by evolved gas analysis and thermal
desorption-GCMS in order to assess their off-gassing potential. A temperature programmable pyrolyzer attached to the gas chromatograph was used in these two
techniques. The combination of sampling methods and techniques has allowed a
better understanding of the chemical nature of off-gassing from the tested materials, as well as an assessment of the suitability of the aforementioned analytical
techniques for this purpose.
99
102
98
Obsidian Spears from the Admiralty Islands with ICP-MS and PXRF
Mark Golitko, The Field Museum of Natural History, 1400 S. Lakeshore
Dr., Chicago, IL 60605, Robin Torrence
Obsidian-tipped spears and daggers from the Admiralty Islands (Papua New Guinea) have been collected by ethnographers, traders, soldiers, and others for both
museums and the private art market since the first examples were acquired by Europeans, including members of the HMS Challenger expedition in 1875. While the production of their distinctive large obsidian blades is known from archaeological sites
dating back at least 2000 years, and despite their ubiquity in museum collections
worldwide, little is known about how, where, and why these objects were produced.
At the time when ethnographers first began collecting these spears and daggers,
a complex system of production and distribution existed in the Admiralties in which
several craftspeople, sometimes living in different communities, may have produced
components of or added work to individual hafted spears and daggers. We report on
analysis protocols and results of chemical analysis by portable X-ray fluorescence
(PXRF) and laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) of obsidian spears and daggers from a number of museum collections in Australia, Europe, and the United States. By chemically characterizing both blades and
pigments applied to hafts, we examine correlations between the compositions of
blades and hafts with blade shape, hafting style, and haft decoration. These analyses shed light on both the organization of production in the Admiralty Islands at the
time of sustained European contact, but also document how production choices
Kinetic Modeling of Batch Slurry Reactions
Paul Gemperline, East Carolina University, Department of Chemistry,
Greenville, NC 27858, Chun Hsieh, David Joiner, Julien Billeter, Mary
Ellen McNally, Ron Hoffman
Slurries are widely used in important chemical and pharmaceutical manufacturing processes. In this presentation we report chemical monitoring and modeling
of batch slurry reactions using optical and calorimetric measurement where key
analytes are present in both the solid and liquid phases. Experimental studies were
performed to: 1) generate supersaturated solutions for the development of kinetic
models of seeded crystallization, crystal growth, and dissolution; 2) generate unseeded crystallization from homogeneous reaction mixtures for the development of
kinetic models of reacting systems, including crystal growth as well as measurement
of crystallization driven by cooling of reaction mixtures including the necessary corresponding solubility curves as a function of temperature; 3) reproduce at laboratory
scale, a commercially relevant slurry reaction starting with heterogeneous reaction
mixture, producing a product slurry. Simultaneous UV/visible attenuated total reflectance (ATR) and near-infrared (NIR) reflectance measurements enabled reliable
characterization of reaction mixtures and detection of the onset of crystallization. Kinetic models for dissolution, seeded crystallization, unseeded crystallization, homogeneous reaction and crystallization driven by cooling were successfully developed
relating the mass of substance in the solution phase and solid phase to UV/Vis ATR
and NIR reflectance measurements. Reactive crystallization experiments with ki-
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2012 EAS Abstracts
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107
changed as obsidian-tipped spears and daggers were increasingly targeted towards
a European market.
Carboplatin Microdosing for Mechanistic and Diagnostic Studies
in Cancer
Paul Henderson, University of California-Davis, 4501 X St., Suite 3016,
Sacramento, CA 95817, George Cimino, Sisi Wang, Tao Li, Ralph de
Vere White, Cindy Lin, Mike Malfatti, Kenneth Turteltaub, Chong-xian
Pan
There is a need for a rapid and accurate test to identify those lung and bladder cancer patients that are likely to respond to chemotherapy. Presently, platinum-based
chemotherapy is the primary therapeutic intervention for these patients, but the
majority of patients do not respond. We are developing the PlatinDx assay, which
can potentially be used to identify those patients which are appropriate for platinumbased chemotherapy. The assay utilizes subtherapeutic “microdoses” (~1/100th the
therapeutic dose) of 14C-labeled carboplatin, which is traced in patient tissues by
accelerator mass spectrometry. We hypothesize that a threshold level of platinumDNA damage is required for cell death and that microdosing is able to predict the
capacity of cancer cells to attain that threshold after exposure to therapeutic doses
of carboplatin. The central objective of our current study is the identification of the
cutoff level at which microdose-induced carboplatin-DNA damage enables the stratification of patient response. We are accruing patients into a feasibility diagnostics
clinical study. The extent of carboplatin-DNA damage is compared to patient response to platinum-based chemotherapy as measured by tumor shrinkage as a
primary endpoint and overall survival as a secondary endpoint.
103
The Effects of Consolidant Treatments and Organic Solvent
Removal on the Stable Carbon, Nitrogen, and Oxygen Isotope
Values of Bone Collagen and Hydroxyapatite
Christine A. M. France, Smithsonian Museum Conservation Institute,
4210 Silver Hill Rd., Suitland, MD 20746, Jennifer Giaccai
The use of stable isotopes from natural materials such as bones and teeth is a
ubiquitous technique that provides valuable information regarding provenance, diet,
and ecology of the remains. Inherent in the use of this technique is the need for wellpreserved material that has been unaltered by post-mortem diagenesis or chemical
preservatives. As researchers look towards older museum collections to provide
comprehensive sample sets, it is necessary to understand the effects of past treatments on the stable isotopic values of the tested material. A variety of glues and
consolidants are commonly used to stabilize bones during excavation or solidify
bones in their original structure to be stored in collections. This study examines
the effects of three common consolidants (PVAc, Butvar B-98, and Paraloid B-72)
on the stable carbon, nitrogen, and oxygen isotopic values of bone collagen and
hydroxyapatite. It also examines the effects of common organic solvents used to
apply and remove the consolidants. Results indicate that collagen and hydroxyapatite phosphate are fairly resistant to isotopic exchange, while the hydroxyapatite
carbonate shows statistical differences between control groups and select samples
with applied consolidants.
108
Evaluation of a LC-MS/MS Approach for Support of Microdose
Clinical Trials: A Case Study
Li Sun, Merck, 770 Sumneytown Pike, West Point, PA 19486, Hankun
Li, Kenneth Willson, Sheila Breidinger, Eric Woolf, Kevin Bateman, Jack
Henion, Yuanyuan Li, Lian Shan, Gary Schultz
Microdosing has emerged as an attractive tool to assist in drug candidate screening,
but this approach necessitates extremely sensitive bioanalytical assays, typically in
the pg/mL concentration range. Currently, accelerator mass spectrometry (AMS) is
the predominant tool for microdosing support. Specialized facility and radiolabeled
compound synthesis are required to apply AMS methodology. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the standard technique for quantitative bioanalysis, but there have been few studies attempted to assess this approach
in the context of microdosing applications due to sensitivity concerns. Herein, the
development of LC-MS/MS methods to quantify five integrase inhibitors in human
plasma is described, with limit of quantitation (LOQ) at 1 pg/mL for raltegravir, and
2 pg/mL for four proprietary compounds. These assays involve double extraction of
plasma (0.9-mL) followed by ultra-pressure liquid chromatography (UPLC)-negative
ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. All
methods were fully validated (precision between 1.20 and 14.1% and accuracy between 93.8 and 107%), and successfully applied to a human microdose study. To
assess the possibility of further sensitivity enhancement using a reduced volume of
plasma, a nanoscale LC coupled with chip-based nanoelectrospray approach was
investigated. Theoretically, a nanoLC-nanoESI-MS/MS system offers opportunity for
sensitivity improvement, as a result of reduced dilution of the analyte zone during LC
separation and improved MS ionization efficiency. Utilizing 2D-LC-nanoESI-MS/MS
with nanoLC as the second dimension, an assay LOQ of 0.5 pg/mL was achieved
for raltegravir using 0.1 mL of plasma. The study results support the use of LC-MS/
MS as a viable tool for microdosing sample analysis.
104
Accelerator Mass Spectrometry (AMS) for Pharmaceutical
Bioanalysis: Technology Fundamentals and Current Applications
Stephen R. Dueker, Vitalea Science, 2121 2nd St., B101, Davis, CA
95618, Le T. Vuong, Jason Giacomo
AMS brought isotope-ratio mass spectrometry (IRMS) methods and benefits to
quantitation of long-lived radioisotopes such as 14C. Amongst these benefits is the
quantitative determination of 14C-tracer content in small bioanalytical samples referenced to the natural elemental and isotope concentrations, removing sources of
error in decay counting due to low recovery or uncertainty in amount of samples
matrix. This talk highlights fundamental of accurate AMS quantitation, including both
the instrument design/operations, bioprocessing best practices and the concept of
universal lower limit of quantitation (LLOQs) and response without specific analyte
knowledge. While AMS is most often associated with microdosing, particularly when
metabolic information is sought, the range of biomedical application is extensive
and growing. Lesser appreciated applications include pediatric pharmacokinetics,
biologic absorption, distribution, metabolism, and excretion n preclinical and human
tests, molecular diagnostics, and standard free metabolite discovery in early phase
clinical studies.
105
Novel Exploratory Approaches to Define Clinical PK and ADME –
Microdoses and Microtracers
Tom Cavalier, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia,
PA 19067, Graeme Young
Advances in technology, the need to conduct decision-making studies earlier in drug
development, and a growing acceptance by regulatory agencies have contributed to
an increase in the number of exploratory clinical investigations that utilize microdose
and microtracer approaches. A summary of these applications is presented and includes both Phase 0 pharmacokinetic (PK) and drug-drug interaction assessments.
Microtracer studies in early and late development to investigate clinical absolute bioavailability and assorted absorption, distribution, metabolism, and excretion (ADME)
assessments are also discussed.
109
Building a Quality Method with Robustness and Design Space
Jianmei D. Kochling, Genzyme, 153 Second Ave., Waltham, MA 02451
As the pharmaceutical industry is embracing quality-by-design in drug development,
it is critically important that the analytical methods are capable of delivering results
which allow scientists to confidently identify critical quality attributes and critical process parameters. Failing to achieving analytical methods performance goals can
result in failure in product quality control. Methods must have built-in specificity,
sensitivity and robustness along with other essential attributes. The most current
version of International Conference on Harmonisation Q2 guidance for method validation states that the robustness of the method should be conducted during method
development. Thus, a meticulous method development approach is essential. However, following traditional approach and varying method parameters sequentially
one-at-a-time, the method development task not only requires a long time, a few parameters that could affect the analytical method performance may also be missed.
Using the design of experiments approach in conducting method development and
method robustness determination, multiple parameters can be evaluated with a finite number of experiments. In addition, comparison of method parameters through
statistical data analysis allows the identification of critical method parameters and
design space. In this presentation, case studies are presented to demonstrate the
use of design of experiments for the determination of critical method parameters to
ensure method robustness and design space.
106
HPLC-AMS Method Validation for Supporting Absolute
Bioavailability Study with Microdosing 14C-Drugs
Xiaomin Wang, Celgene, 86 Morris Ave., Summit, NJ 07901, Mark
Seymour, Ali Arjomand
With the increasing adoption of high-pressure liquid chromatography accelerator
mass spectrometry (HPLC-AMS) in the bioanalytical sphere, and the inclusion of
data generated using this technique in dossiers submitted to regulatory authorities
in support of marketing applications for new pharmaceuticals, practitioners have
begun to turn their attention to analytical method validation. As with any quantitative
method, it is clear that parameters such as accuracy, precision, limits of quantification and run acceptance criteria must be defined. However, there are differences
between LC-AMS and other quantitative methods, such as LC tandem mass spectrometry (LC-MS/MS) that require a distinct approach to that commonly used for
conventional techniques. Two common approaches for determination plasma concentration by HPLC-AMS for supporting absolute bioavailability via 14C-drug micro
tracer are discussed.
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2012 EAS Abstracts
November 2012
110
washed and eluted with methanol/ water/acetic acid. A novel C18 chromatographic
column with a charged surface stationary phase produced peak widths for insulin
that were significantly narrower than traditional C18 columns using formic acid in the
mobile phase. The triple quadrupole used in these experiments produced several
higher m/z (ranging from m/z 700-1400) fragments. This provided a distinct selectivity advantage, significantly reducing endogenous background, relative to use of
lower m/z intense immonium ion fragments. Standard curves prepared in solvent
were linear from 50 pg/mL to 500 ng/mL. The detection limit for extracted human
plasma samples was 0.25 ng/mL for each of the four insulin analogs.
“Validation/Verification” of the Method Operable Design Region
(MODR) and Center Point: Is ICH Q2 Still Appropriate?
James E. Morgado, Pfizer, Eastern Point Rd., Groton, CT 06340,
Kimber Barnett
The tripartite harmonized International Conference on Harmonisation (ICH) Guideline on Methodology Q2B, now incorporated as of November 2005 within Q2(R1)
Validation of Analytical Procedures: Text and Methodology, was approved by the
Steering Committee and recommended for adoption by the three ICH regulatory
bodies on November 6, 1996. It was implemented by each shortly thereafter. It
presents guidance and recommendations on how to consider the various validation
characteristics of a respective analytical method along with recommended ranges,
replicates, and data reporting, (but no recommended criteria). It iterates that the
main objective of a validation is to demonstrate that the procedure is suitable for its
intended purpose. Since these guidelines were implemented, quality risk management (QRM) has taken a center stage in the pharmaceutical industry along with
its inherent concepts of a manufacturing process design space. These QRM concepts have trickled down into the analytical sciences as analytical quality-by-design
(AQbD). Within this framework a method operable design region (MODR) similar to
a design space has been proposed. Boundaries of this MODR are often defined by
multidimensional method factors which impact the separation and performance of
the two dimensional chromatographic separation. Validation of the method in line
with ICH Q2B usually takes place within this MODR. A presentation of how method
validation/verification can be considered or established at these MODR operational
boundaries with respect to an analytical target profile ATP are discussed along with
flexibility and limitations imparted by USP <621> and Ph.Eur. <2.2.46>.
115
Overcoming Bioanalytical Challenges to Enable Reliable
Quantification of Pharmacologically Active Clopidogrel Metabolites
in Human Plasma Samples
Michael T. Furlong, Bristol-Myers Squibb, Route 206 & Province Line
Rd., Princeton, NJ 08543, Pathanjali Kadiyala, Ishani Savant, Mark
Arnold, Anne-Francoise Aubry, Jim Shen, Bruce Stouffer, William Mylott,
Moucan Yuan, Laura Scott, Vikram Roongta
Clopidogrel is a widely prescribed antiplatelet medication. After oral administration
to humans, this drug is rapidly converted to four thiol-containing metabolites, two of
which possess significant pharmacological activity. Thiol metabolite quantification
in plasma samples drawn from subjects taking clopidogrel is challenging for two
main reasons. First, the thiol moiety in the metabolites is highly reactive with other
thiol containing molecules in the plasma sample; this reactivity must be suppressed
immediately after blood samples are drawn from the patient. Second, the four thiol
metabolites are diastereomeric and therefore the assay used for quantification must
be sufficiently selective to ensure that there is no assay interference among these
metabolites. We report herein the successful development of a rugged and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay capable
of simultaneously quantifying all four clopidogrel thiol metabolites in human plasma
samples. Key components of the assay include: 1) stabilization of the unstable thiol
moiety by chemical derivatization at the time of blood collection; and 2) chromatographic separation of the derivatized thiol metabolites. The assay was fully validated
according to Food and Drug Administration and industry standards. The lower limit
of quantification for the thiol metabolites ranged from 101-125 picograms / mL of
plasma sample. A comparison of this assay to a recently published assay is presented.
111
Application of the Quality-by-Design Principals in Formulation and
Process Development
Qun Lu, Merck, 86 Morris Ave., Summit, NJ 07901
No abstract submitted by author.
112
Withdrawn by the author.
113
Causes for Calibration Curve Slope Change and Their Impacts on
Bioanalysis Using LC-MS-MS
Yue Zhao, Bristol-Myers Squibb, Route 206 & Province Line Rd.,
Princeton, NJ 08543, Guowen Liu, Anne-Francoise Aubry
Consistent calibration curve slope is a positive indication of assay performance in
bioanalysis using liquid chromatography tandem mass spectrometry (LC-MS/MS).
It is one of the quality control tools utilized by bioanalytical scientists during the data
review process. However, it is not uncommon that calibration curve slope varies significantly across different analytical runs during sample analysis. Although there is
no specific requirement for the performance of calibration curve slope in regulatory
guidelines, it is very important to understand and identify the causes for such variations and perform investigations when scientifically needed. In this presentation, we
examine the possible causes for slope changes using several case studies. In general, slope change for assays using stable-isotope labeled internal standard (SIL-IS)
may suggest the followings: 1) possible lab errors during sample preparation; 2) IS
working solution instability; 3) interference from other sources; and 4) instrument
variations such as changes in mass spectrometry calibrations. In the case of an
analog internal standard, matrix effect should also be taken into consideration in
addition to the other causes. Specifically, in one case, the calibration curve slope of
a validated assay using an SIL-IS doubled after a regular maintenance of the mass
spectrometry. Systematic investigation revealed that minor shift on MS calibration
led to differential signal intensity changes on the analyte and the SIL-IS. In another
case, the calibration curve slope change was attributed to a differential matrix effect
when an analog internal standard was used. The impact of slope change on data
quality in different situations is also discussed.
116
Conducting Polymer Electrodes for Biomedical Investigations
J. Faye Rubinson, Georgetown University, Department of Chemistry,
Washington, DC 20057, Patrick A. Forcelli, Andew Walls, Anthony
Kammerich, Julia Roberts, Elizabeth Hanna, Cameron Sweeney
Over the last several years our group has focused on optimization of conducting
polymer modified electrodes for use in biomedical applications. To date we have
made substantial progress in three areas: detection of dopamine in the presence
of interferents such as ascorbic acid, development of enzyme-free electrodes for
detection of glucose, and development of high impedance interfaces for stimulation and interfaces characterized by low impedance at the frequencies relevant for
biosignal sensing. In addition to their electrochemical and electrical characteristics,
we also have explored the tissue response to the electrodes. The tissue response
to the electrodes has been explored in a rat model and two polymers have been
identified which result in a statistically significant (p< 0.05) lower astrocyte buildup
near the electrode track.
117
Oligonucleotide Analysis with Different Liquid Chromatography
Methods
Mirlinda Biba, Merck, PO Box 2000, Rahway, NJ 07065, Bing Mao
Different oligonucleotide samples (crude and purified) were analyzed with liquid
chromatography methods, including strong anion-exchange and ion-pair reversedphase liquid chromatography (LC). In anion-exchange LC, the separation is mainly
based on the number of charged groups and this technique is excellent for detection
of single base deletions. In ion-pair reversed-phase LC, in addition to charge-charge
interaction there is also hydrophobic interaction introduced and therefore this technique can better detect any other impurities, such as base switches, depurination,
etc. In addition, mixed-mode (anion-exchange and reversed-phase) columns were
also evaluated. Overall, the results show the complementary nature of these two
different methods and support the need to use both separation techniques for oligonucleotide analysis and characterization.
114
Development of an SPE-LC-MS/MS Method for Quantitation of Four
Synthetic Insulins in Human Plasma: Challenges of Working with
Large Peptides
Erin E. Chambers, Waters, 34 Maple St., Milford, MA 01757
Insulin is perhaps one of the best known and earliest peptide therapeutics. Although
historically quantified using ligand blot analysis, recently there has been a trend toward the analysis of large molecules by liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS has the advantage of shorter development times,
greater accuracy and precision, the ability to multiplex, and can readily distinguish
between closely related biotherapeutics and their metabolites. Intact insulins are difficult to analyze by LC-MS/MS however due to poor sensitivity and poor fragmentation. Insulin and its analogs also suffer from non-specific binding and poor solubility,
making LC and sample preparation method development difficult. The solid-phase
extraction (SPE) LC-MS/MS method described here was successfully used to quantify insulin glargine, insulin detemir, insulin aspart, and insulin glulisine in human
plasma. 250 µL human plasma was loaded onto a reduced-bed format 96-well plate,
118
Polysialic Acid : Resolving Homologues with Higher Degree of
Polymerization
Kirk Chassaniol, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale,
CA 94087, Peter Bodsky, Lipika Basumallick, Jeff Rohrer
Polysialic acid, a collective name for linear polymers of sialic acid, are widely expressed in nature (in animal and human brains, bacteria, fish, and a variety of cancers) and modulate cell-cell interaction, neural plasticity, and tumor metastasis.
There is a need for sensitive methods to accurately determine degree of polymeriza-
18
2012 EAS Abstracts
November 2012
tion (DP) for polysialic acid as it is critical to its function. This presentation describes
a High Performance Anion Exchange – Pulsed Amperometric Detection based
method to achieve better resolution for polysialic acids. A CarboPac PA200 column specially developed to provide high resolution separation of oligosaccharides
was used to analyze colominic acid (homopolymer of N-acetylneuraminic acid). The
proposed method resolves homologues with DP up to 100 in 70 minutes (with acetate gradient) and 140 in 90 min (with nitrate gradient) - an improvement over the
maximum DP (about 60 for acetate and 80 for nitrate) that could be achieved using
other columns.
the strategy used to develop, validate and maintain the NIR and Raman fingerprints
are discussed. These devices can potentially be utilized as a screening tool in the
field and the decision making process in identifying a counterfeit drug can be vastly
improved in terms of time, cost and efficiency.
122
Untargeted Detection of Novel Abused Substances Using High
Resolution Time-of-Flight Mass Spectrometry with Comprehensive
Collision Induced Dissociation
Kevin Siek, LECO Corporation, 3000 Lakeview Ave., Saint Joseph, MI
49085, David Alonso, Joe Binkley, Jeffrey S. Patrick
To accurately map the rapidly evolving landscape of chemical substance abuse requires analytical technologies that can detect targeted and untargeted analytes with
equal facility. High resolution time-of-flight mass spectrometry can offer the forensic
scientist such capabilities, but in practice, extracting signals of untargeted drugs
from volumes of high-resolution spectrometric data proves challenging. Coupling
comprehensive collision induced dissociation (CID) with high-resolution time-offlight mass spectrometry enables advanced signal processing algorithms and data
interrogation tools to rapidly detect and confirm novel abused substances present
in urine or seized materials. The mass spectrometric technology and the data processing strategies discussed in this presentation were used to identify a designer
psychostimulant and its metabolites present in human urine, and to identify an unknown drug and an unknown metabolite present in racehorse urine. High resolution
time-of-flight technology aided the identification of structurally diagnostic fragment
ions for the synthetic cannabimimetic JWH-018. Extending high resolution time-offlight mass spectrometry with comprehensive CID to detection of designer steroids
is also briefly discussed.
TLC-SERS Study of the Main Alkaloid Constituents of Peganum
Harmala Seeds
Federica Pozzi, City College of New York, 138th St. at Convent Ave.,
New York, NY 10031, Marco Leona, Nobuko Shibayama, John R.
Lombardi, Elena Platania
Surface-enhanced Raman scattering (SERS) has found increasing acceptance
in art conservation and forensic science for its great potential in detecting trace
amounts of material. However, SERS is not a separation technique and; therefore, it
is not suitable for distinguishing different components in a mixture. Coupling of thin
layer chromatography (TLC) and SERS has been investigated here as a tool for the
separation and identification of four alkaloids, namely harmalol, harmaline, harmane
and harmine, from the seed extract of Peganum harmala. These compounds were
historically used as a dye and for medicinal purposes, and have recently drawn
attention due to their antitumor activity. The use of TLC over high-pressure liquid
chromatography (HPLC) is a convenient way to reduce the amount of material, sophisticated equipment and time needed for the analysis, and coupling of TLC with
SERS allows gaining of vibrational information on each compound in the mixture.
Complementary HPLC analyses were also performed as a test of our technique
to ascertain the composition of Peganum harmala extract and validate the results
obtained from TLC-SERS. In addition, Peganum harmala extract and its commercial
alkaloid components were characterized by Fourier transform Raman, Raman and
SERS spectroscopies, in order to provide reference data to be used for identification
purposes. It is interesting to notice that harmalol, harmaline, harmane and harmine
have analogous molecular structures and therefore, in most cases, provided very
similar spectra. However, we have identified discriminant bands which can be used
to differentiate among them, which is one of the main issues in forensic applications.
120
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119
Cracking Bath Salts: Use of Direct Deposition GC-IR for Complex
Controlled Substances Mixtures Identification
Ming Zhou, Spectra Analysis Instruments, 257 Simarano Dr.,
Marlborough, MA 01752, Tom Kearney, Sid Bourne
Many countries are starting to ban bath salts because of incidents linked with their
consumption. This ban requires new identification methods as current analytical
techniques such as gas chromatography mass spectrometry (GC-MS) are not offering the discrimination power required by criminal proceedings. Indeed, mass spectrometry the forensic workhorse, cannot be used when synthetic cannabinoids, and
“bath salts” are handled as they all produce samples which require the ability to
discriminate between isomers in a complex mixture. In order to meet this stringent
requirement, most forensic labs currently follow the recommendations outlined by
SWGDRUG (scientific working group for the analysis of seized drugs). For conclusive qualitative analysis, SWGDRUG requires 1) a minimum of two analytical
methods to form an acceptable analytical method, and 2) that each method must
yield conclusive and definite results (precluding false positive). The hyphenation of
infrared spectroscopy and gas chromatography using direct cryo-deposition meet
both requirements, and, enables the differentiation of both isomers of position or
configuration. Indeed, after exiting the GC column via a heated transfer line, the
eluents flow through a deposition tip, and are deposited as a collimated jet onto a
cryogenically cooled disk. The infrared (IR) transparent disk rotates/translates under
the deposit tip to result in a spiral deposit track of samples. As analytes elute from
the capillary and onto the cold disk they form a solid phase deposit on the disk. An
IR beam from an interferometer passes through the disk along the deposition track,
and transmission solid phase infrared spectra are collected onto a Mercury cadmium telluride detector. This poster provides examples of the use of GC-IR technology in a forensic environment with bath salts and cannabinoids mixtures analysis.
Non-Destructive Characterization of Counterfeit Designer
Handbags by Vibrational and Atomic Spectroscopy
Gene S. Hall, Rutgers University, 610 Taylor Rd., Piscataway NJ 08854,
Christine Perna, Robyn Seigler
There are numerous counterfeit (“knock off”) designer handbags being sold on the
street, the internet, and at flea markets. Some of the handbags are well designed
and difficult to distinguish between the genuine handbags. Since handbags have
several different components ranging from metallic to animal skin, these different
components can reveal the tradesmanship of the counterfeiter and perhaps determine the country or location of manufacture. With this in mind, we have used a
combination of non-destructive complimentary spectroscopic techniques such micro
energy dispersive X-ray fluorescence (M-EDXRF) and attenuated total reflectance
Fourier transform infrared (ATR-FTIR) spectroscopy to determine differences between different components of counterfeit and genuine handbags. EDXRF (atomic
spectroscopy) showed that the metal components of the counterfeit handbags were
of a different alloy than the genuine. In addition, ATR-FTIR (vibrational spectroscopy) showed that the counterfeit handbag material was made of polyvinyl chloride
(PVC) to simulate leather (“pleather”). This presentation describes in detail sample
collection, instrumentation setup, data collection, and data interpretation. Results
are presented to show differences between counterfeit designer handbags and
genuine designer handbags from Coach, Gucci, and Burberry.
124
Raman Characterization of Molecular Orientation in Trace Evidence
Fibers
Fran Adar, HORIBA Scientific, 3880 Park Ave., Edison, NJ 08820, John
A. Reffner, Dale K. Purcell
Trace evidence, as collected from crime scenes, provides a means for law enforcement to identify and prosecute criminals. Infrared absorption microscopy provides
identification of molecular composition for matching trace evidence to suspect samples, and visible absorption microscopy enables color matching. In addition, fibers
exhibit molecular orientation to varying degrees. This includes natural fibers such
as cellulose and silk, as well as man-made fibers. By controlling the fiber orientation
relative to the Raman instrument configuration, including the polarization conditions,
it is possible to quantitate the degree of orientation. This provides an additional
parameter for characterizing trace evidence and for differentiating samples. For
instance, it has been shown that Raman can clearly document increasing orientation and crystallinity in polyethylene terephthalate fibers that correlate with spinning
take-up-speed, and draw ratio and temperature[1]. This information provides a valuable addition to the arsenal of physical measurements of trace evidence.
121
Screening “Small and Big” Pharmaceutical Molecular Counterfeits
Ravi Kalyanaraman, Bristol-Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08903
Counterfeit and adulterated drugs have caused a serious threat to patients in the
United States after the recent Avastin® injection incident and they have entered the
pharmaceutical supply chain due to increasing international trade and sales via the
internet. Due to this infiltration, pharmaceutical manufacturers are finding ways to
authenticate and identify their product by various technologies. One of the effective ways to authenticate and verify the contents in a pharmaceutical product is
by using portable near-infrared (NIR) and Raman spectrometers to obtain unique
fingerprints of the drug product itself. Using these product fingerprints, it is possible
to test a suspect sample and identify if it is a counterfeit and also to authenticate
if it is a legitimate product. Solid (tablets, capsules and powders) dosage forms
were analyzed directly through packaging, such as glass and blister packs and
were non-destructive with no sample handling or preparation required. Examples
of counterfeit drugs identified using portable NIR and Raman spectrometers and
[1] F. Adar and H, Noether, Polymer 1985, 26, 1935-1943; and M. Richard-Lacroix
and C. Pellerin, Macromolecules 2012.
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The Latest Development of the On-line Tablet Characterization
System of Tandem for Content Uniformity
Yan Wang, Bruker Optics, 19 Fortune Dr., Billerica, MA 01821, John
Richmond
An update is presented on the latest development of the Fourier transform nearinfrared (FT-NIR) system of Tandem, the fully automated on-line process analytical
technology tool that provides both physical and chemical characteristics of pharmaceutical tablets. The Tandem provides real time information on tablet content uniformity (CU), weight, thickness and/or hardness for better process understanding. The
implementation enhances the capability of pharmaceutical manufacturing in quality
assurance of the finished products of tablets, and at the same time eliminating operator contact with high potency drugs for safe operation. The tablet handling and
positioning becomes more precise with the renovated engineering mechanism, ensuring no sampling errors, and thus generating reproducible and reliable tablet CU
results in real time. The long term instrument stability is guaranteed by the patented
rock solid interferometer and precision in spectrometer engineering. The FT-NIR
spectrometer employed in the Tandem is identical to the mostly installed FT-NIR
Multi-Purpose Analyzer for off-line tablet analysis in pharmaceutical manufacturing. In addition, robustness of analysis results is discussed from different aspects:
instrument performance and stability, design of experiment of calibration tablets,
chemometric modeling, and implementation of the developed calibration models.
target concentration need to be carefully prepared and analyzed with NIR. Typically
calibration sets with 75% to 125% potency have to be prepared. This procedure is
time consuming since samples of various concentration of active pharmaceutical ingredient (API) and different excipients need to be prepared. This can introduce a lot
of errors, especially for low concentrations. For example, the weighing may not be
accurate enough and the blending of API and excipient may not be homogeneous.
In this study we come up with a different approach. We collect spectra of pure API
along with spectra of each pure excipient. Then spectra of the calibration set were
generated mathematically from the pure spectra. In this presentation, we discuss
how the calibration spectra are generated and the performance of the calibration
developed based on mathematically generated spectra when it was used to predict
the real sample.
130
Physical Stability and Recrystallization Kinetics of Amorphous
Ibipinabant Drug Product by FT-Raman Spectroscopy
Wayne E. Sinclair, Bristol-Myers Squibb, Reeds Lane, Moreton CH46
1QW, United Kingdom
The solid-state physical stability and recrystallization kinetics during storage stability
are described for an amorphous solid-dispersed drug substance, ibipinabant, at a
low concentration (1.0% w/w) in a solid-oral dosage form (tablet). The recrystallization behavior of amorphous ibipinabant-polyvinylpyrrolidone solid dispersion in the
tablet product was characterized by Fourier Transform (FT) Raman spectroscopy.
A partial least-square analysis used for multivariate calibration based on Raman
spectra was developed and validated to detect better than 5% (w/w) of the crystalline form in the low-dosage tablet (equivalent to less than 0.05% of the total mass
of the tablet). It was determined that exposure to moisture had a significant impact
on the crystallinity of amorphous ibipinabant and dissolution testing demonstrated
the predicted crystallinity had a direct correlation on this physical property of the
drug product. Recrystallization kinetics evaluated by application of the JohnsonMehl-Avrami (JMA) kinetic model to determine recrystallization rate constants and
Avrami exponent (n=2). The analysis showed that the JMA equation could describe
the process very well, and indicated that the recrystallization kinetics observed was
a two-step process with an induction period (nucleation) followed by rod-like crystal
growth. The type of placebo excipients used in the formulation is found to have a
significant influence on the re-crystallization kinetics. The application of FT-Raman
spectroscopy to the detection and characterization of ibipinabant solid-state form
in such a low dose drug product would be difficult to achieve by any other method.
126
Improved Process Understanding and End-Point Determination of
High Shear Wet Granulation Process at Different Scales Using InLine NIR
Hua (May) Ma, Hoffmann-La Roche, 340 Kingsland St., Nutley,
NJ 07110, Yu-E Zhang, Hung Tian, Duk Soon Choi, Navnit Shah,
Dharmendra Singhal, Hitesh P. Chokshi
In-line near-infrared (NIR) spectroscopy is now widely considered as an efficient tool
for expeditious extraction of physical and chemical information during pharmaceutical processes. Using this approach, better process control is made possible through
improved process understanding. In this study, in-line NIR was applied for real time
monitoring and end-point determination of high-shear wet granulation process at
difference scales with a placebo formulation. By applying multivariate data analysis
on the NIR spectra collected in real time and correlating the process trajectories
acquired from different scales, in-depth process information at each wet granulation
sub-phase was obtained and the wet granulation mechanism was elucidated to support the accuracy of the process end-point determination. Results from this study
demonstrated the applicability of using in-line NIR for high shear wet granulation
process monitoring and end-point determination. Furthermore, it also revealed the
great potential of using this technique to enable the identification of critical process
parameters and facilitate formulation and process optimization and scale-up.
131
Understanding the Dynamic Process of Dissolution Using Infrared
Spectroscopy
Vrushali M. Bhawtankar, Seton Hall University, 400, South Orange Ave.,
South Orange, NJ 07079
Dissolution studies are critical tests for measuring the performance of a drug product. In the past few years, the importance of the dissolution test has increased. Using in-situ attenuated total reflection Fourier transform infrared spectroscopy spectroscopy we developed a methodology of analyzing and monitoring dissolutions of
active pharmaceutical ingredients. The accuracy of this technique was found to be
± 3% relative to high-pressure liquid chromatography and UV using salicylic acid
calibrator tablets and acetaminophen tablets. Additionally, research was conducted
on multi-component drugs such as Excedrin and extended release acetaminophen
tablets. We are still working on multicomponent tablet. In fact, we also performed the
hydrolysis of aspirin and studied the chemistry behind the dissolution by react IR. In
the calibration study of acetaminophen we observed that at higher concentration its
behavior is non-linear, and out hypothesis is due to dimerization of acetaminophen.
With the study of hydrolysis of aspirin we are trying to study the non-linear behavior
of acetaminophen. With improved sensitivity, this is a promising method for monitoring dissolution of pharmaceutical tablets with an excellent automated capability for
distinguishing individual components.
127
Application of Quality-by-Design Principles to Near-Infrared
Multivariate Analytical Methods
Claudia C. Corredor, Bristol-Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08901, Ruben Lozano, John Bobiak, Dongsheng Bu, Tim
Stevens, Gary McGeorge, Douglas Both
Analytical quality-by-design (AQbD) enhances method understanding and robustness. It helps identify and control sources of variability and facilitates continuous
improvement. In this talk we present applications of AQbD for near-infrared spectroscopic multivariate methods. Specific examples of AQbD are presented applied
to near-infrared models for blending uniformity and potency and uniformity dosage
units of tablets. An overview of the analytical target product profile, risk assessment
and risk control, and the method operable design region are presented.
128
On-Line Monitoring Powder Blending Process Using Antaris Target
NIR Analyzer
Herman He, Thermo Fisher Scientific, 4410 Lottsford Vista Rd., Lanham,
MD 20706, Michelle Pressler
For pharmaceutical and food manufacturers who produce solid dosage forms, mixing or blending processes are critical. It is the blending step that assures the resulting dosage forms will be uniform. In this study, a microelectro-mechanical-system
based near-infrared (NIR) spectrometer was investigated as a tool to monitor the
dynamics of production blending processes. Several common math processing
algorithms were used to evaluate the blending data and the results of this study
demonstrate that the Thermo Target NIR analyzer is a good approach for in-process
confirmation of blending. The excellent triggering and sampling capability of the
Target analyzer was deemed critical for this application.
132
Pharmacokinetics of 40kDa PEG in Rodents Using High-Field NMR
Spectroscopy
Purnima Khandelwal, Bristol-Myers Squibb, 311 Pennington-Rocky Hill
Rd., Pennington, NJ 08534, Lisa Zhang, Anjaneya Chimalakonda, Janet
Caceres Cortes, Christine Huang, Punit Marathe, Michael D. Reily
A quantitative nuclear magnetic resonance (NMR)-based method was developed
and successfully applied to measuring double branched polyethylene glycol 40kDa
(PEG40) in in-vivo serum samples, enabling determination of pharmacokinetic (PK)
parameters of PEG40 in preclinical species. Conjugation of macromolecular drugs
to PEG improves their therapeutic potential by altering their PK profile, but literature
on complete PK profiling for PEG40 itself is lacking. Other analytical techniques
have difficulty measuring PEG40 due to its large, sticky nature which frequently
causes contamination of mass spectrometer (MS) sources; and the fact that PEG is
a polymer which contains many individual analytes, complicating chromatographic
or MS-based measurement. NMR is ideal for measuring such polymers because,
among other advantages, a single, sharp peak is obtained for all the equivalent
129
Simulation Approach for Calibration Development
Hui Li, Bruker Optics, 19 Fortune Dr., Billerica, MA 01821, John
Richmond
Near-infrared (NIR) is widely used in pharmaceutical industry for blend uniformity
and content uniformity testing. In order to get a good prediction of the active ingredient concentration, a calibration set with multiple samples in a range larger than the
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2012 EAS Abstracts
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136
methylene protons which is insensitive to polymeric heterogeneity. High-field NMR
(600 MHz) with cryoprobe technology allowed for analysis of samples in 250nM
range. CD-1 mice serum samples collected after PEG40 IV dosing were analyzed
for PEG40 using the NMR method and PK parameters were calculated using noncompartmental analysis. The volume of distribution at 0.17 L/kg indicated limited
distribution to the plasma and interstitial space. A low clearance and a half-life of 18
h for PEG40 are consistent with literature.[1] Compared to the 125I radioactive assay
in,[1] the current NMR technique is quite user-friendly. Additionally, this is the first
report, to our knowledge, of NMR spectroscopy application to PK profiling in serum.
Taking the Laboratory to the Sample – Handheld Instrumentation
Today and Tomorrow
M. Bonner Denton, University of Arizona, Department of Chemistry, PO
Box 210041, Tucson, AZ 85721
For many years innovators have dreamed of a handheld technology capable of
providing high quality chemical analysis of an unknown object. The television series
“Star Trek” featured the fictional “Tricorder” device for achieving this goal. Recent
developments in miniature precision optical components, solid-state lasers, detector
arrays, microcomputers and a host of other innovations have provided the technology necessary to develop a variety of high-pressure instrumentation capable of
providing many of the desired abilities of the fabled Tricorder. Currently available
and new instrumentation under development are discussed which provide the ability
to identify and analyze a wide variety of materials. Performance considerations and
design concepts, including tradeoffs and near term realizable levels of performance
are presented. Another important factor contributing to the power of the latest generation of instruments is both the incorporation of large spectral data bases coupled
with powerful search engines that provide high quality search and match abilities.
This presentation discusses how these advances are being implemented in new
generations of handheld instruments and demonstrate some of these instruments’
current and near term future abilities.
[1] Yamaoka et al, J. of Pharmaceutical Sciences 83 (4), 1994, 601.
133
Highly Efficient Protein Separations in Submicrometer Particles
Mary Wirth, Purdue University, Department of Chemistry, 560 Oval Dr.,
West Lafayette, IN 47907, Benjamin J. Rogers, Bingchuan Wei, Owen
Rehrauer
We are investigating reversed-phase liquid chromatography (RPLC) for particles
between 0.1-µm and 1-µm in diameter to improve protein and peptide separations.
This is new ground because particles of commercial columns exceed 1-µm in diameter. One might think that submicrometer particles would be impractically small because the backpressure would be excessively high, but there are two factors that alleviate the backpressure issue. The first is that the plate height becomes two orders
of magnitude smaller by alleviating broadening from packing and mass transport;
therefore, the separation length can be shortened to a few cm yet still provide many
more theoretical plates. The second is that there is a phenomenon in nanofluidics
called slip flow, whereby flow rate is enhanced when the particle diameters goes
below 1-µm. With slip flow, the flow rate decreases in proportion to particle diameter
over the range of 0.1 to 1-µm, rather than decreasing with the square of the particle
diameter. Studies of slip flow and protein and peptide separations using the media
in nanoLC are presented.
137
Analytical Applications of GUMBOS
Isiah Warner, Louisiana State University, Department of Chemistry, 434
Choppin Hall, Baton Rouge, LA 70803, Bishnu Regmi, Sergio de Rooy,
Indika Galpouthdeniya, Paul Magut, Susmita Das
My research group has been exploring the analytical applications of room-temperature ionic liquids (RTILs) for several years. Recently, we have extended the range
of these materials to include analytical applications of similar solid materials, i.e.,
organic salts with melting points of solid ionic liquids (25 °C to 100 °C) up to melting points of 250 °C. To contrast these new materials with RTILs, we have created
the acronym, GUMBOS (group of uniform materials based on organic salts). These
GUMBOS have the tunable properties frequently associated with RTILs, including
tunable solubility, melting point, viscosity, thermal stability, and functionality. Thus,
when taken in aggregate, these properties allow the production of solid materials
which have a wide range of applications in measurement science. In this talk, I highlight the applications of GUMBOS which we have recently explored for measurement science, including GUMBOS as sensors, imaging agents, stimuli-responsive
materials, and for production of nanoGUMBOS. In regard to nanoGUMBOS, we believe that our methodology represents an extremely useful approach to production
of nanomaterials since our materials are designed and assembled for specific uses,
rather than adapted for use as is done for many nanomaterials. Selected applications, including sensor applications, are highlighted in this talk. Particular emphasis
is placed on a novel quartz crystal microbalance sensor for measurement of volatile
organic compounds with simultaneous molecular weight determination
134
Imaging of Single-Molecule Binding Kinetics at Liquid-Solid
Interfaces
Joel M. Harris, University of Utah, Department of Chemistry, 315 S.
1400 E, Salt Lake City, UT 84112, Grant A. Myers, Eric M. Peterson
Quantitative imaging of fluorescent molecules at surfaces is a powerful method to
characterize equilibria and dynamics of interfacial processes. Internal-reflection
confines laser excitation to an interface, allowing imaging of molecules at surfaces in contact with liquids. We have applied this method to count membraneactive peptides in lipid bilayers on glass surfaces. Fluorescence imaging reports
the membrane-binding equilibrium constants through counting of individual bound
peptides versus the solution concentration. The membrane residence times of individual molecules can also be determined, and rates of unbinding calculated from
a histogram of residence times. We have used this method to characterize the lipid
membrane affinity of glucagon-like peptide-1 (GLP-1), a 30-residue peptide involved
in glycemic control. Residence-time data were interpreted using a sequential twostep, three-state binding model, where microscopic rates connecting membraneadsorbed and -intercalated states of the peptide could be resolved. Single molecule
imaging was also used to monitor individual complementary strand DNA hybridization events. Target single-stranded DNA (ssDNA) was immobilized at an interface,
and its surface density and association constant were determined from the binding
isotherm of fluorescently-labeled complimentary strand ssDNA. Dissociation rate
constants of the DNA duplex were determined from the dissociation times, and association rates were calculated from the association constant and the dissociation
rate assuming a two-state binding model. From the dependence of association constants, association rates, and dissociation rates on ionic strength, an Eyring model
was used to determine the electrostatic contributions to the free energy of the transition state and the fully hybridized double-helix.
138, 139
140, 141
Designer Drugs 2012: The Problem, Identification, and the
Future
Christopher Guglielmo, Andrea Placke, Babita Joseph, Yuriy
Uvaydov, Drug Enforcement Administration (DEA), Northeast
Laboratory, 99 Tenth Ave., Suite 721, New York, New York 10011
The objective of this workshop is to present the problem that all controlled substance laboratories are currently facing: the identification of designer drugs and
the determination of their controlled status. Are these compounds controlled substances? Do they fall under Food and Drug Administration regulations? Are these
compounds analogues of controlled substances, and if so which ones? What law
applies to these compounds? Where can reference standards be obtained in order
to identify these compounds in accordance with current requirements? Designer
drug threats in the past, including PCP analogs in the late 1970’s, Fentanyl Analogs
in the 80’s, and MDMA/Ecstasy analogs in the last ten years, have only involved a
half dozen or so new compounds. Once these substances were able to be identified,
it was relatively easy to move to structure laws to control them.
135
Neuroscience Research: A Target Rich Environment for
Measurement Driven Breakthroughs
Tim Harris, HHMI Janelia Farm Research Campus, 19700 Helix Drive,
Ashburn, VA 20147
The understanding of brain structure and function is among the today’s most challenging research problems. Even a drosophila brain contains approximately 100,000
neurons of 500-5000 types and approximately 100 million connections. Static structure of so complex a system remains beyond our reach, but understanding requires
measurement of both structure and activity at this scale. I illustrate the current frontier with two examples: a fluorescence imaging microscopy platform to determine
connections with sub-diffraction resolution in three dimensions. Electrophysiology
monitoring activity in hundreds of neurons with millisecond time resolution is shown.
That situation began to change when dozens of new steroid (pro-hormone) drugs
were developed and introduced in early 2000’s. We now have a perfect storm where
the internet and the global chemical development market have combined to introduce hundreds of new designer drugs which are a threat to public safety. The current designer drugs encountered regularly in the northeast encompass broad drug
types from synthetic cathinones to designer steroids to synthetic cannabinoids to
compounds contained within plant materials, and others.
We begin with a discussion of the drug trends in the northeast, followed by a timeline, tracing the development and distribution of these drugs, along with the current status of the laws regarding their controlled status effects. We then discuss
the particular difficulties facing laboratories regarding these compounds, to include
the analogue issue, as well as the issues of obtaining reference standards. The
laboratory identification of these compounds with specific lectures directed at each
particular drug group and their analysis are also covered.
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2012 EAS Abstracts
November 2012
The many challenges associated with the ever-changing use of drugs in today’s
world will forever present difficulties to forensic laboratories as they continue to allocate resources in an attempt to keep pace with the analysis and identification of all
controlled substances. A goal of this workshop is to share as much information as
possible to assist in the identification and to ensure that all lines of communication
remain open to continue to work together to address these compounds
methods, such as liquid chromatography (LC) or ion mobility (IM) spectrometry. Interactive LC is ideally suitable for the separation of oligomer mixtures with constituents of different polarities. This capability is illustrated for amphiphilic copolymers
and nonionic surfactants. IM-MS may be also viewed as a chromatographic method
that disperses post-ionization according to size, charge, and shape. Since it does
not involve interactions with a stationary phase, it is particularly useful for weakly
bonded species. Our group has applied IM-MS to characterize supramolecular polymers, self-assembled from designed building blocks. Self-assembly generally creates many isomers and conformers (architectures); these have identical masses but
unique shapes and, thus, can be separated and identified by IM-MS, as is shown for
a variety of supra-macromolecules. Further insight about the binding interactions in
these materials is gained by their dissociation energetics, assessed through gradient IM-MS/MS experiments.
142
How Isomers Make Life of a Chemist Interesting: Selected
Examples of LC-MS Analysis of Oligonucleotides, Peptides, and
Glycopeptides
Martin Gilar, Waters, 34 Maple St., Milford, MA 01757, Ying-Qing Yu,
Joomi Ahn, Sean M. McCarthy, Vera B. Ivleva
We live in age of ultra high-pressure liquid chromatography (UHPLC) and mass
spectrometry (MS). While high peak capacity of UHPLC provides tools for advanced
separation of compounds of interest, MS can identify and quantify the compounds
that are incompletely resolved by chromatographic methods, because they have
different mass (m/z). Yet, one class of analytes present challenge even for stateof-the-art LC-MS instrumentation. Optical or positional isomers are hard to resolve
chromatographically and have isobaric mass, so it is difficult to differentiate them by
MS as well. In this presentation we discuss several applications where isomerism
complicates analysis. For example, cis-trans proline isoforms complicate separation of proline rich peptides and cause severe tailing of selected peptides in mAb
tryptic digests. Another example is analysis of phosphorothioate oligonucleotides,
which consists of multiple diastereomers. We illustrate how partial separation diastereomers complicates the LC-MS analysis of DNA/RNA based drug candidates.
Examples of oligonucleotide MS/MS sequencing is shown. Finally, we discuss the
implications of positional isomerism on LC and LC-MS analysis of glycans and
glycopeptides. Because glycans are comprised of relatively few building monosaccharide blocks (hexoses, deoxy-hexoses, NAc-hexoses…) it is challenging to distinguish among their isomers even when using LC-MS(MS). We show the utility of
hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography separation modes for analysis of both glycan and glycopeptide isoforms.
146
Perspective on Outsourcing Strategies and Challenges – in
Regulated Bioanalysis
David Evans, Janssen Research & Development, 1000 US Highway
202, Raritan, NJ 08869
Once viewed as tactical partners to offset peaks in activity, contract research organizations now represent an integral part of drug development, allowing Pharma
to evolve strategies that provide resource flexibility and allow focus on value-add
activities. Implicit in this trend is the perspective that functional execution of work
by pharmaceutical companies is not a differentiating competitive advantage, and
financial resources, once used to support such functions, are better deployed to
support innovative basic research or compound acquisitions to stoke sparse drug
pipelines. But is there agreement around these statements, does this represent an
over simplification, or are there hidden risks in this shift of emphasis as it pertains,
in this instance, to regulated bioanalysis? Judging from the broad spectrum of pharmaceutical organizational models, there are clearly varying opinions on this matter,
not least because there are less tangible variables to consider when honing in on
an outsourcing model. These include an organization’s perspective on how to innovate, where competitive differentiation can be found, how to influence or participate
in regulatory guideline discussions, how to assure scientific partnership, and how
to create the talent pipeline of scientists that drives all of these vital activities while
managing the external relationships and work. Collectively, these varying, often
competing, perspectives are discussed in the context of an outsourcing strategy
that conserves internal “value” balanced with overall financial prudency and long
term sustainability within a contemporary research and development organization.
143
The Implementation of a Non-Labeled, Gel Free, MS-Based
Proteomics Method to Evaluate Endocrine Disrupting Compounds
in Small Fish Species
Kimberly Ralston-Hooper, Duke University, Department of Chemistry,
Durham, NC 27708
No abstract submitted by author.
147
The Other Side of the World in Outsourcing
Frank Li, Alliance Pharma, 17 Lee Blvd., Malvern, PA 19355, Yifan Shi,
Michael Zhang
Both pharmaceutical and agrochemical companies are in line with the current trend,
to form alliance partners with external resources, such as contract research organizations (CROs) to increase efficiency and reduce the cost. However, there are
tremendous challenges for both industries. There are also great challenges behind
the opportunities for CROs, such as regulatory compliance, scientific excellence,
professional ethics, and also wining the trust from industrial sponsors. The difference between bioanalytical services and agrochemical services are very obvious
in regard to methodology, technical skill and regulatory requirements. However, the
principals are the same. Data quality, quick turnaround time and pricing are always
the topics on the table from both sponsors and CROs.
144
Sequence Confirmation of Phosphorothioate Containing siRNAs
by Chemical and Enzymatic Digestion and Liquid Chromatography
Mass Spectrometry Approach
Jiong Yang, Merck, 126 East Lincoln Ave., Rahway, NJ 07065, Fanyu
Meng, Huimin Yuan, Bing Mao
Small interfering RNAs (siRNAs) have shown promise as novel therapeutics. Similar
to other oligonucleotide based drugs such as antisense based therapeutics, the
incorporation of phosphorothioate backbone has been widely used to improve the
stability of siRNAs. While this technique is quite effective, it also created an analytical challenge for complete sequence confirmation of siRNAs. In the past, we have
shown that enzymatic digestion coupled with mass spectrometry is a useful method
for siRNA sequence confirmation. However, reports indicated that phosphorothioate
(PS) linkages are resistant to enzymatic digestion. In this study, we synthesized
siRNA sequences with varying PS content in the 3’ region, and explore different
digestion methods to efficiently sequence these siRNAs. We analyzed the digestion mixture of the sample containing PS and w/o PS. The result clearly showed
PS can play a major role in resisting the enzymatic digestion of siRNAs and we
optimized the oxidation condition to convert PS to phosphodiester and the digestion
of oxidized siRNA is very helpful in confirming the sequences to meet the regulatory
requirements.
148
Challenges with Immunoanalytical Method Transfers to CROs
Dennis Stocker, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ
08543, Zoe Tzogas
Immunoanalytical methods used for bioanalytical support (pharmacokinetic and immunogenicity) of biologic drugs in pre-clinical and clinical studies often pose unique
challenges when transferring to contract research organizations (CROs). These are
typically plate based ligand binding assays that require multiple manual steps and
several unique reagents that are specific to the analyte being measured and instrument platform. These circumstances can pose many challenges to achieve concordant and consistent performance and data quality at the CRO over the course of
the transfer, validation and study sample analysis. Continuous oversight of the CRO
during method transfer, validation and implementation is often required to mitigate
problems early before they can have significant impact on study results. Data quality and consistency needs to be assessed and compared to in-house data at each
step in the process, and cross-validation is recommended. Another challenge with
long-term support of these methods at the CRO is the required bridging of new lots
of reagents over several years. Good quality systems, thorough standard operating
procedures, appropriate staff training and instrument availability are also important
in the selection of the appropriate CRO. The Bristol-Myers Squibb’s perspective on
these challenges when outsourcing methods for bioanalytical support of biologic
drugs are discussed and some examples are presented.
145
Mass Spectrometry Methods for the Characterization of New
Synthetic Polymers and Supramacro-Molecules
Chrys Wesdemiotis, University of Akron, Department of Chemistry,
Akron, OH 44325
Matrix-assisted laser desorption ionization and electrospray ionization have enabled
mass spectrometry (MS) analyses for a wide variety of synthetic polymers; however,
considerable challenges still exist. Polymerizations often create complex mixtures
that are impossible to characterize by single-stage MS because of superimposed
compositions or discrimination effects. These problems can partly be resolved by
combining MS with fragmentation and a second stage of mass analysis, i.e., by
tandem mass spectrometry (MS/MS). This is demonstrated for styrene homo- and
copolymers, whose MS/MS characteristics reveal unequivocally the corresponding
architecture, functionality location, and comonomer sequence. For the characterization of more complex systems, MS and MS/MS must be combined with separation
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153
149
Waiter, What is this Fly Doing in my Soup?” – 30 Years of Consumer
Complaint Analysis
John Lemmo, Johnson & Johnson, 199 Grandview Rd, Skillman, NJ
08558
This is an interesting and entertaining review of several case studies involving consumer complaints, raw material defects, and process problems – from the viewpoint
of the analytical chemist. The presentation discusses a few key analytical techniques used to probe these problems, and what you can and cannot infer from
the results. More importantly, the presentation addresses some of the pitfalls of
analytical/investigative process. Pitfalls such as: What is the real question I need
to answer? What can I learn from inconclusive results? How do I deal with external
labs and internal customers?
Keys to a Successful Relationship with your CRO
Steve Unger, Worldwide Clinical Trials, 8609 Cross Park Dr., Austin, TX
78754, Greg Poch, Edward Wells, Melvin Tan
Establishing, building and maintaining a relationship with a contract research organization (CRO) must consider: your needs (user requirements), lab capabilities
(people, training, equipment, compliance history) capacity, business model (innovator, generic, specialized vs. soup-to-nuts), scientific diversity (bioanalytical, drug
metabolism pharmacokinetics, chemistry, manufacturing, and control, toxicology,
clinical), quality processes (standard operating procedures, quality control/quality analysis and reporting), small (liquid chromatography mass spectrometry) and
large (ligand blot analysis) molecule capabilities, experience with other validated
assays (biomarker, DDI and comed), ability to trouble-shooting science and partner
in drug development, and cost. Most effective relationships have core experts on
both sides that manage the work. Project teams define drug development plans
which are translated into action at CRO. Within bioanalytical science, details are
important. From sample collection to pharmacokinetic analysis, all aspects of bioanalysis must be planned in advance of the final protocol. Special requirements
such as chromatographic figures of merit may be needed. Opportunities should be
thoroughly reviewed and potential problems resolved. Details such as non-specific
binding, stability, detection specificity, assay selectivity and sensitivity need to be
well understood. Due diligence is required to fill any knowledge gaps at the assay
transfer stage. This can include blood stability or red blood cells partitioning and
sample condition (hemolyzed and lipemia). Examples of heme-mediated oxidation
show how stability can be impacted in hemolyzed plasma. Incurred sample reanalysis has become a common investigational tool over the past five years. An example
shows how an isomeric interference resulting from a degrading metabolite impacted
both pro-drug and active assays.
154
Concepts for Spectral Engines: Platforms for Handheld Instruments
John Coates, Coates Consulting, 12 North Branch Rd., Newtown, CT
06470
In the past five years there has been significant growth in the market for small
handheld spectrometers. A large number of products now exist that provide measurements in the visible and near- and mid-infrared spectral regions, which include
Raman methods of detection. Generally, these instruments are similar in price to
traditional laboratory spectrometers, and sometime they are even more expensive.
With miniaturization and size reduction there has been an expectation that the role
of spectroscopy would be expanded, and the cost of implementation would be reduced. A way to achieve this is to consider the heart of the system and to place an
emphasis on producing a miniaturized spectral engine that can be adapted to a
broader market and to different, even non-conventional areas of application. This
paper reviews progress in the development of miniaturized spectral engines that can
be used for embedded, portable and handheld applications. The spectral platforms
and spectral regions covered include the visible-shortwave NIR (360nm to 1100nm),
the medium/longwave NIR (900nm to 2500nm) and the mid-IR (2.0microns to 15microns). The enabling technologies involve the use of advanced thin-film coatings,
microelectro-mechanical-system (MEMS) components and micromachining, and
less conventional approaches to the system design. The use of MEMS extends
to both emitting (sources) and detection (single element and arrays) systems. The
presentation discusses the technologies used and their implementation in practical
measurement systems.
150
Consumer Complaint Analyses
Doug Knawby, McNeil Consumer Health, 200 Great Valley Pkwy,
Malvern, PA 19355
Consumer complaints for odor or taste are relatively common in the food and beverage industries, but are relatively rare in the pharmaceutical and consumer health
care industries. However, with regulatory scrutiny of complaint trends and with
multiple recalls of pharmaceutical and consumer healthcare products due to taints
since 2009, there has been an increasing need for the quick identification and quick
quantitative determination of potential taints. Towards this end, analytical and olfactory methodologies that can detect taints with concentrations ranging into the low
part-per-trillion are described. Examples illustrate how methodologies may be used.
155
Innovative Handheld Raman Analyzer with Dual Excitation
Wavelengths: Advantages for Real World Applications
Claire Dentinger, Rigaku Raman Technologies, 1101 McKay Dr, Suite B,
San Jose, CA 95131, Jim Pasmore, Elizabeth Yarbrough, William Yang,
Steve Pullins
Raman spectroscopy is a powerful analytical technique for material identification.
Recent advances in lasers, detectors and optics have allowed the development
of handheld portable Raman spectrometers. Rigaku Raman Technologies recently
introduced a portable Raman spectrometer with two excitation wavelengths. A portable instrument with 785 and 1064 nm excitation is a powerful tool for analyzing a
wide variety of materials with different characteristics and being handheld provides
ultimate flexibility to “take the lab to the sample.” This dual wavelength spectrometer
is a nearly ideal tool for real world sample analysis because many samples fluoresce when 785 nm (or shorter wavelength) excitation is used. This fluorescence
can obscure the Raman signal. However, when 1064 nm excitation is used many of
these fluorescence problems can be avoided, very significantly improving sample
identification capability. Some examples of materials that can be successfully analyzed with 1064 nm but not with 785 nm excitation include: many highly colored materials, pharmaceuticals such as some diet medications and their counterfeits and
methylphenidate, Haz Mat surrogates such as cinnamon, flour and street drugs. For
samples which do not show fluorescence interference, the 785 nm excitation has
a higher Raman scattering cross section which can lead to more sensitive material
analysis which is especially important if one is interested in lower concentrations.
The advent of a two wavelength small portable Raman spectrometer expands the
identification capability to a wider variety of materials or a higher sensitivity, for the
optimum sample analysis capabilities in the field.
151
UPLC Beyond the Marketing: Using Increased Peak Capacity as a
Tool in Complex Multi-Active Separations
Travis Jones, McNeil Consumer Healthcare, 7050 Camp Hill Rd., Fort
Washington, PA 19034
We have all seen the brochures from ultra high-pressure liquid chromatography
(UHPLC) vendors showing amazing separations of related compounds in under a
minute, but what happens when the sample to be analyzed contains four to six nonrelated actives at varying label claims? What happens when those same samples
include degradation products related multiple actives along with a placebo containing colors, flavors, gums, now a matrix exists that will not be separated in less than
a minute. The traditional approach to these separations has been multiple methods
with complex buffers and long analysis times that are a strain on a quality control
(QC) laboratory’s resources as well as the environment due to increase solvent
needed. This discussion focuses on strategies used in developing robust, analyst
friendly methods that maximize UHPLC technology in order to decrease analyst’s
preparation time and the amount of resources a QC laboratory must divert to a
multi-active product.
152
Supporting Analytical Development of OTC Drug Products
Globally: Challenges and Opportunities
Jonathan Zeszotarski, McNeil Consumer Healthcare, 7050 Camp Hill
Rd., Fort Washington, PA 19034
As many companies look to emerging markets as a source of growth, developing over-the-counter (OTC) drug products for global markets can be an attractive
strategy. This presentation examines some of the advantages of leveraging global
development and the positive analytical chemistry impacts. It also calls attention to
some of the difficulties - both obvious and surprising - in executing analytical development through an international network of laboratories for emerging markets. The
presentation concludes with the author’s outlook on best practices for leveraging
analytical development through global laboratories.
156
New Handheld Near Infrared Spectrometer for Pharmaceutical
Applications
Nada A. O’Brien, JDS Uniphase, 2789 Northpoint Pkwy, Santa Rosa,
CA 95407, Charles A. Hulse, Donald M. Friedrich
Near-infrared spectroscopy is a powerful analytical tool for fast and non-destructive
evaluation of materials. With the advent of miniaturization of optical and electronic
components, the evolution of taking the spectrometer to the sample is starting to
become a reality. In this paper, we report on an ultra-compact near-infrared (NIR)
spectrometer built with a linear variable filter (LVF) as the dispersing element covering the wavelength range of 950-1650 nm or 1150-2150 nm. LVF is a thin-film
Fabry Perot bandpass filter whose thickness is intentionally wedged in one dimension such that the center wavelength of the bandpass is continuously varying across
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2012 EAS Abstracts
November 2012
the length of the filter. The NIR spectrometer is built the with the LVF component
attached to a multi-pixel indium gallium arsenide (InGaAs) detector array, an integrated light source, custom collimating optics, and readout electronics, resulting in a
spectrometer that weighs less than 60 grams and measures 45 mm in diameter and
42 mm in height, with power consumption of less than 2.5 Watts. The spectrometer
can be used in transmission or diffuse reflection for the measurements of liquid or
solid compounds. We demonstrate the capability of the spectrometer in a number
of quantitative and qualitative applications of interest to the pharmaceutical industry. Some of the applications include raw material identification and pharmaceutical
counterfeit detection. The quantitative study reports on determining the concentration of multiple active pharmaceutical ingredients in specific pharmaceutical formulations. Additionally, the thermal and mechanical stability results of the spectrometer
are shared.
activities. This paper gives examples of where a wide range of spectroscopic techniques have supported quality-by-design (QbD) and real-time release (RTR).
161
Correlating Blend End-Points and Tablet Content Uniformity via
Near-Infrared Spectroscopy
Benoit Igne, Duquesne University, 410C Mellon Hall, Pittsburgh, PA
15282, Sameer Talwar, James K. Drennen III, Carl A. Anderson
Critical quality attributes (CQA), as defined by the US Food and Drug Administration, should be set with respect to patients’ safety and drug efficacy. To achieve
these clinically relevant CQAs, designs of experiments can be conducted to determine their relationship to critical process parameters, and thus ensure drug quality.
While it is conventional to include in experimental plans, formulation and process
parameters, it is less common to evaluate the appropriateness of algorithms used
to achieve a certain material attribute. In this study, the relevance of various blend
end-point criteria was tested for a five-component direct compression formulation.
Blends mixed for varying amounts of time were compressed and tablets active
content was evaluated. A mixing time after which resulting tablets were considered
within specifications was determined. Using independent test batches, the ability of
quantitative end-point criteria to determine homogeneity was tested. Techniques involving prediction residuals, model error and prediction distribution were compared.
Significant differences were found based on the nature of the information taken
into account by the end-point algorithm. Specifically, criteria that included other parameters (e.g., major excipients) were found to require longer mixing times and
agreed with more traditional qualitative approaches to blend monitoring. The study
concluded that the nature of the formulation and the mechanical properties of the
delivery form were to guide the choice of blend end-point criterion.
157
Recent Advances in Handheld Raman, FTIR and NIR and their
Applications
Michael Hargreaves, Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury,
MA 01876, Robert Green, Lin Zhang, Wayne Jalenak, Craig Gardner
Every single day we interact with technology in multiple forms. Consistently new
technologies emerge, or are further enhanced, improving the end-user experience,
with easier, smarter and faster tools, than the previous generation. This evolving
generation of analytical devices is striving to offer ever great capabilities, ease of
use and portability in order to afford an overall improvement in the analysis experience, with handheld vibrational spectroscopic tools following the same trend. In this
paper we present handheld Raman, Fourier transform infrared and near-infrared
analyzers, discussing several applications, spanning the safety and security, pharmaceutical/chemical and narcotics application arenas. Specific examples from each
application area with be presented, highlighting, in several cases the complementary nature of these vibrational spectroscopic technologies.
162
Visualizing Drug Release and Component Distribution from
Medical Devices by Confocal Raman Microscopy
Karin Balss, Johnson & Johnson, Welsh and McKean Rds., Spring
House, PA 19447, Mary Ellen Serianni, Maureen F. Chisholm, Sarah A.
Nielsen, Cynthia A. Maryanoff
Confocal Raman microscopy was used to visualize and monitor the drug release
from a medical device during in-vitro studies at simulated physiological conditions
and from an in-vivo porcine model. Specifically, calibration curves are generated
from formulation standards built from varying ratios of drug to the polymer matrix. The spectral images from the formulation standards were then used to build
quantitative spatial models by partial least-squares (PLS) regression analysis after
spectral image preprocessing was performed. The quantitative models were tested
on independent sample sets to gage the accuracy and precision of the predictions
(against an analytical assay method). The calibration models were then applied
to predict the quantitative spatial distribution of drug as well as other components
on each device as a function of time. Reproducible drug release profiles calculated by PLS predictions was observed for both in-vitro and in-vivo model samples.
During a 180 day in-vivo porcine study, we observed the drug distribution change
luminally to abluminally on the device, and visualized the majority of drug release
to occur by day 60. Excellent agreement was found between the concentration of
drug predicted by PLS and a bulk analytical assay for one in-vivo study. Spectral
interference from porcine explants material was observed at later time points after
the majority of the drug had been released. The implications of quantitative spatial
information on understanding bulk performance properties such as in-vivo drug elution are discussed.
158
Learning from the Application and Approval of Real Time Testing
Douglas Both, Bristol-Myers Squibb, PO Box 191, New Brunswick, NJ
08903, Ambarish Singh, Omar Sprockel, Jatin Patel, Gary McGeorge,
Tim Stevens, Pankaj Shah
While application of quality-by-design (QbD) principles is not new to non-pharmaceutical industries, recent years have seen an explosion of discussion on QbD,
quality risk management, process analytical technology (PAT) application and control strategies based on PAT application. Many pharmaceutical companies have
begun to engage health authorities in discussion on application of QbD principles
and PAT in drug development. The Bristol-Myers Squibb Company has been very
active in this area and this presentation provides an overview of quality-by-design
principles in conjunction with the application of PAT to develop a cogent overall
control strategy which justified reduced end-product testing (real-time release testing) and to share what has been the feedback from various health authorities on
such strategy.
159
Spectroscopy Enabling Quality-by-Design (QbD) and RTR in a Low
Dose Product
Steve Hammond, Pfizer, 100 Route 206 North, Peapack, NJ 07840,
Mark R. Smith
The use of spectroscopic techniques such as near-infrared spectroscopy to deliver
greater process understanding, either providing difficult to obtain compositional data
or just lots more data has been a key factor in the expansion of QbD. The potential
for condition monitoring of a unit operation in real-time during the manufacturing
process adds enormously to the ability to understand, and reduce the risk of any
unit operation. This talk describes the spectroscopic techniques used to assess the
process during qualification, and the measurements applied to develop a real-time
release (RTR) filing for a low dose product. The RTR approach has been approved
in Europe and by the Food and Drug Administration, as well as in many other countries around the world.
163
Chronicling Wound Healing with Raman Spectroscopy
Nicole J. Crane, Naval Medical Research Center, 503 Robert Grant
Ave., Silver Spring, MD 20910, Rajiv Luthra, Emily Valaik, Jonathan
Forsberg, Eric Elster
Predicting wound outcome during the management of modern traumatic war
wounds remains a significant challenge for inexperienced clinicians. Though the
ensuing inflammatory response that follows injury determines the pace of wound
healing and tissue regeneration, the timing of wound closure is often subjectively
based. Vibrational spectroscopic techniques such as Raman spectroscopy offer the
potential to evaluate the molecular environment of acute wounds throughout the
debridement process while in the surgical arena. In a recent Department of Defense
clinical trial, we collected over 200 tissue biopsies from 75 combat-wounded warfighters and subjected them to non-invasive analysis using Raman spectroscopy.
During treatment for patients, the wounds are debrided in the operating room every
two to three days until the wound is closed. For each patient, we examined the tissue biopsies obtained at each surgical debridement (first, second, third, final, etc.)
during the wound healing process, chronicling each wound with Raman spectroscopy. All spectra were analyzed with curve fitting. Band area ratios were calculated
from the spectra to monitor molecular changes in the tissue. For instance, deconvolution of the Amide I band in the Raman spectrum can provide an indication of the
presence of collagen by calculation of the 1660:1445 cm-1 band area ratio. These
findings have been corroborated by a decrease in the fold change of expression of
160
The Role of Spectroscopy for Facilitating QbD and Enabling RTR
Martin Warman, Vertex, 130 Waverly St., Cambridge, MA 02139
In many ways the current quality initiatives of pharmaceutical manufacturing can be
broadly broken down in to 5 steps: 1) Identifying critical process steps, that are the
processing steps producing intermediates those attributes impact final product quality. 2) Description of product design space, defining boundaries for acceptable product. 3) Defining process model, linking intermediate attributes to final product attributes. 4) Identifying control model, describing what process parameters to change
to cause the change in the intermediate attribute. 5) Implementing and maintaining
the process and control models. This systematic approach to first define what are
the critical steps in making an acceptable product, define the process space within
which we should operate and ensure the process stays within that process space to
ensure product quality is driven by an end for process understanding and process
control - and spectroscopy has been shown to have a pivotal role in both these
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2012 EAS Abstracts
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168
collagen I and III genes. Thus, Raman spectroscopy may provide a means for objective evaluation of the wound environment during the healing process.
Art and Industry: Development of Novel Cleaning Systems for
Acrylic Emulsion Paints
Melinda Keefe, The Dow Chemical Company, 727 Norristown Rd.,
Spring House, PA 19477, Christopher Tucker, Anastasia M Behr, Cory
Peitsch, Tom Learner, Alan Phenix, Bronwyn Ormsby
This study reports on the development of novel cleaning systems designed for acrylic emulsion paints. A range of new cleaning systems were designed to meet different
challenges in art conservation. The cleaning systems and challenges include: 1) a
buffered aqueous cleaning formulation with high cleaning efficacy and low pigment
transfer; 2) a solvent formulation with intermediate cleaning efficacy; 3) a solventcontinuous micro-emulsion for water-sensitive paintings with heavy soiling; 4) a
solvent-continuous micro-emulsion for water-sensitive paintings for which a less
aggressive cleaning system is sufficient. A number of the new systems were identified via a high-throughput (HTP) automated approach used to prepare hundreds of
formulations. A separate HTP approach was used to evaluate the cleaning efficacy
of a large number of water- based, solvent-based, and micro-emulsion systems for
artificially soiled artists’ acrylic emulsion paints. The most promising candidates from
the HTP evaluations were then assessed by researchers and practitioners at Tate,
London, and by conservators participating in a series of workshops organized by
the Getty Conservation Institute under the title Cleaning of Acrylic Painted Surfaces
(CAPS). A few case studies highlighting the use of these new systems on acrylic
paintings and oil paintings are also reviewed. This report is part of an ongoing collaboration between Dow, Tate and the Getty Conservation Institute into the development of improved cleaning systems for acrylic emulsion paints.
164
Biomedical Probes for Raman Spectroscopy of Bone
Francis W. L. Esmonde-White, University of Michigan, 930 North
University, Ann Arbor ,MI 48109, Michael D. Morris
Many tissue properties can be non-invasively measured using spectroscopy. Raman spectroscopy is well-suited to measuring the inorganic mineral component and
collagen matrix component of bone. Raman spectroscopy of bone is particularly
exciting because bone mineral is a unique spectroscopic target. The unique bone
Raman signal and use of commercially-available probes allows for direct translation for in-vivo bone measurements in the clinic. We discuss several approaches
to probe design, including regulatory and sterility concerns. A limitation of optical
diagnostics is the requirement for developing custom optical probes for particular
anatomic sites and tissues. For in-vivo Raman measurement of bone this means
maximizing the Raman signal from the bone while minimizing the fluorescence and
Raman signals of overlaying tissues, subject to constraints such as limitations on laser power-density and acquisition time. We have developed methods for designing
enhanced optical probes based on simulations, experiments with tissue phantoms,
and experiments with human cadavers. Our current applications include measurements of infected bone in diabetic osteomyelitis, and testing equivalence between
transcutaneous Raman measurements and exposed bone measurements during
anterior cruciate ligament reconstruction surgery. We use commercially available
Raman probes for our ongoing clinical projects, which simplifies the regulatory pathway. Moreover, we use probes which can be sterilized for use with patients. Together, these features enable clinical studies. We have developed a flexible platform
for testing custom fiber optic probe geometries. We are also currently developing
(potentially disposable) miniaturized optical probes for biomedical spectroscopy and
microfluidics.
169
Cleaning Acrylic Painted Surfaces: Research into Practice
Tom Learner, Getty Conservation Institute, 1200 Getty Center Dr., Suite
700, Los Angeles, CA 91436, Bronwyn Ormsby
Investigating the effects of cleaning systems on any work of art is a near impossible task. Although highly sophisticated analytical techniques can be used to detect
minute changes in chemical, physical and optical properties of well-characterized
test paint-outs, knowing what happens in a cleaning treatment of an actual work is
a significant challenge. To date, the conservation profession has used two main approaches to make more informed decisions: first, to establish basic trends/concepts
on test paint systems using scientific methods; and second, to utilize the empirical
knowledge built up from the hands-on experience of conservators. Acrylic emulsion paints have remained one of the most widely-used synthetic paints by artists since their introduction in the mid-1950s, and therefore represent an important
class of materials found in modern and contemporary works of art. To date, they
have displayed excellent optical, mechanical and aging properties, but conservators
have faced challenges in developing safe and effective cleaning strategies for them,
largely because they appear very sensitive to most cleaning systems used on Old
Master paintings. This paper focuses on the problem of translating cleaning theory
and research into practice, drawing on the salient findings of recent work improving
our understanding of the sensitivities of acrylic paints to cleaning materials, and in
developing new systems designed to increase efficacy of dirt removal. A particular
attempt has been made to ensure the research being carried out remains as relevant as possible to the real case scenario in the conservation studio, via a series of
workshops, in which conservators can assess the more promising materials being
identified by the scientific studies.
165
Theory-Enhanced Raman Imaging for Tissue Histopathology
Rohit Bhargava, University of Illinois-Urbana-Champaign, Department
of Chemistry, Urbana, IL 61801, Matthew Schulmerich, Thomas van
Dijk, Paul Carney
Infrared (IR) spectroscopic imaging was widely believed to be a simple combination
of spectroscopy and optical microscopy. Our recent theoretical work has demonstrated that this is not the case. There is a non-trivial coupling of the structure and
spectral content of a sample. Understanding the data obtained in IR spectroscopic
imaging is considerably more complicated than previously believed and presents a
rich area of investigation. Here, we report on the extension of a scalar wave theory
model for IR imaging to understand complex samples in a variety of microscopy
configurations. We predict the performance of microscopy systems and resulting
images from first principles, resulting in several new insights. Microscope configurations based on these predictions were set up and performance was found to agree
well with predicted response. We then extended the work to a variety of samples,
optical configurations and sources, leading to the development of instruments with
new capability. Specifically, we show how unprecedented spatial resolution can be
obtained and describe the fundamental limits of information extraction based on
rigorous theory. Finally, we demonstrate the experimental realization of the theory
and simulation to demonstrate the recording and subsequent reconstruction of accurate spectral data.
170
In-Situ Solubility Measurements of Ionizable Drugs and
Precipitation Behavior in the Presence and Absence of Plasdone
Polymer Crystallization Inhibitors
Jon Mole, Sirius Analytical, 100 Cummings Center, Suite 231c, Beverly,
MA 01915, John Comer, Karl J. Box, Robert Taylor
Drug compounds were studied using the CheqSol solubility method which allows
for the determination of the solution concentration of the free drug during an acidbase pH titration. Compounds were dissolved at an appropriate pH, with and without
Plasdone® polymers, and titrated towards the samples pKa until the neutral species
precipitates. The precipitation behavior is then monitored to provide a picture of
the crystallization tendency of the drug. Results: The effects of different Plasdone®
crystallization inhibitors were evaluated with ketoprofen and metoclopramide. PVP
grades K-12, K-17, K-25, K-29/32 and K-90, at 1:1 (w/w) polymer:drug, all kept
ketoprofen in solution, for more than three hours, at concentrations between 286
– 293 µg/mL, well above the crystalline solubility of this compound. By contrast,
under aqueous conditions ketoprofen descended within 20 – 30 minutes towards
its crystalline solubility of 114 µg/mL. PVP grade K-25 was able to sustain ketoprofen in solution in a more soluble (amorphous) form for sustained periods even at
polymer:drug ratios of 1:5w/w (>three hours), 1:10 (100 minutes) and 1:50 (45 minutes). Metoclopramide solubility was significantly improved with Plasdone® S-630,
a PVP/VA 60/40 copolymer. At polymer:drug ratios of 1:1 and 5:1 (w/w) metoclopramide was sustained in a high solubility form (493 - 505 µg/mL) for 40 minutes
and 60 minutes respectively before solubility decreased towards the crystalline solubility of 93 µg/mL. Conclusion: A technique for studying precipitation behavior and
166
Acrylic Paint, Its Ingredients and Durability
Stuart Croll, North Dakota State University, PO Box 6050, Fargo, ND
58108
Acrylic paint used by artists these days is usually a water-borne paint that has a
great deal in common with acrylic latex house paint. The differences are usually that
it is more viscous than house paint and it has much stronger and purer colors, but
otherwise the ingredients are very similar and serve the same purpose. An exemplar
paint formulation is used to demonstrate the type and number of ingredients, as well
as discuss their function. Typically, there are many candidate materials available to
the paint manufacturer to fulfill each of the roles required of the paint. Each material affects the immediate application properties and appearance and, ultimately,
the durability of the painting and its long-term appearance. Durability is discussed
according to the impact of the environment and the properties found within raw
material classes.
167
Applied DESI-MS Techniques for Monitoring Surfactant Segregation
Phenomena
Richard Wolbers, University of Delaware, Winterthur Museum Program,
Newark, DE 19716
No abstract submitted by author.
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2012 EAS Abstracts
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174
monitoring the potential of different Plasdone® polymers as crystallization inhibitors
has been described. Useful insights into drug precipitation kinetics can be gained.
Characterization of Intravaginal Ring with Raman Imaging
Eunah Lee, HORIBA Scientific, 3880 Park Ave., Edison, NJ 08820, Philo
Morse, Jeffrey Bodycomb, Robert Lee, Andrew Whitley
The goal of the quality-by-design (QbD) is to predict the performance of the formulation by understanding the correlation between the performance and the formulation.
The importance of QbD is emphasized in an extended and targeted drug delivery
system for potent drugs. An example is the intravaginal ring that is designed to
deliver AIDS drug over weeks. The traditional way of characterizing intravaginal
ring is an iterative procedure where designing the candidate formulation is based
on the drug release tests from the previous cycle. Raman imaging visualizes the
spatial distribution (at a given time) and migration (over a finite duration of time) of
active pharmaceutical ingredient (API), and helps to characterize the behavior of the
particular formulation in a subject. This allows more effective design of the rings and
fewer design iterations for faster and less expensive development. In this paper, we
investigate intravaginal rings that contain varying amounts of API, and have been
immersed in elusion buffer varying amount of time with Raman imaging. The results
show that high API concentration and long immersion in the elusion buffer may
cause recrystallization of API, and thus influence the drug release rate.
171
Validation and Uncertainty of a Karl Fischer Method: Impact of
Residual Water on Reference Materials for Quantitative Applications
Bryan Dockery, Cerilliant Corporation, 811 Paloma Dr., Suite A, Round
Rock, TX 78665, Isil Dilek, Uma Sreenivasan
A thorough characterization of neat reference materials is essential to determine an
accurate mass balance purity factor for preparation of quantitative reference solutions. In addition to chromatographic purity, residual moisture, residual inorganics
and residual solvents should be assessed when developing reference standards.
Changes in residual water content of a reference material over time can significantly
impact the concentration of analytical solutions prepared from the neat material.
Ability to control and accurately measure the residual moisture levels in reference
materials is a challenge in testing laboratories. Karl Fischer is a widely used technique that utilizes coulometric or volumetric titration methods to determine residual
amounts of water. Coulometric Karl Fischer analysis is particularly effective to detect
the residual moisture levels when limited amounts of materials are available. In
this study, we report the development and validation of a coulometric Karl Fisher
method based on the ‘USP <921> Water Determination’ chapter. The experimental design of this study consisted of these modules/studies: accuracy, verification
of limit of quantitation (this was determined by regression analysis using standard
error), upper limit of quantitation linearity, robustness, and intermediate precision.
Precision, two neat materials were used in the validation with each representing a
particular moisture content level in order to establish the higher end sample analysis
conditions and the lower end sample analysis range. The two neat materials are as
follows: 1) Naloxone HCL dihydrate, Cerilliant Brand; Lot # PN060209-01; used in
the “high end” moisture content analysis’. 2) Atenolol, Sigma Aldrich Brand; Product
# A7655-25G, Lot # BCBB5166. This material was used in the “low end” moisture
content analysis.
175
An Approach to Dissolution Method Optimization Using Fusion AE
Lilly Lo, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08502,
Xujin Lu, George Cooney
Fusion AE is a quality-by-design based software platform that provides automated
design of experiments for product and process research and development. This
platform has been successfully applied to liquid chromatography method validation and in-vitro inhaler testing method development. A new module is currently
in development to facilitate the dissolution methods development in pharmaceutical applications. This module is designed to transparently manage complex statistics and models for method optimization, build experiments, analyze data, and
present results as visual and numeric method predictions. The use of this module
has helped in establishment of the operational space of dissolution methods. Case
studies are discussed in this presentation for the use of Fusion AE for optimization
of the dissolution parameters and detection parameters of UV fiber optic (UVFO)
dissolution methods. The first case study was to evaluate the dissolution parameters that could affect the drug release and dissolution profile of a model capsule
formulation (100mg), including the pH of dissolution medium, the paddle speed,
and the concentration of the surfactant. The second was to optimize the UVFO
instrument operating parameters that could affect the testing results (concentration
of released drug) of a model tablet formulation (0.25 mg), including selection of
detection wavelength, charge coupled device detector scan number, background
correction location and range. A Distek 6100 dissolution bath and a Leap Technologies UV fiber optic spectrometer were used for the studies. Fusion AE was used for
the data processing and statistical analysis. The pros and cons of using the software
are discussed.
172
Residual Solvents Testing of Packaging Materials per ASTM F
1884-04 with the Versa and HT3 Automated Headspace Samplers
Nathan Valentine, Teledyne Tekmar, 4736 Socialville Foster Rd., Mason,
OH 45040, Roger Bardsley, Tammy Rellar
Printing inks are used extensively to enhance the visual appearance of packing
materials of consumer products. These inks are typically comprised of colorants and
pigments, binder systems, solvents, and additives. American Society for Testing and
Materials (ASTM) International is recognized for developing and delivering international voluntary consensus standards. They provide packaging material suppliers
with a standardized test method, ASTM F 1884-04: Standard Test Methods for Determining Residual Solvents in Packaging Materials, to determine residual solvents
remaining in their packaging materials from numerous printing processes utilizing
solvent-based printing inks. Test method A, outlined in ASTM 1884, recommends
an auto-sampler to perform the headspace-gas chromatography examination of residual solvents in packaging materials. This poster demonstrates the capability of
the Teledyne Tekmar Versa and HT3 automated headspace samplers to meet the
rigorous demands of ASTM F 1884-04. These scientific instruments can provide
packaging material suppliers with quality analysis following ASTM F 1884-04 with
minimal sample preparation.
176
Withdrawn by the author
177
Acidic and Basic Fractionation to Improve Analysis of Trace
Volatile Compounds in Cured Vanilla Bean Extracts
Suying Zhang, Firmenich, 250 Plainsboro Rd., Plainsboro, NJ 08536,
Jana Pika
The complexity of natural products like vanilla beans often requires some kind of
fractionation for comprehensive analysis. Vanillin is the major constituent of vanilla
beans along with many other compounds including other substituted phenols, which
contribute to the elegant and complex vanilla flavor. Due to the slight acidity of phenolic compounds, sequential acidic and basic washes are practical for fractionating
distilled vanilla bean extracts. In this poster, a detailed scheme of acidic and basic
washes is presented as well as the major categories of compounds in each fraction.
Compound identification was based on linear retention index and comparison of
mass spectra with spectra databases after peaks were deconvoluted using Automated Mass Spectral Deconvolution and Identification software developed by the
National Institute of Standards and Technology. Some compounds which were not
identified in the non-fractionated distilled vanilla extracts and belonged to a few
different categories including hydrocarbons, esters, ketones, aldehydes, and heterocyclic compounds, were found in the neutral fraction after acidic-basic washes.
173
Quantifying Anions and Cations as Pharmaceutical Salts
Bruce Bailey, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Christopher A. Crafts, Marc Plante, Mark Netsch
The need to accurately quantify both inorganic ions and organic salts stretches
across several fields including environmental, pharmaceutical, and food and beverage. Although highly sensitive and selective analytical methods such as ion chromatography-conductivity or liquid chromatography mass spectrometry are required
for trace analysis in environmental water samples, the needs of the pharmaceutical
industry can be different. Presented here is work that focuses more on the pharmaceutical industry both during early salt selection along with methods to monitor salt
release from a capsule. The use of novel column technology along with charged
aerosol detection enables the simultaneous analysis of the active pharmaceutical
ingredients and counter ions along with low level inorganic impurities. The analytical figures of merit of this approach for several common drug substances are discussed. A general screening approach to test for salt stoichiometry of many potential
salt candidates is presented. The limits of detection for the technique were typically
in the low nanogram on column level. This permits detection of the parent drug, the
appropriate counter ion (e.g., sodium or chloride), and potential salt impurities to <
0.1 w/w %. A modified dissolution test which measures for both sodium and naproxen from an over the counter pain relief tablet during several time points is discussed.
The simplicity of this approach provides significant advantage to for testing the quality of bulk counter-ion starting materials and early stage product formulations.
178
Selective and Sensitive Analysis of Organophosphorus Pesticides
in Baby Food Using an Inexpensive GC Detector
Gary Engelhart, OI Analytical, PO Box 9010, College Station, TX 77842,
Phil Griffiths, Scott Hazard
Organophosphorus (OP) pesticides are a group of cholinesterase-inhibiting insecticides, which have been widely used in agriculture due to their broad spectrum
activity and effectiveness. Widespread usage has led to frequent exposure in human populations, and studies have demonstrated that consumption of food items
containing pesticide residues is the primary pathway of exposure in children. The
26
2012 EAS Abstracts
November 2012
National Research Council has reported vulnerability of infants and children to
pesticide toxicity are attributable to their immature physical development and high
consumption of fruits and vegetables. This dietary exposure has driven development and refinement of analytical methods for OP pesticide residues. Many analytical methods for OP pesticide residues are based on the QuEChERS (quick, easy,
cheap, effective, rugged, and safe) approach followed by gas chromatography mass
spectrometry (GC-MS) or GC-MS/MS for detection and quantitation. The presence
of pigments, fats, proteins and other matrix interferences can obscure MS peak
identification even with extensive sample clean-up steps. The high cost of a triple
quadrupole mass spectrometer is inconsistent with the criterion of a “cheap” analytical method. A Pulsed Flame Photometric Detector (PFPD) is a highly selective GC
detector that costs a fraction of a triple quadrupole mass spectrometer. Configured
for phosphorus detection the PFPD responds exclusively to the phosphorus in the
pesticide, completely eliminating matrix interferences that plague and complicate
MS analyses. This poster describes use of a PFPD for sensitive, selective detection
of OP pesticide residues in baby food. Comparative PFPD and MS chromatograms
for a variety of baby food extracts are presented.
product. The hops themselves contain little bitterness in their raw form. However,
once they are heated, a chemical conversion takes place. Insoluble alpha (α) acids
in the hops are thermally isomerized to soluble, flavorful iso-α acids. Our efforts
are aimed at measuring the iso-α acid content in hopped wort during the industrial
whirlpool process to assess whirlpool efficiency over time. Published high-pressure
liquid chromatography methods for the analysis of wort have been adapted for use
in our laboratory. Efforts continue to identify and quantify the individual iso-α acids
present in the wort samples during the whirlpool process. The results of these studies are presented.
182
Maximizing Hop Flavor Profiles Through Supercritical Fluid
Extraction
Rudy Baskette, Supercritical Fluid Technologies Inc., 1 Innovation Way,
Suite 303, Newark, DE 19711, Kenneth J. James
Supercritical fluid extraction (SFE) has proven to be successful in oil extraction from
natural products. The technique is useful to produce oils of standardized concentration of active ingredients and products with much higher concentration (higher yields
and purity) and quality (with less creation of artifacts) than traditional methods. Hops
can add remarkable depth to the flavor profile of a beer by amplifying fruity, spicy,
woodsy, or citric flavors. To extract maximal flavor profiles, a two-step supercritical
fluid extraction (SFT-110) was performed on four uniquely flavored types of hops.
Gas chromatography mass spectrometry analysis identified the concentration of
chemicals that comprises a distinctive hop aroma. We exhibit evidence that SFE
extraction produces purer fractions and higher yields of pure hop oil than traditional
extraction methods for each unique flavor profile.
179
The QuEChERS Sample Preparation Approach with Dispersive and
Cartridge SPE Cleanup, GCxGC-TOFMS, and LC-MS/MS for the
Analysis of Pesticides in Tobacco
Julie Kowalski, Restek, Bellefonte, PA 16823, Michelle Misselwitz, Chris
Rattray, Jason Thomas, Jack Cochran
The production of tobacco, a high-value crop for the United States, is increased
by the use of pesticides that are specifically approved for use on tobacco by the
Environmental Protection Agency (EPA). Even after the processing of tobacco,
some pesticide residues remain on the product and under its pesticide registration
program EPA is charged with assessing risks to smokers from exposure to these
residues. Because tobacco is such a complex matrix, the challenges associated
with determining pesticides at trace levels are substantial. This includes pesticide
extraction from tobacco, and the instrumental analysis step, which can be complicated by matrix interferences. We used the quick, easy, cheap, effective, rugged,
safe (QuEChERS) sample preparation approach with to isolate pesticide residues
from tobacco leaf, ranging from such small, polar pesticides such as methamidophos, to relatively involatile, non-polar, large pesticides like deltamethrin. We also
explored two extract cleanup methods, dispersive solid phase extraction (dSPE)
and cartridge SPE (cSPE), monitoring their efficiency at pesticide recovery and matrix reduction. Comprehensive two-dimensional gas chromatography with time-offlight mass spectrometry (GCxGC-TOFMS) and liquid chromatography with tandem
mass spectrometry (LC-MS/MS) were compared and contrasted for their ability to
determine pesticide residues in the resulting extracts.
183
Water Analysis System with Titration and Ion Chromatography
Tore Fossum, Mettler Toledo, 1900 Polaris Pkwy, Columbus, OH 43240
For potable water analysis pH, conductivity and alkalinity are important, and can
be obtained with an automatic titration system. Other ions may be analyzed with
ion chromatography (IC). In the present system, a Mettler Toledo T90 titrator with
Rondo 30 sample changer was coupled to a Dionex ICS RFIC Anion/Cation IC. The
utility of the system is extended to include the anions fluoride, chloride, bromide,
nitrate, sulfate, nitrite and phosphate. It also determines by IC the cations sodium,
ammonium, potassium, magnesium and calcium. The reagent free ion chromatography (RFIC) automatically produces the reagents needed by the IC. The titrator
and its sample changer brings the sample to the titration station for analysis of pH,
conductivity and alkalinity or acid value, and it delivers sample to the IC in both parts
cationic and anionic for analysis. Results are automatically computed and printed
for each sample.
184
Analysis of Caprolactam and Residual Vinyl Caprolactam Monomer
in Soluplus® with a Mixed Mode Separation, Using a Fast Gel
Permeation Chromatography Column
Ashish G. Soman, Boehringer Ingelheim, 900 Old Ridgebury Rd.,
Ridgefield, CT 06877, Madhuri Jefry
The technique of high-throughput or fast gel chromatography was used to separate and quantify related residual vinyl caprolactam monomer present in Soluplus
(copolymer of polyethylene glycol, vinyl caprolactam and vinyl acetate). The chromatography exploits both size-exclusion and adsorption modes of separation. This
methodology offers a single step analysis of residual vinyl caprolacatam monomer
as compared to multi-step and time-consuming reverse-phase chromatography
measurements, yielding similar results. Fast gel permeation chromatography and
reverse-phase analyses are quantitatively compared and reported. The results of
this study demonstrate that fast gel permeation chromatography presents a viable
option to traditional reverse-phase chromatography in a quantitative analysis of residual vinyl caprolactam monomer.
180
Determination of Fructose Units in Inulin by Flow Injection Analysis
Lucia Hernandez-Garciadiego, Facultad De Quimica Unam, Ave.
Universidad 3000, 04510 Mexico, Humberto Gomez-Ruiz, Mariana Soto
Acevez
Fructans or fructooligosaccharides (FOS) are fructose polymers synthesized in
nature. They are classified as inulins when bearing predominantly b2-1 glycosidic
linkages between fructose molecules or as levans when predominantly joined by
b2-6 bonds. FOS are widely distributed in nature. They are found as reserve carbohydrates in 15% of flowering plants, representing the second-most common mechanism of energy storage (after starch). Agave plants accumulate between 13%-17%
(w/w) fresh weight fructan in mature plants, which is similar to the amount found in
chicory (15.2–20.5% (w/w) fresh weight), the most important source of industrial
fructans. Mexico has been considered the center of origin and biodiversity of the
AgaVe genus. Of the 310 species reported, about 272 can be found in this country.
Today inulin is still considered as the reference exogenous marker for glomerular
filtration rate, in addition, these compounds have multiple implications on health
and represent an important agro-industrial option for their technical, productive, and
nutritional applications. In addition, and on the same concept, inulin hydrolysis has
been identified as an alternative for fructose-rich syrup production. Fructose-rich
syrups have received increasing attention as food additives due to their beneficial
health effects. Determination of fructose units on inulin is therefore important on
the nutritional, health and agro-industrial point of view. In this work, a flow injection
system for the determination of fructose units on inulin from agave is presented.
This method is based on the hydrolysis (acid or enzymatic) of inulin. The fructose
obtained is measured spectrophotometrically by the reaction with 3,5-dinitrosalicylic
acid.
185
Extending the Usefulness of HPLC with Electrochemical Detection
Ian N. Acworth, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Michael Hvizd, Marc Plante
High-pressure liquid chromatography with electrochemical detection (HPLC-ECD)
is typically chosen for its extreme sensitivity, with low femtogram (pM) limits of detection readily achievable. Unfortunately, when compared to UV detection, ECD is
very selective with relatively few organic compounds capable of undergoing redox
reactions. A number of approaches can be used to extend the range of compounds
detected by ECD while maintaining sensitivity. In this poster we evaluate four such
approaches: 1) Pre-column derivatization: application to amino acid analysis. 2) Use
of immobilized enzymes to indirectly measure electrochemically inert species: the
measurement of acetylcholine in microdialysis perfusates is presented. 3) Use of
on-line pre-electrode high-energy photolysis to generate transiently electrochemically active species from inert compounds: a global method for the measurement
of explosive residues is shown. 4) Use of novel working electrodes that can render
inert compounds electrochemically active through electrotagging: use of a borondoped diamond working electrode to measure genotoxins, thiols/disulfides, and
polyaromatic hydrocarbons.
181
Monitoring Iso-α Acids of Hopped Wort to Evaluate Whirlpool
Efficiency
Kelsey A. Packard, University of New Hampshire, Department of
Chemistry, Durham, NH 03824, Elizabeth A. Brady, Sterling A. Tomellini,
Austin Gregoire
Hops are used in the brewing process of beer to add bitterness, aroma, and beer
stability. Hops provide the characteristic bitter, tangy flavor and aroma to the finished
27
2012 EAS Abstracts
November 2012
186
Optimization of Microemulsion Electrokinetic Chromatography for
the Separation of Charged Pharmaceutical Enantiomers
Erin J. Ennis, Drexel University, 3141 Chestnut St., Philadelphia, PA
19104, Joe P. Foley
Electrokinetic chromatography (EKC) is a technique used primarily to separate neutral compounds via a pseudostationary-phase (PSP) into which the compounds can
differentially partition. Charged compounds, including charged enantiomers, can
also be separated by EKC via such differential partitioning, although these separations have received less attention. The separation of charged enantiomers via
microemulsion EKC is examined at various conditions in order to optimize microemulsion formulation and experimental methods. Experimental emphasis is on optimization of resolution, migration time, and enantioselectivity by factorial design for
pharmaceutical compounds of varying hydrophobicity. Chromatographic figures of
merit are compared for all parameters and applications are explored for future work.
demonstrating the advantages of capillary IC with 4-µm columns are presented for
the analysis of anions and organic acids in fruit juices, for fast screening of ions in
different drinking water samples and amines in biological and food samples.
190
Fundamentals of Capillary Ion Chromatography
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94087, Frank Hoefler
Ion chromatography (IC) has become one of the preferred technologies to analyze
ionic species in any type of matrices from ultra-high purity water to drinking and
waste water and from fruit juices to body fluids. Recent developments in ion chromatography include capillary IC which was brought to the market in 2010 and has
generated great interest. In capillary IC packed columns with internal diameters of
0.4 mm are typically used with flow rates of 10 µL/min, thus enabling continuous
operation with less than 15 mL of mobile phase per day. Further advantages include
that less sample volume is required due to increased mass sensitivity, better fit for
interfacing with mass spectrometry due to lower flow rates and increased sensitivity in two-dimensional separations using a 4 mm inner diameter column in the first
dimension and a capillary column in the second dimension. Recent developments in
capillary IC allow continuous operation at pressure up to 5000 psi thus favoring the
use of new 4-µm resins instead of the conventional 7-µm materials.
187
Unique Stationary Phases for Sub-2 Micron Chromatography
Columns for SFC Applications
Matthew Przybyciel, ES Industries, 701 South Rt 73, West Berlin, NJ
08055
Reversed-phase high-pressure liquid chromatography (HPLC) is widely used for
separation and analytical analysis of many pharmaceutical compounds. Unfortunately, there are mixtures that are not well separated by HPLC leading to incomplete
analytical analysis. An alternative separation technique maybe required such as
supercritical fluid chromatography (SFC) to effect a complete separation of many
mixtures. In addition, SFC can be utilized as an orthogonal separation technique to
HPLC for many separations. SFC provides many unique features including producing high-pressure/high-speed separations. These features suit SFC well to utilizing
columns packed with 1.8-µm particles. It is the purpose of the work to develop sub2 micron column that have been engineered to specifically for SFC. We provide
examples and applications on the chromatographer can benefit from these types of
stationary-phases using the 1.8-µm particle size format. We demonstrate how these
SFC columns can provide for the high resolution separations over a wide variety
flow rate conditions and mobile compositions.
191
Effect of Cationic Surfactant Concentration on the Analysis of
Analytes Using Capillary Electrophoresis
Jinmo Huang, The College of New Jersey, PO Box 7718, Ewing, NJ
08628, Jessica Ramsden
Cationic surfactants are usually added to running buffers to reverse the electroosmotic mobility when analyzing anions using capillary electrophoresis. The addition of surfactant affects the quantitative determination of analytes. In this research,
various concentrations of cetyltrimethylammonium bromide (CTAB) are added to
phosphate running buffers in the analysis of benzoate, terephthalate, and dimethyl
sulfide. The effect of CTAB concentration on the analysis of the three analytes is
studied. The results including migrating times, peak shape, detection limit, and sensitivity for the three analytes are compared and discussed.
192
188
Applications of Type-C Silica Based HPLC Columns in
Pharmaceutical Analyses
Joshua E. Young, MicroSolv Technology, PO Box 4, South New Berlin,
NY 13843, Maria T. Matyska, Joseph J. Pesek
High-pressure liquid chromatography (HPLC) columns based on Type-C Silica™
were used for separations of a variety of pharmaceuticals. The unique nature of
the Silica-C surface leads to many advantages in pharmaceutical analyses where
run time, specificity, and method robustness are of high importance. This study investigates separations of several pharmaceutical formulations and describes how
the characteristics of the columns may be advantageously used to address these
issues. Because the columns have most of the hydrophilic surface silanol moieties
replaced with silicon hydride groups, they do not have a tendency to attract a strongly associated water shell as in conventional silica-based materials. This leads to
faster equilibration and better robustness. Furthermore, the unique properties of
the material allow for retention of hydrophilic compounds by aqueous normal phase
chromatography.
Chemical Reactions Monitored Using High-Pressure Ion Mobility
Spectrometry Technology
Clinton A. Krueger, Excellims Corporation, 20 Main St., Acton, MA
01720, Carol A. Moraff, Ching Wu
Ion mobility spectrometry (IMS) is a gas phase technique that separates molecules
based on their size and shape whereby the molecule’s drift time can be correlated
to the molecule’s mass weight and structure. An advanced data process mechanism
based on molecular information and measured ion mobility enables rapid target molecule identification based on ion mobility measurement. For these chemical reaction monitoring studies, an electrospray ionization source was used with Excellims
high-pressure IMS (HPIMS) to provide an accurate reading on reaction progress
in a millisecond to second time frame. Consumption of starting materials and the
formation of the desired product can be identified using sub-nanogram quantities
in the reaction mixture. Rapid analysis with no or minimal method development
improves the drug discovery time cycle (drug discovery, design, and development).
The technique can be used to analyze chromatographically sensitive molecules
and molecules without a UV-chromophore. Various reaction solvents such as: DMF,
DMSO, THF, Toluene, ACN, MeOH, were investigated for method viability. The merits of analysis were investigated: qualitative, semi-quantitative, and quantitative for
various synthetic organic reactions.
193
How to Develop a Method for Hydrophilic Vitamins Using Silica
Hydride HPLC Columns
Joshua E. Young, MicroSolv Technology, PO Box 4, South New Berlin,
NY 13843, Maria T. Matyska, Joseph J. Pesek
A method was developed for the separation of several common hydrophilic vitamins with the goals of finding liquid chromatography mass spectrometry compatible
method conditions, keeping the retention in the range 1 < k < 10, and obtaining
baseline separation of the critical peak pair. An iterative method development process is described in which the final conditions are obtained utilizing six steps comprising both reversed-phase and aqueous normal phase retention modes. Selection
of the appropriate pH and buffer capacity are essential factors to evaluate in arriving
at the optimal method. Finally, a suitable gradient must be developed in order to
obtain adequate resolution in the minimum analysis time. For completeness, the
robustness and intermediate precision of the optimized procedure should be evaluated if a method is to be used routinely and/or validated.
189
New Developments in Capillary Ion Chromatography Using 4-µm
Columns
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94087, Frank Hoefler, Teri Christison, Fei Pang, Cathy Tanner
Recent developments in ion chromatography (IC) include capillary IC which was
brought to the market in 2010 and has generated great interest. In capillary IC
packed columns with internal diameters of 0.4 mm are typically used with flow rates
of 10 µL/min, thus enabling continuous operation with less than 15 mL of mobile
phases per day. Further advantages include that less sample volume is required
due to increased mass sensitivity, better fit for interfacing with mass spectrometry
due to lower flow rates and increased sensitivity in two-dimensional separations
using a 4 mm inner diameter column in the first dimension and a capillary column in
the second dimension. On capillary scale it is technically also easier to use columns
with smaller particles which are operated at higher backpressures and higher flow
rates. Reasons to use smaller particle size stationary phases are two-fold; first to
increase chromatographic efficiency and resolution by maintaining column length
or to accelerate the separation with smaller particles and reduce the column length
to maintain the chromatographic resolution. Recent developments in capillary IC
allow continuous operation at pressure up to 5000 psi thus favoring the use of new
4-µm resins instead of the conventional 7-µm materials. Real-world applications
194
Isolation and Identification of Phase I and Phase II Metabolites
from Rat, Dog and Monkey Urine
David A. Schenk, Merck 126 East Lincoln Ave., Rahway, NJ 07065, Jim
Small, Rebecca B. White, Roy Helmy
The structural identification of drug metabolites is an important component of drug
discovery and development. A common/useful method to identify drug metabolites
is high-pressure liquid chromatography (HPLC) coupled with mass spectrometry;
however, often the exact position of the biotransformation cannot be determined.
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2012 EAS Abstracts
November 2012
When a more definitive structure is required, the metabolites can be generated by
chemical synthesis, electrochemical oxidation, biologically through microsome, hepatocyte, or recombinant enzyme incubations, or by isolation from biological fluids.
Isolation of metabolites from biological fluids or biological production has the advantage of avoiding the synthesis of the chemical families of theoretical metabolites.
Nevertheless, the purification of drug metabolites from either large scale in-vitro
incubations or biological fluids is challenging because the metabolite(s) of interest
are typically minor components in a complex matrix. Presented here are examples
of isolation procedures that afforded metabolites suitable for NMR analysis and how
the use of radiolabeled parent can assist in the isolation.
Results are presented for the analysis of adjuvants including mixtures of squalene
oil-in-water emulsions, monophosphoryl lipid A, plant derived saponins, and lipids
including cholesterol and dipalmitoyl phosphatidyl choline. Typical figures of merit
include low-nanogram on-column sensitivity, four orders of magnitude of dynamic
range, and peak area precision typically less than two percent relative standard
deviation.
198
Method Development for the Determination of Stereoisomers of
L-Hydroxylproline Using Corona Charged Aerosol Detection (CAD)
Yan Ma, Bristol Myers Squibb, One Squibb Dr., New Brunswick, NJ
08903, Yan Zha, Jason Huang
Trans-4-hydroxyl-L-proline is a common non-proteinogenic amino acid. It has been
widely used as a chiral synthon for the chemical synthesis of pharmaceuticals. It is
important to critically monitor and control the impurities of trans-4-hydroxyl-L-proline
to ensure the quality of the downstream intermediates and final product. The compound contains two chiral centers; therefore, three stereoisomeric impurities are
possible trans-4-Hydroxyl-D-proline, cis-4-Hydroxyl-L-proline and cis-4-Hydroxyl-Dproline. This presentation describes the challenges faced during the development of
a high-pressure liquid chromatography (HPLC) method for the determination of the
stereoisomers of trans-4-hydroxyl-L-proline with sensitivity suitable for International
Conference on Harmonization Q3A levels. Trans-4-hydroxyl-L-proline and related
impurities are very polar analytes, which do not have retention on reversed-phase
HPLC columns. A Chirobiotic T Column (4.6 × 250 mm, 5-µm) with polar ionic mode
was utilized to achieve the desired retention for trans-4-hydroxyl-L-proline and its
stereoisomers. Mobile-phase composition, column temperature, as well as organic
modifiers were optimized to achieve baseline separation between trans-4-hydroxylL-proline and its stereoisomers. In addition, trans-4-hydroxyl-L-proline and its related impurities have no UV chromophores. Therefore, UV detection is not sensitive
enough to monitor the impurities at low levels (0.1% w/w). Both charged aerosol
detection (CAD) and mass spectroscopy have been evaluated and established as
feasible detection techniques for this quantitative method. CAD was pursued further due to its high sensitivity (0.1% w/w for all stereoisomers), good reproducibility,
and ease of operation. This is the first HPLC-CAD method to our knowledge to
quantitate trans-4-hydroxyl-L-proline and its related impurities without using any derivatization technique. The method was validated for specificity, linearity, sensitivity,
accuracy, and robustness.
195
Isolation of Pharmaceutical Degradents Using SFC
Jeff Kiplinger, Averica Discovery Services, 1 Innovation Dr., BIO3,
Worcester, MA 01605, Paul Lefebvre, Mickey Rego, John Tipping
Chromatographic isolation of degradents from stressed lots of pharmaceutical compounds can be a painstaking process for the analytical laboratory. Stress degradation conditions must be assessed, peaks of interest selected based on chromatographic signatures, and finally the degradents – which may be trace components
in the mixture – isolated in sufficient quantity for structure elucidation by tandem
mass spectrometry (MS/MS) and nuclear magnetic resonance. Generally a suite
of analytical instrumentation and multiple chromatographic methods are required,
and the final isolation can be a slow process of accumulating one fraction over
many chromatographic injections. We demonstrate that using supercritical fluid
chromatography (SFC) in place of traditional reversed-phase and normal phase
high-pressure liquid chromatography (HPLC) timelines can be greatly reduced by
leveraging the rapid method development cycle of SFC and the high efficiency of
preparative separations. In addition, the lability of degradents during the isolation
process may be minimized in common SFC solvent systems and by the collection
of highly concentrated fractions.
196
Using High-Pressure Ion Mobility Spectrometry for Pharmaceutical
Content Uniformity Measurements
Clinton A. Krueger, Excellims, 20 Main St., Acton, MA 01720, Carol L.
Moraff, Ching Wu
Several pharmaceutical products were analyzed using an electrospray ionization
high-pressure ion mobility spectrometer (ESI-HPIMS) for content uniformity. This
study selected capsules or tablets at random and assayed the individual content of
the active ingredient in each capsule or tablet. Various pharmaceutical drugs were
tested with varying molecular weight, structure, and functional groups to demonstrate the precision and sensitivity of the ESI-HPIMS. The samples were prepared
using existing common extraction procedures that are currently used for high-pressure liquid chromatography (HPLC) based content uniformity studies. This HPIMS
based method can separate and quantify active pharmaceutical ingredients in the
drug products in seconds compared to the HPLC based assays that require a 12
minute analysis time per sample. ESI-HPIMS is more efficient in terms of both time
and resources and it does not require any organic solvents during the separation
process. The ESI-HPIMS can be used at production line due to its speed of analysis
and field portability. More importantly, the recent development of HPIMS enables the
system to deliver 2-4 orders of magnitude linear response range and 1-5% relative
standard deviation measurement precision. The quantitation aspect of the analytical
performance of HPIMS is comparable to common UV detectors.
199
Improving Pharmaceutical Laboratory Throughput in the Analysis
of Trace Impurities and Residual Solvents with Liquid/Headspace
Unattended Switching and Automated Standard Preparation
Massimo Santoro, Thermo Fisher Scientific, Strada Rivoltana, Rodano,
20090 Italy, Dwain Cardona, Silvia Gemme, Eric Phillips, Matthew
Lambing
United States Pharmacopeia (USP) method <467> details the procedures for the
identification, control and quantification of Class 1 and Class 2 residual solvents
through the use of headspace gas chromatography (GC). Some pharmaceutical
laboratories have also to analyze solvent impurities as part of the incoming raw
material testing process according to the various solvents’ monographs. For this
purpose, liquid GC injection is normally chosen. Because of the limitations of the GC
instrumentation currently available, most of these laboratories dedicate one instrument to headspace and another one to liquid injection analyses even if their configuration is exactly the same. This impacts labs productivity and spending. This poster
demonstrates the use of an innovative robotic platform able to switch automatically
from liquid to headspace mode and also to use different syringe volumes in the same
run or sequence onto a single GC, thus to enhance overall lab’s productivity. Test
results of residual solvents quantification, where sample and standard preparation
steps according to the USP<467> procedures are carried out by the auto-sampler
prior the headspace analysis are shown as well as the lack of carryover achieved
by the use of an headspace gas-tight syringe, heated and flushed between injections. Modern laboratories benefit from this automation in terms of time required
for sample preparation, increased results accuracy and reduced chance for errors.
197
Direct Analysis of Multicomponent Adjuvants by HPLC with
Charged Aerosol Detection
David Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Marc Plante, Daniel Kutscher, Ian N. Acworth
A vaccine adjuvant is any substance that helps promote the effectiveness of a vaccine by reducing the amount or frequency of the required dose, by prolonging the
duration of immunological memory, or by modulating the involvement of humoral
or cellular responses. This functional definition of adjuvants encompasses a very
diverse group of substances whose chemical structures and mechanisms of action vary widely. Adjuvants for human or animal vaccines are typically subjected to
rigorous standards of analysis including quantification of strength, purity, stability
and degradation behavior, even though they are not currently regulated in the same
manner as active pharmaceutical ingredients in the United States. Complicating
such analysis, many adjuvants under investigation contain components that are not
readily analyzed by traditional high-pressure liquid chromatography (HPLC) with
UV detection. These include various mixtures of lipids, fatty acids, and glycosides
that lack suitable UV chromophores. In this work, the lack of a detectable chromophore in several adjuvant compounds and degradation products was overcome
by using HPLC with charged aerosol detection, a detector that can measure any
non-volatile compound. As response is similar for all compounds and independent
of chemical structure, charged aerosol detection is able to measure intact adjuvant
species along with degradation products and potential impurities, yielding accurate
estimates of relative concentration even in the absence of pure primary standards.
200
Evaluation of an Acetonitrile Recycler as a Green Chemistry
Initiative
Kristine L. Cappuccio, Merck, 126 East Lincoln Ave., Rahway, NJ
07065, Brian C. Jung, Robert Hartman
As part of Merck’s commitment to Green Chemistry we are exploring new technologies that allow us to lower the environmental impact of high-pressure liquid
chromatography (HPLC) in analytical laboratories. Currently, ultra-pure acetonitrile
is the preferred solvent for HPLC analysis. This extremely pure acetonitrile is run
through the HPLC once and then comingled with all the laboratory solvent waste
and sent for disposal. Since a large majority of our HPLC research does not require
this ultra-pure acetonitrile there is an opportunity to recover this solvent as a high
purity azeotrope and employ it for non-good manufacturing practice development
work. A case study is presented from our evaluation of an acetonitrile recycling unit.
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2012 EAS Abstracts
November 2012
201
comparable; as were two of the continuous spectral instruments from company “B”;
yet they were significantly different from each other. In order to rely on color data obtained from a colorimeter, it is very important to specify the vendor, the type of instrument and the parameters by which specifications and/or analyses are to be tested.
GPC-IR Analysis of Acrylic Copolymers
Dana Garcia, Arkema, 900 First Ave., King of Prussia, PA 19406,
Stephan Moyses, Tom Kearney, Ming Zhou, Sidney Bourne
Hyphenated techniques coupling gel permeation chromatography (GPC) with structurally sensitive Fourier transform infrared (FTIR) spectroscopy afford the opportunity to study chemical composition in copolymers as a function of molecular weight.
This paper details results obtained for commercial acrylic copolymers employing
the DiscovIR-LCTM from Spectra Analysis Inc. We have shown that copolymers of
methyl methacrylate (MMA), butyl acrylate (BA), butyl methacrylate (BMA) and ethyl
acrylate (EA) exhibit increased MMA content at high molecular weight and increasing EA and BA concentration as the molecular weight decreases. These results
were achieved with a very high resolution, IR spectra collected every 0.5 seconds
along the chromatography time axis. Such compositional analysis is critical to process optimization and material property tuning.
205
In-Line Moisture Analysis in a Lab Scale Fluidbed Dryer Using
Diode Array NIRS
Robert A. Mattes, FOSS NIRSystems, 7703 Montpelier Rd., Laurel, MD
20723
One of the elements of the process analytical technology initiative is to use in-line
analysis to increase process understanding and control and to verify product quality
and release it for subsequent processing without delay. Using near-infrared spectroscopy the drying process in a fluid bed dryer can be monitored for residual moisture level for better process understanding, control and end-point determination.
This experiment demonstrated the use of a diode array based near-infrared instrument to monitor the moisture in a lab scale fluid bed dryer. The fluid bed dryer was
charged with Lactose monohydrate and microcrystalline cellulose. Spectra were
collected every 50 seconds and a sample thief was used to collect loss-on-drying
samples every 5 minutes and correlated with spectra acquired at the same time. A
partial least squares calibration model was developed using two factors over the
spectral region 1100 nm – 1650 nm. A standard error (size exclusion chromatography) of 0.7358 % and an R2 of 0.9519 was achieved from the model.
202
Noise and Spin-Noise: Cable Dependence of Optimal Tuning/
Matching Conditions in NMR Spectrometers
Eli Bendet-Taicher, New York University, 24 Waverly Pl., New York, NY
10003, Norbert Muller, Alexej Jerschow
Previous studies have shown that tuning/matching conditions optimized for transmission and detection can be significantly different for a variety of commercial
nuclear magnetic resonance (NMR) probes. In addition, it was also shown that by
optimizing reception tuning (as opposed to typical transmission or reflection tuning)
one may in some cases obtain sensitivity enhancements by as much as 25-50%. In
earlier work, spin-noise and absorbed circuit noise signals have also been used to
characterize reception optima. In this work, we show how the length of the coaxial
transmission line cable between the pre-amplifier and the probe affects the positions
of the reception tuning optimum, the radiation damping strength, induced frequency
shifts, as well as, the shape of the spin-noise and absorbed circuit noise line shapes
206
Characterizing Copolymer Compositional Heterogeneity by GPCFTIR Combined System
Ming Zhou, Spectra Analysis Instruments, 257 Simarano Dr.,
Marlborough, MA 01752, William Carson, Tom Kearney
Full-spectrum mid-infrared Fourier transform infrared (FTIR) is a powerful technique
for determining structural and compositional information of polymers. Hyphenating
gel permeation chromatography (GPC) size separation with FTIR analyzer through
an automated solvent removing interface allows compositional and structural characterization throughout the separation process of a complex polymeric mixture.
GPC-FTIR was used to characterize copolymer samples of SBR, SEBS, Copovidone PVP/VA, and PLGA for compositional variations across molecular weight
distributions. Significant amount of compositional drifts were observed for SBR,
Copovidone PVP/VA samples, smaller levels of compositional drifts for SEBS while
no compositional drift for PLGA copolymer. GPC-IR was also used to investigate a
complicated copolymer system based on PMMA/BA (~6:1 ratio) main backbone but
incorporated with small amount of methacrylic acid, styrene and DAAM co-monomers to improve various copolymer end-use properties. Different patterns of compositional heterogeneity of those copolymers with the 3-5 co-monomers were found for
structure-property correlation study. GPC-IR hyphenated technology is well suited
to characterize copolymer compositional drifts, lot-to-lot / supplier-to-supplier variations, polymer modification and polymer degradation from thermal processing.
203
XPS Surface Characterization of Disposable Laboratory Gloves
and the Transfer of Glove Components to Other Surfaces
Brian R. Strohmeier, Thermo Fisher Scientific, 5225 Verona Rd.,
Madison, WI 53711, Alexander Plasencia, John D. Piasecki
Disposable elastic gloves are ubiquitous in scientific laboratories and are also widely used in many industries during handling of critical surfaces. Disposable gloves
offer users protection from various aqueous acids and bases, biological fluids, organic solvents, and other potentially harmful chemicals. A second application of
disposable gloves is to protect manufactured products and analytical samples from
contamination caused by the transfer of skin cells, oils, salts, or other residues resulting from contact with bare hands. However, disposable gloves can also be a
potential source of contamination. In addition to the primary polymer structure, many
types of common laboratory gloves also contain a variety of inorganic materials,
mold-release agents, and organic surface coatings that improve one or more glove
properties. Contamination transfer resulting from surface residues on gloves can
adversely affect materials used in industries where surface cleanliness is essential
for optimum product performance and can also interfere with the analysis of samples depending on the specificity and sensitivity of the analytical technique. X-ray
photoelectron spectroscopy (XPS) is a qualitative and quantitative surface sensitive technique that can be used to evaluate the surface composition of disposable
gloves and to determine if contamination transfer occurs from gloves in a specific
process. In this study, XPS was used to characterize the surface compositions of a
variety of common laboratory gloves, evaluate the transfer of surface components
from gloves to other material surfaces and to determine changes in the surface
composition of gloves following exposure to several common laboratory solvents.
207
De-Formulating Complex Polymer Mixtures by GPC-IR Coupled
System
Ming Zhou, Spectra Analysis Instruments, 257 Simarano Dr.,
Marlborough, MA 01752, William Carson, Tracy Phillpott, Tom Kearney
Gel permeation chromatography infrared (GPC-IR) coupled system separates copolymer samples or polymer mixtures according to their hydrodynamic sizes (molecular weight (MW) distributions) and then continuously takes Fourier transform
infrared (FTIR) snapshot pictures of polymer chemical compositions (chemical compositional distributions) at each MW slice after the solvent removal and a controlled
film deposition on Zn-Se disc. The high-quality IR spectra are database searchable
for identification (de-formulation) and can be processed with GRAMS software for
many applications including three-dimensional GPC-IR spectrum mapping (CCD vs.
MWD), copolymer compositional analysis across MWD, polymer blend ratio analysis and polymer degradation study. The system is compatible with GPC or highpressure liquid chromatography with all solvents and solvent gradients, including
highly corrosive Hexafluoroisopropanol solvent for high performance plastics and
1,2,4-trichlorobenzene solvent at high temperature (150 ºC) for polyolefin products.
Three case studies are presented to illustrate GPC-IR capabilities to de-formulate
complex polymer mixtures for competitive analysis, IP protection and analytical
trouble-shooting in polymer-related industries: 1) De-formulation of a hot melt adhesive involved with three different polymers (EVA, glycerol ester rosin and paraffin)
for adhesive industry; 2) de-formulation of a flexible conductive ink involved with
three different polymers for ink industry; 3) de-formulation of a lubricant oil formula
involved with two copolymer additives (PIBS and styrene-acrylate copolymer) for
specialty chemical industry.
204
Colorimeters - Be Very Careful, Not All Instruments Give the Same
Results
Walter A. Roy, McCormick and Company, Technical Innovation Center,
204 Wight Ave., Hunt Valley, MD 21031, Roman Grypa, Charles M. Zapf
Colorimeters are spectrometers that measure reflected light in the visible spectrum,
yet not all colorimeters report the same results, there are significant differences
among them based on the technology used. This study uses five different instruments and focuses on two technologies: tristimulus and continuous spectral formats. Six standardized tiles were measured by each instrument. The colored tiles
were white, beige, blue, green, red and yellow. The color space used was Hunter
L*a*b; the parameters were set for a source illuminant of C (6,000 degree Kelvin)
and a 2 degree observer. Of the five instruments, number 1, a tristimulus format,
was chosen to be the “target” to which all the other instruments would be compared.
Each tile was read seven times at different spots and averaged. Standard deviations, relative standard deviations, and Delta-E values were calculated for each color and each technique and instrument. The results indicate that there is a significant
difference between tristimuli and continuous spectrum meters; as well as significant
differences among several instruments from the same company using the same
technology. The two high end tristimulus instruments from company “A” were very
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2012 EAS Abstracts
November 2012
208
kJ. The apparent structure of this complex is given. Also the health implication of the
Cr-curcumin complex is discussed.
Intermolecular Dipolar Coherences as a Tool for Morphological
Studies by NMR Spectroscopy
Alexander A. Marchione, DuPont Central Research and Development,
PO Box 80328, Wilmington, DE 19880
This presentation describes the extension of the CRAZED class of nuclear magnetic
resonance (NMR) pulse sequences to apply NMR spectroscopy as a morphological
probe of analytes on the micron to millimeter scale. These experiments observe
dipolar couplings between NMR-active nuclei within a radius selected by simple
adjustment of the power and duration of the applied pulsed-field gradients. With
common commercial probes, radii between ca. 2-µm and ca. 3 mm can be chosen.
Both homo-nuclear (1H to 1H) and hetero-nuclear (1H to 19F, 13C, 31P) experiments are
discussed. In addition to applying previously-published two dimensional sequences
with this new goal, frequency-selective one-dimensional versions are explored for
the first time, offering a substantial time savings in most systems. This technique enables the application of NMR spectroscopy to morphological problems in a new way.
Examples with emulsions and systems containing fullerene-type nano-particles are
given.
213
FT-NIR Study of Protein Denaturation
Erin Morgan, Iona College, 715 North Ave., New Rochelle, NY 10801,
Sunghee Lee, Emil Ciurczak, Cynthia Kradjel
Protein denaturation has important consequences in food manufacturing, most often brought about by the action of heat in the production environment. Agitation or
pressurization is another physical cause of denaturation. Addition of acid or salt can
result in chemical denaturation. This study was designed to monitor the change in
Fourier transform near-infrared (FT-NIR) spectra as proteins were subject to physical and chemical denaturation. In particular, the frequency shift as hydrogen bonds
are destroyed in the denaturation process was a key area of focus.
214
An Inexpensive, Portable, LED-Based Instrument for Fluorescence
Anisotropy Measurements Applied to Monitoring the Curing of
Epoxy
Stephanie M. Myers, Kutztown University, PO Box 730, Kutztown, PA
19530, Thomas A. Betts
Epoxy-based adhesives and coatings are as diverse as the applications in which
they are used. The performance of these materials often depends upon cure time
and the degree to which the epoxy cures. In order to follow the epoxy curing process we have constructed a portable, T-Format, LED-based, anisotropy fluorometer
(T-LEAF) that is capable of measuring fluorescence anisotropy comparable to a
research-grade, steady-state fluorescence spectrophotometer. At a fraction of the
cost, approximately $100, the T-LEAF is powered by a 9-V battery, and uses a
custom interface designed in LabView to collect and process fluorescence intensity
measurements. Fluorescence anisotropy depends upon molecular rotations, which
slow as an epoxy cures. An instrument like this dramatically reduces the cost of
fluorescence anisotropy measurements, and takes this capability outside of the lab.
209
Response of Intracellular Sodium NMR to Hyperthermia of T-Cells
Lizheng Zhu, New York University, 24 Waverly Pl., New York, NY 10003,
Xiang Xu, William Eng, Lara Mahal, Alexej Jerschow
Magnetic resonance imaging (MRI) is a powerful and non-invasive method for the
clinical diagnosis of diseases. 23Na MRI provides an important insight into cell and
tissue viability. Especially the balance between intracellular and extracellular sodium is a critical factor in both normal and pathological cell function. The sodium
concentration in extracellular space is about 10 times higher than that in intracellular
space. It has been shown that the change of intracellular sodium concentration is
strongly correlated with the onset of certain diseases, such as cancer and muscle
dysfunctions. Unfortunately, the two sodium compartments share the same resonance frequency, complicating the selection of intracellular sodium signals. In this
project, we demonstrate a significant increase in intracellular sodium concentration
in JURKAT cells after hyperthermic treatment. We demonstrate that the extracellular
and intracellular sodium can be differentiated by their intrinsic longitudinal relaxation
properties and multiple quantum coherences.
215
Direct Analysis of Volatile Gas Mixtures by Broadband Rotational
Spectroscopy
Brent Harris, University of Virginia, Chemistry Department, McCormick
Rd., PO Box 400319, Charlottesville, VA 22904, Amanda Steber, Matt T.
Muckle, Brooks H. Pate
Advances in the design of spectrometers for rotational spectroscopy of room-temperature gases provide a new method to directly analyze complex gas mixtures
without chromatographic separation. These new chirped-pulse Fourier transform
mm-wave spectrometers use digital electronics and solid-state devices to generate
the molecular excitation pulses. This novel light source is composed of a high-speed
arbitrary waveform generator (AWG) and a series of mm-wave/THz frequency multipliers. The 2-4GHz output range of the AWG is able to generate any frequency in
the operating range of the spectrometer (260-290 GHz). The rotational spectrum
is detected by digitizing the coherent emission following sample excitation. Fast
Fourier transform produces the frequency domain spectrum for subsequent analysis. For unbiased analysis of the gas sample, the full spectrum can be acquired
in a measurement time of 150 microseconds. For analysis of a predefined list of
molecules, the AWG-based system can cycle through molecule-specific detection
ranges in 2 microseconds (in any order). The frequencies and intensities of the rotational spectrum are directly related to the three-dimensional geometry of the molecule and, therefore, provide unambiguous identification of isomers. Each rotational
transition has a line width of about 2 MHz and the spectrum of a volatile species can
contain on the order 50 strong transitions in the 260-290 GHz frequency range. As a
result, mixtures of volatile gases with 10-100 species can be quantitatively analyzed
without prior separation. Results for detection limits, quantitative accuracy, speed,
complex mixture analysis, and applications extending into other analytical uses are
discussed.
210
Using Machine Learning Techniques to Classify Plastics, Organics,
and Documents by Near-Infrared Spectroscopy
Sunghee Lee, Iona College, 715 North Ave., New Rochelle, NY 10801,
Kevin Towler, Alexander Soderberg, Smiljana Petrovic, Emil Ciurczak
This study investigates use of variety of machine learning methods to classify various plastics, food extracts and proteins, and postage stamps by near-infrared (NIR)
spectroscopy. NIR spectroscopy has the advantage of simple and quick data collection and analysis. Spectra of several kinds of each sample group were collected
and analyzed by three supervised learning techniques, with a purpose of finding
parameters that allow for simple and quick identification of plastic from its NIR spectra. Artificial neural networks demonstrated good results in several other studies. We
extend those studies to use of support vector machines and bayesian networks and
provide comparative results. Selection of the most relevant wavelengths is evaluated.
211
FT-NIR Multi-Level Evaluation of Ingredients, In-Process Blends
and Final Products of Dry Powder Blending Manufacturing
Processes of Dietary Supplements
Jaclyn Robustelli, Iona College, 715 North Ave., New Rochelle, NY
10801, Allyson Moffat, Alex Soderberg, Sunghee Lee, Cynthia Kradjel,
Emil Ciurczak
Dietary supplement manufacturers with dry blending operations face challenges
that range from managing the variability of incoming raw materials to ensuring homogeneous blends to verifying the final product quality. This research designed an
analytical scheme using Fourier transform near-infrared (FT-NIR) to minimize the
risk of accepting adulterated or sub-par ingredients, to qualify suppliers and to test
incoming raw materials for key parameters that can be used to feed forward to
improve production efficiency. Conformity index measures the standard deviation
between spectra and is used to determine when a blend is homogenous and that is
meets target specifications. The final link in the analytical chain is the verification of
quality of final products.
216
Implementation of New Technology for In-Process Determination
of Protein Concentration
Keyur Soni, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ
08901, Scott Huffman, Joe Ferraiolo
The process of assaying protein containing samples for concentration is vital to
determining the level of protein present in drug substance or drug product solutions. Proteins in solution absorb ultraviolet light at various frequencies, with different proteins and nucleic acids having varying absorption characteristics. One of the
most prevalent techniques currently used for determining protein concentration is
based on UV-Vis spectrophotometry called A280. Traditional A280 analysis utilizes
the Beer-Lambert law and a fixed optical path length, usually 1 cm. Under these
conditions the sample must be diluted to a target concentration which falls within
the linear operating range of the instrument. The process of diluting protein containing samples in preparation for a traditional A280 analysis is time consuming,
highly dependent on analyst technique, and prone to analyst error. However, the
solo variable path length extension (Solo VPE) for protein concentration analysis
increases throughput and assay robustness in support of clinical manufacturing. An
212
The Stability Constant Determination of Cr-Curcumin Complex by
Differential Pulse Voltammetry
Maurice O. Iwunze, Morgan State University, 1700 E. Coldspring Ln.,
Baltimore, MD 21251
Both differential pulse voltammetry (DPV) and the UV-Vis techniques were used in
the determination of the stability constant of chromium complexed with curcumin.
The obtained results by both techniques agree reasonably well within limits of experimental error. The formation constant thus found was 2.74 x 10-8 for 1:3 ratio of
Cr-curcumin. With this value the free energy of formation is calculated to be 48.13
31
2012 EAS Abstracts
November 2012
221
In-Situ Exploration of the Interior of a Nanopore Single Molecule
Sensor
Cameron M. Frament, University of Rhode Island, Department of
Chemistry, 143 Whipple Ave., Warwick, RI 02889, Jason R. Dwyer
Nanopores enable a variety of single-molecule biosensing applications, from drug
detection to DNA sequencing. Nanopore devices typically consist of two electrolyte
solutions separated by a single nanoscale hole in an insulating membrane that allows the passage of the electrolyte ions and analyte molecules. The presence of
an analyte molecule can be detected when it perturbs the ion current through the
pore. The particular nanopore size and shape determine the performance of these
devices. Characterization of the nanopore shape by time- and expertise-intensive
electron microscopy-based techniques can result in permanent distortion of the
pore. A non-destructive characterization method was recently proposed, using routine conductance measurements already being performed during experiments. It
leverages the effect of the pore surface chemistry and the pore shape on the measured nanopore conductance. We developed a framework in which this approach
could deliver reasonable pore geometries in spite of technical challenges that would
otherwise limit the method. We found that the method is particularly sensitive to
the accuracy of the surface chemistry, to the point that it is possible to determine
the surface chemistry parameters from the conductance of a well-characterized
nanopore. Augmenting this conductance-based characterization with additional
measurements may allow for more detailed modeling of nanopore shape and will
underpin the use of this single-molecule detector for biosensing.
A280 analysis utilizing a UV-Vis spectrophotometer equipped with the Solo VPE can
usually be conducted using a neat sample, with no additional dilutions required. This
is accomplished through variation of the optical path length by the Solo VPE attachment. The use of the Solo VPE is therefore faster, easier, and less prone to error.
Productivity also increases when in-process samples analyze in Solo VPE compare
to traditional A280 analysis. A study was conducted to evaluate the effectiveness
of a Solo VPE and A280 in performing the protein concentration analysis. The data
generated through the execution of this study demonstrates the speed, accuracy,
consistency, and reliability of the Solo VPE system.
217
Raw Materials ID without Opening the Container Using Spatially
Offset Raman Spectroscopy
Matthew Bloomfield, Cobalt Light, The Electron Building, Fermi Ave.,
Harwell Oxford, Oxfordshire, OX11 0QR United Kingdom, Darren
Andrews, Pavel Matousek
Incoming materials verification in the pharmaceutical industry is both mandatory
and resource-intensive. For a lot of incoming goods the containers are opaque,
meaning that non-invasive spectral analysis is difficult or impossible using common
techniques such as Raman or near-infrared. To enable the use of these techniques
the containers have to be opened individually and either sampled or probed invasively. A new technique called spatially offset Raman spectroscopy (SORS) is able
to identify materials through unopened sacks, tubs and bottles whether plastic, paper or glass. This talk introduces SORS and show performance on identifying active
pharmaceutical ingredients and excipients through real-world containers.
222
Examination and Dating of Silver Gelatin Fiber Based Photographic
Papers using Near-Infrared Spectroscopy
Ana Martins, Museum of Modern Art, 11 West 53rd St., New York,
NY 10019, Lee An Daffner, Chris McGlinchey, Paul Messier, Donald
Dahlberg
Gelatin silver developing-out papers were introduced in the 1880s and became the
most prevalent photographic papers until their popularity faded with the advent of
color processes followed by the digital era. Fiber based gelatin silver papers were
produced throughout the whole 20th century up-to this day, and their composition is
expected to reflect changes and developments in the paper and photographic paper
industries. In return, knowing the composition of a photographic paper may provide
meaningful information on its manufacturing process and print date. Preference is
given, in the field of Cultural Heritage, to non-invasive analytical methods, which
is particularly restrictive in the case of photographic papers. Although not yet very
popular in this field, near-infrared spectroscopy has an established status for nondestructive and quantitative evaluation of chemical and physical properties of many
materials and a number of applications have been developed for the paper industry.
We have therefore been exploring the use of this technique for the characterization
of photographic papers, not only directly from the composition stand point, but trying
to establish direct correlations between spectra and attributes such as manufacturing date, manufacturer, finish, etc. We also report on a chemometric model with and
without generalized least squares weighting data pretreatment.
Mass Spectrometric Analysis of Fish Oil Dietary Supplements
Christina Onorato, Rutgers University, 23 Dryden Terrace Phillipsburg,
NJ 08865, Gene S. Hall
Fish oil dietary supplements are becoming popular in supplying consumers with
what is thought to be an adequate amount of essential fatty acids, namely DHA and
EPA. Mass spectrometry was used to study the backbone structure and fatty acid
composition of various over-the-counter fish oil dietary supplements. The results
were used to determine the accuracy of each supplement label. Each supplement
ran as a lithiated adduct through a Finnigan LCQ-Duo mass spectrometer. The
backbone structures, mainly triacylglycerols or diacylglycerols, were determined
based on the precursor ions’ mass-to-charge ratios. Subsequently, the identity and
positioning of each fatty acid was estimated using the product ions’ mass-to-charge
ratios. It was found that although each sample contained the essential fatty acids
DHA and EPA, many of the supplements did not have these fatty acids positioned
accurately for complete absorption. Furthermore, many supplements had saturated
fatty acids positioned such that they would be absorbed, which may be a health
risk to consumers. There were a few supplements, denoted here as “designer,”
that were made up of primarily DHA and EPA. The designer supplements also had
DHA and EPA positioned correctly for these fatty acids to be properly absorbed.
These results showed that the backbone structure and fatty acids in fish oil dietary
supplements are still not completely understood. The study also showed that mass
spectroscopy can be used to further study and understand the composition of overthe-counter dietary supplements, which is essential for consumer health.
219
223
218
Withdrawn by the author.
Conductive Hybrid Nanoparticle Films for Toxic Gas Sensing
Uduak Udoeyo, Temple University, Department of Chemistry, 1901 N.
13th St., Philadelphia, PA 19122, Devika Sil, Jacqueline Hines, Eric
Borguet
Hydrazines, e.g., monomethyl hydrazine, (N2H3CH3), are used as rocket fuels.
These materials are toxic and explosive. Human exposure or atmospheric release
of these chemicals may pose serious health, safety and environmental concerns.
In order to minimize hazards and potential exposure to personnel, it is essential to
rapidly identify and contain accidental releases. Hence the need arises for fast, reversible and reliable sensors capable of detecting leaks at concentrations far below
regulatory exposure limits. Palladium (Pd) is known to expand and undergo changes in electrical conductivity in the presence of hydrogen. Pd nanoparticles, in part
because of their high surface area, have been used as the basis of hydrogen sensors. As hydrazine adsorbs onto Pd, decomposing to release hydrogen atoms, Pd
is suitable for both hydrogen and hydrazine detection. We hypothesized that a thin
film of Pd and Pd doped with TiO2 nanoparticles could be used as a sensing layer
in detecting hydrogen and hydrazine via changes in electrical conductivity. Pd and
Pd doped with TiO2 nanoparticles were prepared via a sol-gel route. Transmission
electron microscopy (TEM) and energy-dispersive X-ray spectroscopy were used to
characterize nanoparticle sizes and elemental composition. Thin films of Pd and of
Pd doped with TiO2 nanoparticles were prepared by drop casting. The morphology
of the thin film of nanoparticles was determined using atomic force microscopy. Real
time, in-situ electrical conductivity measurements of these films showed reversible
changes due to hydrogen exposure.
220
Investigating Mobility Correction Methods for Individual Organelle
Analysis by CE
Jack Doenges, University of Minnesota, 207 Pleasant St. SE,
Minneapolis, MN, 55455, Gregory G. Wolken, Vratislav Kostal, Edgar
A. Arriaga
The reproducible analysis of electrophoretic mobility distributions of individual organelles by capillary electrophoresis with laser induced fluorescence detection (CELIF) is largely affected by the coating quality and changes in separation conditions
between trials. The complex combination of these factors and the nature of biological particles result in variations in migration times of the organelles detected, making
it difficult to quantify electrophoretic mobility changes of the sample even when one
or two internal standards are used for mobility corrections. Development of a more
robust correction method is highly needed for applications where the electrophoretic
mobility of an organelle is used for assessing the changes in sample composition,
organelle surface quality or degradation. Here, we investigate mobility correction
methods that are based on the mobility distribution of individually detected synthetic
carboxylate beads using simultaneous CE-LIF dual detection of two different bead
populations: one used as standards and the other used as particles to be corrected.
Using the observed differences in mobility percentiles in the bead standards, correction methods were applied to the second population of beads and to isolated
mitochondria. Once further developed, we intend for these methods to be applied to
any sample in which particles are individually detected, providing increased precision in the mobility distributions collected from multiple trials.
32
2012 EAS Abstracts
November 2012
224
228
Synthesis and Characterization of Di-μ-oxo Dimanganese
Complexes: Development of Catalysts for Solar Water Oxidation
Jessica Ziegler, Rensselaer Polytechnic Institute, The Baruch ’60 Center
for Biochemical Solar Energy Research, 110 8th St., Cogswell 201,
Troy, NY 12180, Christopher S. Coates, Ruchira Chatterjee, Sergey
Milikisiyants, K.V. Lakshmi
The light-driven process of photosynthesis oxidizes water to molecular oxygen in
the following charge-transfer reaction: 2H2O à O2 + 4e- + 4H+. The four-electron
water oxidation reaction is catalyzed by a tetranuclear manganese-calcium-oxo
(Mn4Ca-oxo) cluster in the oxygen-evolving complex (OEC) of Photosystem II
(PSII). It is thought that the Mn4Ca-oxo cluster forms a MnV=O intermediate during
the water oxidation reaction. In order to mimic the function of the Mn4Ca-oxo cluster,
we have synthesized mixed-valence dimanganese (III/IV) di-μ-oxo complexes with
a variety of ligands. We have determined the X-ray crystal structures of the dimanganese (III/IV) di-μ-oxo complexes that confirm the presence of bound substrate
water molecules. We characterize the electronic structure and oxygen-evolving
properties of the complexes using hyperfine sub-level correlation spectroscopy and
oxygen assays. In detailing the structure and function of these complexes, we develop a clearer understanding of the catalytic activity of the Mn4Ca-oxo cluster in
the OEC of PSII.
Spectroscopy with Frequency Comb Stabilized Lasers
Trevor Sears, Brookhaven National Laboratory, Stonybrook University,
Chemistry Department, Upton, NY 11973
Originally developed for applications in metrology such as optical clocks, frequency
combs can provide stable and accurate frequency markers allowing the stabilization
of other laser sources for precision spectroscopy. In particular, fiber laser-based
combs based on telecommunications technology are now readily available and
convenient to use. The frequency stability and accuracy of laser spectrometers referenced to frequency combs can be used to make precise measurements of the
positions of very narrow spectral lines, for example in cold molecular samples, for
the investigation of fundamental physical properties. They are also very useful for
characterizing spectral line shapes, and the measurement precision can challenge
existing models and provides new avenues for the investigation of inter-molecular
forces via collisional effects on line shapes. I discuss recent measurements of line
broadening and shifts caused by collisions in gaseous samples and the implications
for accurate species concentration retrievals from absorption measurements.
229
Drugs of Abuse
Christopher Benintendo, Drug Enforcement Administration, 99 Tenth
Ave., Suite 710, New York, NY 10011, Kiana Nurideen
Drugs of abuse are defined in the Controlled Substances Act (CSA) which places
all substances which were in some manner regulated under existing federal law
into one of five schedules. This placement is based upon the substance’s medical
use, potential for abuse, and safety or dependence liability. The Act also provides a
mechanism for substances to be controlled (added to or transferred between schedules) or decontrolled (removed from control). The Comprehensive Crime Control
Act of 1984 and the Anti-Drug Abuse Act of 1986 furthered the CSA by allowing for
emergency scheduling of drugs of abuse and defined controlled substance analogues. This classification scheme and particular drugs of abuse are discussed.
225
Cavity-Enhanced Spectroscopic Measurement of Greenhouse
Gases
Joseph T. Hodges, National Institute of Standards and Technology, 100
Bureau Dr., Gaithersburg, MD 20899
A rigorous understanding of the absorption of visible and infrared radiation by atmospheric greenhouse gases is central to many observations and models in climate science. In particular, the need for remote sensing from ground-, aircraft- and
satellite-based platforms and the desire to accurately predict radiative forcing motivates the development of experimentally validated, first-principles spectroscopic
models of greenhouse gases. I discuss how recent advances in laser-based cavity
ring-down spectroscopy and photo-acoustic spectroscopy have been exploited to
yield accurate line-by-line reference data for relatively weak near-infrared bands
of H2O, CO2, CH4 and O2. By comparison to traditional Fourier-transform spectroscopy, these methods generally provide superior sensitivity and spectral resolution
and give exceptional measurements of line parameters (e.g., positions, pressure
shifts, intensities, shape coefficients) as well as line mixing and collisional-induced
absorption effects.
230
Concealment Methods/Trends
Michelle Camilleri, Drug Enforcement Administration, 99 Tenth Ave.,
Suite 710, New York, NY 10011
In keeping up with demand of various controlled substances in the United States, it
is the goal of well-funded drug organizations to devise ingenious ways to get their
product across our borders successfully undetected. This is a visual account of attempted concealment techniques including a brief discussion on the drug extraction
and analysis results.
231
226
Fakes and Counterfeits
Jeffrey Comparin, Drug Enforcement Administration Special Testing and
Research Laboratory, 22624 Dulles Summit Ct., Dulles, VA 20166
After this presentation, attendees will have been informed of various topics concerning counterfeit and fake drugs. Discussion includes working definitions, some
statistical information, and ideas on the magnitude of this problem. For example, at
the time of this writing, simple Google searches on the terms “Counterfeit Drugs,”
and “Counterfeit Medicine” returned 1.28 million and 6.14 million results, respectively. Counterfeiters target controlled and non-controlled (prescription) medication
with emphasis on “quality of life” drugs and powerful pain medicines. The forensic
chemistry of counterfeit and fake drugs along with some analytical methodology will
be discussed. These types of materials may be found to contain the correct ingredients, with insufficient amounts of active pharmaceutical ingredients (API), excessive
amounts of API, incorrect ingredients, or any combination thereof. Counterfeit or
fake drug products may be detected by their packaging. Variations on packaging
include mislabeling, altering expiration dates, incorrect fonts or incorrect product
logos. Product inserts may also be indicative of counterfeit products. Lastly, an
overview of some Drug Enforcement Administration analyses of counterfeit or fake
products is discussed.
Improving the Short and Long Term Stability of Continuous Wave
Cavity Ring-Down Spectroscopy
Kevin Lehmann, University of Virginia, Chemistry Building,
Charlottesville, VA 22932
My laboratory has been trying to increase the sensitivity of continuous wave cavity
ring-down spectroscopy (cw-CRDS), which requires achieving the highest possible
stability of the cavity decay rate. We have attacked this problem both from a theoretical and experimental approach. We have also done theoretical analysis of the
theoretical sensitivity of several related techniques. In this talk, our work in the area
is presented.
227
Trace Gas Detection Using a Broadband Continuous Wave-Cavity
Ring-Down Spectrometer
Erika Coyne, Tiger Optics, 250 Titus Ave., Warrington, PA 18976, Yu
Chen, HongBing Chen
Continuous-wave cavity ring-down spectroscopy (cw-CRDS) is a field-proven analytical technique for trace gas detection that directly derives the absolute optical loss
due to molecular absorption inside the cavity from a simple time-based measurement, independent of laser intensity noise and environmental conditions. cw-CRDS
provides an ideal analytical solution that is fast, sensitive, accurate yet extremely
robust and simple to operate. Tiger Optics developed a new generation of cavity
ring-down spectrometers that transcends the bandwidth limitation of high-reflectivity
mirrors by employing Brewster’s angle prisms, which exploit total internal reflection,
to form a broadband, high finesse cavity. Integration of multiple diode lasers, combined with such a cavity, allows real-time detection of multiple analytes with very high
sensitivities. The Prismatic is best suited to serve applications requiring detection of
multiple analytes in a single gas stream, as well as applications which need measurements of a single species spanning a larger dynamic range than can be covered
using a single molecular absorption line. Multi-species detection is required in a wide
range of applications like quality control of high purity gases, hydrogen analysis for
fuel cells and emission monitoring. Performance data is presented illustrating simultaneous detection of CO2, CO, CH4 and H2O, achieving detection limits of 150, 100,
1 and 10 parts-per-billion (ppb), respectively. Furthermore, the Prismatic enables
an expanded dynamic range (from ppb to percent levels) for moisture analysis by
measuring the moisture concentration using several absorption lines, each with differing line strengths, to cover this broad concentration range with a single analyzer.
232
Tamperability/Extractability of Prescription Drugs
Robert P. Bianchi, Prescription Drug Research Center, 4031 University
Dr., Fairfax, VA 22030
Prescription drug abuse is second only to marijuana in the United States. Prescription opioid abuse is a major, rapidly growing problem, having surpassed cocaine
and heroin as drugs of abuse. Experts offer several reasons for this, including the
introduction of high dosage, extended release formulations that have proven easy
to manipulate. The regulatory approach to controlling the availability of prescription
opioids is the drug scheduling process mandated in the Controlled Substances Act
(1970) in which drugs with abuse potential are assigned to a progressively higher
schedule (I-V) as the perceived abuse liability increases. One of the factors considered in evaluating the abuse potential of a prescription drug, and therefore its
schedule, is the ease with which active ingredients can be extracted from the drug
product (“extractability”). Neither the Food and Drug Administration (FDA) nor Drug
Enforcement Administration has developed a standard battery of tests designed to
evaluate abuse resistant properties of delivery systems. The in-vitro experiments
33
2012 EAS Abstracts
November 2012
discussed in this paper present a foundation on which sponsors can present information to the FDA on their formulation’s abuse resistance properties.
tion, is excellent for drug analysis including natural products. Applications for routine
forensic drug analysis using sub-2-µm fully porous particles, including the analyses
of phenethylamines and related compounds, anabolic steroids, coca leaf, and for
general drug screening are presented.
233
Higher Throughput and Acuity for Biotherapeutic Characterization
via Novel Column Technology
Nathan Lacher, Pfizer, 700 Chesterfield Pkwy West, Chesterfield, MO
63017, Qian Wang, Melissa R. Thompson, Laura A. Bass
The Pfizer Biotherapeutic portfolio includes therapeutic monoclonal antibodies, antibody drug conjugates and recombinant proteins as well as vaccines (therapeutic
and prophylactic) that can range from 30 kDa up to 3 MDa. As a result of the structural complexity as well as the high degree of product heterogeneity that may exist,
it is often necessary to employ an extensive series of assays to ensure product quality and safety. Depending on the molecule of interest, it may be desirable to study
post-translational modifications such as oxidation, de-amidation, glycosylation or
conjugation properties which will generally only be possible with extensive sample
preparation that may include a proteolytic digest. It has now become possible in
some cases to eliminate the sample preparation as a result of improved instrumentation as well as by chromatographically separating peaks of interest. Enhanced
separations can be achieved by developing new column chemistries as well as by
enhancing classical phases to improve mass transfer which can improve the resolution between the peaks of interest as well as significantly decrease run time. We
discuss several applications where we have been able to directly introduce novel
column technologies to improve the acuity of the separation as well as the throughput of the analytical assays which are both a necessity to achieve a greater understanding of the product.
237
Effect of Peptide Structure on Measured MALDI TOFMS Signal
Intensity
Kevin G. Owens, Drexel University, Chemistry Department, 3141
Chestnut St., Philadelphia, PA 19104, Andrew Mahan
This presentation describes two series of experiments designed to gain a better understanding of the effect of peptide structure on the matrix-assisted laser desporption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) signal response.
It is generally understood that increases in the hydrophobicity of the peptide leads
to a decrease in the MALDI signal, while an increase in the number of basic groups
present (lysine, arginine, histidine and the amine terminus) leads to large increases
in the observed MALDI signal. This work goes a step further by investigating how
the proximity of and exact location of multiple basic sites in the peptide chain affects
the MALDI signal. The initial hypothesis was that increased separation of the basic
sites would lead to the observation of higher signal by increasing the sphere of capture for protons in the desorbing MALDI plume. Rudimentary molecular modeling
is used to rationalize the results obtained. The second experiment investigates the
effect of the acid/base properties of both the peptide and matrix on the number of
positive versus negative ions observed in the MALDI spectrum. This work explores
the two-step model of ionization in MALDI as described by Knockenmuss.[1] In both
cases electrospray deposition (rather than the conventional dried droplet method) is
used to produce highly homogeneous MALDI samples that yield highly reproducible
analyte ion signal. These results can be important to those seeking to understand
the observed sequence coverage obtained in peptide mass mapping or ladder sequencing type experiments.
234
Applications of UHPLC in Regulated Bioanalysis
Guowen Liu, Bristol-Myers Squibb, Route 206 & Province Line Rd.,
Princeton, NJ 08543, Yue Zhao, Jian Wang, Anne-Francoise Aubry
Ultra high-pressure liquid chromatography (UHPLC) using columns packed with
sub-2 micrometer particles for chromatographic separations in the pharmaceutical
industry has become the trend in recent years due to its improved resolution and
shorter run times. However, in regulated drug bioanalysis, where most bioanalytical
assays have only one or two analytes and the analytical run time for routine assays was less than five minutes already using regular HPLC, the implementation
of UHPLC has had relatively low impact on workflows. In many applications, the
UHPLC system has replaced the HPLC system and a sub-2 micrometer column, the
3 micrometer column, but other experimental conditions (flow rate, gradient time,
and equilibration time) have not been optimized to maximize its potential. In this presentation, we have summarized the current status of UHPLC use in regulated laboratories, based on published literature and our own experience of using UHPLC.
Different parameters, such as extra-column volume, injection volume, and flow rate,
etc. have been investigated on the performance of an UHPLC method. The benefits
and productivity gains of using the technique to its full potential are also discussed.
[1] Int. J. Mass Spectrometry, 2008, 273, 84-86.
238
New Ionization Approaches for Mass Spectrometry: From Laser
Spray Ionization to Matrix Assisted Vacuum Ionization
Sarah Trimpin, Wayne State University, Chemistry Department, 5101
Cass Ave., Detroit, MI 48202
Laser spray ionization (LSI) uses the same matrix/analyte sample preparation and
laser as atmospheric pressure matrix-assisted laser desporption/ionization (AP
MALDI) but produces multiply charge ions nearly identical to electrospray ionization (ESI). Fundamental research led to the understanding that in LSI, ionization is
initiated in the inlet of the mass spectrometer. A laser was not necessary and simply
introducing particles of matrix/analyte into the inlet produces identical mass spectra
(matrix assisted inlet ionization (MAII)). Ion abundance increases with increasing
inlet temperature suggesting that matrix droplets/particles become charged in the
heated inlet and desolvation by evaporation/sublimation creates sufficiently dense
surface charge to field evaporate analyte ions by a mechanism proposed for ESI.
Numerous small molecules act as matrix compounds in MAII suggesting that an
important function of a matrix is to act as a solvent, which is supported by solvent
assisted inlet ionization in which analyte is introduced into the inlet in a solvent.
The understanding that producing charges on particles or droplets is common in
nature and that desolvation of these charged droplets/particles is key to observing
bare ions in mass spectrometry, led to the production of highly charged ions by
laser ablation of volatile matrices in vacuum MALDI ion sources. We were able to
demonstrate that these are not MALDI processes because using a volatile matrix
produces highly charged ions of even proteins in a vacuum MALDI source without
the laser (so called matrix assisted vacuum ionization). These highly sensitive ionization methods are discussed along with our current understanding of the ionization
mechanism.
235
An Industrial Perspective on the Evolution of Fast LC – Selected
Applications for Small Molecule, Oligomer and Polymer
Separations
Matthias Pursch, Dow Deutschland, Anlagengesellschaft mbH,
Industriestr. 1, Rheinmuenster, 77836 Germany
Fast liquid chromatography (LC) has become very popular over the past ten years
after higher pressure instrumentation and small particle column technology have
been introduced. The technology has enabled many laboratories to increase
throughput, obtain faster results and/or get higher information content or sensitivity.
This talk highlights some of the developments and applications for fast LC in chemical industry. High-pressure / ultra high-pressure liquid chromatography (HPLC/
UHPLC) instrumentation is discussed, sub-2-µm particle and core-shell columns
are reviewed, along with applications of the chemical industry including herbicides/
insecticides, additives, epoxy oligomers and polymer separations. Select examples
using two-dimensional LC are presented.
239
Applications of MALDI-TOF in Biopharmaceutical Analysis and
Quality Control
Gary H. Kruppa, Bruker Daltonics, 40 Manning Rd., Billerica, MA 01821,
Matt Willetts
Recent advances in software and instrumentation have resulted in the adoption of
matrix-assisted laser desporption/ionization time-of-flight (MALDI-TOF) and electrospray ionization time-of-flight (ESI-TOF) mass spectrometry for routine biopharmaceutical analytical and quality control applications. These applications include verification of biopharmaceutical product molecular weight, sequence, and verification
of post-translational modifications such as glycosylation. Software suitable for use
by bench level quality control (QC) technicians and biopharmaceutical researchers
with limited experience in mass spectrometry has been developed that makes this
technology suitable for deployment in a wide range of environments, including QC
laboratories. Instrumentation solutions have also become more robust, and suitable
for deployment in 24/7 GMP environments. Software and results for the development of routine biopharmaceutical applications are presented. Another emerging
application of MALDI-TOF is the routine identification of microorganisms using the
MALDI Biotyper. The MALDI Biotyper identifies microorganisms based on protein
236
UHPLC and UHPLC-MS for the Analysis of Seized Drugs
Ira S. Lurie, United States Department of Justice, Drug Enforcement
Administration, Special Testing and Research Lab, 22624 Dulles
Summit Ct., Dulles, VA 20166
The analysis of seized drugs is important for both legal and intelligence purposes.
Chromatographic and electrophoretic separation methods used in forensic drug
analysis must have the ability to separate a wide variety of controlled substances,
many of which can be structurally similar and or be present in complex matrices.
Ultra high-pressure liquid chromatography (UHPLC) offers significantly improved
limits of detection and peak capacity with the added benefit of higher speeds of
analysis, and is well suited for this purpose. Hydrophilic interaction chromatography (HILIC) provides for highly efficient and rapid isocratic UHPLC separations of
seized drugs. In addition, operating at elevated temperatures can provide for even
faster analyses. Tandem mass spectrometry (MS/MS) detection via single reaction
monitoring (SRM), which provides higher selectivity and sensitivity than UV detec-
34
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2012 EAS Abstracts
November 2012
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2012 EAS Abstracts
November 2012
243
mass fingerprints and has many advantages over other phenotypic and genotypic
methods commonly employed in pharmaceutical microbiology labs. Results can be
obtained in minutes after culture at a consumable cost of about $0.30 per sample.
The MALDI Biotyper instrument is specifically designed for 24/7 operation in GMP
and clinical environments, for use by technicians with no previous experience with
mass spectrometry. An overview of the MALDI Biotyper technology and some results
from pharmaceutical microbiology applications of this technology are presented.
Understanding Sampling and the Impact on PAT Method
Performance for Solid Dosage Manufacturing
Gary McGeorge, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, Dongsheng Bu, Dimuthu Jayawickrama
Non-invasive spectroscopic methods have become a foundation of the measurement of critical material attributes for oral solid dosage manufacturing. Consideration
of what the measurement is and how to sample one’s process are vitally important
to the success and validity of the specific control, and ultimately into the overall
product control strategy. We share our findings on the topic with specific regard to
sampling of powder blends using on-line near-infrared (NIR) spectrometers. Here
the volume of information is discussed and its relationship to filtering approaches is
evaluated. This area has seen significant inconsistencies in the literature regarding
how to estimate the sample mass being tested in such measurements and it often
causes confusion with the health authorities when relating it to the mass of a dosage
unit. Moving along in the process we evaluate options for sampling of tablets using
at-line NIR or Raman for both potency and content uniformity assessment. With the
capability to test an increased number of tablets it is necessary to redefine the release criteria for content uniformity and link the specification specific sampling plans
to a product. We work though an example product where a modified large-N sampling procedure was adopted and presented to global health authorities. Without
such careful consideration in these areas it is not possible for the pharmaceutical industry to see the real benefits of in-process control and ultimately ensure improved
product quality and real-time-release. These will clearly be even more important as
the industry adopts continuous manufacturing.
240
Advancing the Development of RNA Therapeutics with Innovative
Mass Spectrometry Based Assays
Mark Cancilla, Merck, 770 Sumneytown Pike, West Point, PA 19486
Modulation of messenger RNA (mRNA) expression by RNA interference (RNAi) is a
break-away technology with tremendous potential for rapid in-vivo target validation
and development of therapeutic small interfering RNA (siRNAs) against “non-drugable” targets. Effective design of chemically modified siRNAs remains a key ratelimiting technological step in realizing this value. Despite the advancement of RNA
inhibitors into the clinic, the pharmacokinetics and metabolism of RNAi therapeutics
is poorly understood. Currently, quantitative polymerase chain reaction (qPCR) is
a primary analytical method but has limitations distinguishing between the parent
siRNA and its metabolites. In order to understand siRNA metabolism and develop
best-in-class RNAi therapeutics, a robust and sensitive high resolution liquid chromatography mass spectrometry (LC-MS) method for characterizing siRNA metabolites was developed from various biomatrices. The trials and tribulations of analyzing
siRNAs from biological matrices using LC-MS techniques are discussed. The talk
is separated into three parts: 1) rapid sample preparation of siRNAs from complex
biological matrices (the foundation of any biological analysis); 2) overview of rapid
and sensitive LC-MS methods of oligonucleotide therapeutics that provide simultaneous qualitative and quantitative data utilizing Orbitrap mass spectrometers; and
3) attempts at overcoming the bottleneck of data analysis using various commercial
and in-house software platforms.
244
Raman as a Multipurpose Sensor for Biopharmaceutical Processes
Saly Romero-Torres, Pfizer, 100 Route 206 North, Peapack, NJ 07977
No abstract submitted by the author.
245
Structural Insights into Integrin Signaling
Olga Vinogradova, University of Connecticut, 69 North Eagleville Rd.,
MS: U-3092, Storrs, CT 06269
Integrins comprise a large family of cell-surface adhesion receptors found in many
animal species. Cells in multi-cellular organisms have to interact with each other
and their extracellular surrounding in order to survive. This adhesion is primarily
mediated by integrins. Integrins control cell migration and anchorage and are, therefore, crucial for many biological processes including embryogenesis, haemostasis,
immune response and maintenance of the tissue integrity. In addition integrins are
involved in bone development and remodeling, extracellular matrix assembly, blood
clotting, wound healing and fighting infections. The aberrant stickiness of the cells,
caused by the integrin mutations or mis-regulation, contributes to a number of serious human disorders, including rheumatoid arthritis, heart attack, stroke and cancer.
Certain viruses, including HIV, use integrins as a portal of entry into the cells. Therefore, it is important to understand the mechanisms of integrin function from both
physiological and pathological perspectives. Significant progress has been made
in molecular understanding of integrin activation, referred as inside-out signaling.
However, much less is known about the early intracellular events following integrin
mediated extra-cellular matrix engagement, a process termed as outside-in signaling. Among the earliest biochemical responses related to outside-in signaling are
tyrosine kinases activation and phosphorylation of β integrin cytoplasmic tails. We
have used solution nuclear magnetic resonance in concert with other biophysical
techniques to investigate and are going to present how tyrosine phosphorylation
affects the structure of β3 integrin cytoplasmic tail and its interaction with several
intracellular target proteins.
241
Process Analysis by Vibrational Spectroscopy: Polymer and
Pharmaceutical Applications
Heinz W. Siesler, University of Duisburg-Essen, S Schuetzenbahn 70,
Essen, D 45127 Germany, Sven Borchert
Increasing cost pressure and legal requirements with reference to quality control,
processing conditions and environmental compatibility of chemical, pharmaceutical,
petrochemical, food and agricultural products have enforced the consideration of
new specific, non-destructive, rapid and flexible process-control techniques to meet
these demands. In this context, light-fiber coupled near-infrared (NIR) and Raman
spectroscopy in combination with special probes and statistical evaluation procedures have become indispensable analytical tools for industrial process monitoring
in a wide field of applications. The present communication focuses on selected application examples for both techniques. Thus, the potential of Raman spectroscopy
is demonstrated with reference to a styrene-butadiene-styrene triblock-copolymerization reaction and its continuous concentration monitoring of the starting materials
and the final polymeric product. In further examples the implementation of NIR spectroscopy for the 100% on-line inspection of blend uniformity on a continuous mixer
and the subsequent content uniformity control on a tablet press in a pharmaceutical
production process are discussed in some detail.
242
Real-Time Determination of Drug Concentration in a Continuous
Powder Mixing Process
Rodolfo Romañach, University Puerto Rico-Mayaguez, PO Box 9000,
Mayaguez, PR 00682, Yleana Colon, Jenny Vargas
This talk describes efforts to implement real-time near-infrared (NIR) drug concentration measurements in continuous powder mixing. Method validation is discussed with the objective of making these measurements a reality in pharmaceutical
manufacturing. The determination of drug concentration in a pharmaceutical mixing
process is definitely not a pioneer research effort. A high number of high-quality research studies have been done in this field. The time has come to pull together this
knowledge and transform pharmaceutical manufacturing. Two research efforts are
discussed: one conducted in an academic environment using a 10% (w/w) target
concentration, and an industrial process. The validation of both methods has been
performed while visualizing their industrial application with the goal of developing
robust methods that will require minimal updating. The different steps taken to make
the method robust are discussed. This includes variation in excipients and active
pharmaceutical ingredient, as well as development in more than one instrument.
Both methods also include outlier testing, which will be capable of detecting malfunctions in the blending process. Thus, the conference includes significant emphasis on method implementation and moving beyond method validation.
246
Transient Substrate and Domain Interactions in Non-Ribosomal
Peptide Synthetases
Dominique Frueh, Johns Hopkins School of Medicine, Hunterian 701,
725 N Wolfe St., Baltimore, MD 21205, Bradley Harden, Andy Goodrich,
Subrata Mishra, Scott Nichols
Non-ribosomal peptide synthetases (NRPSs) are multi-module, multi-domain enzymes, that synthesize important natural products in bacteria and fungi, many of
which have pharmaceutical applications (e.g. antibiotics, antitumor agents, immunosuppressants). NRPSs use an assembly line organization to covalently load chemical substrates onto each module and catalyze peptide bond formation between substrates loaded on adjacent modules. These multiple catalytic steps require a series
of sequential domain/domain and domain/substrate interactions, which are currently
poorly understood. We have used nuclear magnetic resonance (NMR) to study transient interactions between domains and between domains and chemical substrates.
We present novel NMR experiments to rapidly probe protein dynamics or to assign resonances of the larger (50-60 kDa) domains. We show that many NRPS
domains recognize both chemical and protein substrates and we discuss structural
and dynamics effects during molecular interactions. Understanding the dynamic
mechanism of NRPS domain communication may open the venue to efficient NRPS
assembly line reprogramming and the production of novel pharmaceuticals.
39
2012 EAS Abstracts
November 2012
247
250
Uncertainty of NMR-Derived Parameters to Characterize Protein
Dynamic Equilibrium Systems
Rieko Ishima, University of Pittsburgh, Biomedical Science Tower 3,
3501 5th Ave., Pittsburgh, PA 15260
Nuclear Magnetic Resonance (NMR) relaxation is a highly advantageous technique that provides information on fluctuation of local conformation and equilibrium
constants of individual sites in proteins. 15N longitudinal and transverse relaxation
rates and {1H}-15N heteronuclear NOE are often recorded to characterize sub-nano
second motion, as well as milli-micro second motion of protein backbone amides
in solution. The slower time-scale motion can be further studied by recording CarrPurcell-Meiboom-Gill (CPMG) relaxation dispersion that is sensitive to chemical and
conformational exchange and the involved rate constants. Slow and fast exchange
profiles of ligand titration experiments on proteins enable to determine ligand-dissociation constants at individual sites. Estimation of uncertainty of these parameters is
important when these NMR-derived parameters are compared with those obtained
by other methods. Thus, we evaluated these errors based on the self-consistency
of the data.
EPR Spectroscopy of Historic Iron Gall Ink Manuscripts: Monitoring
Fenton-Type Reactions through Selective Spin-Trapping
Richard Wolbers, University of Delaware, Winterthur Program in Art
Conservation, Newark, DE 19716
No abstract submitted by the author.
251
Elastic Polymer-Borate Cleaning Systems for Art Conservation Properties, Applications, and Analysis of Cleaning Mechanism
Lora V. Angelova, Georgetown University, Reiss Science Building 240,
37th and O Streets NW, Washington DC 20001, Richard G. Weiss,
Barbara H. Berrie
Polymeric gel-like systems have been designed and examined for their potential applications as surface cleaning agents for cultural heritage works. The systems consist of partially hydrolyzed poly(vinyl acetate), with boron-based cross linkers. Initial
formulations contained predominantly aqueous liquid compositions.[1] The polymer
and cross linker have since been modified to accommodate higher volume fractions of organic liquids.[2,3] Systems with entirely organic liquid compositions have
recently been produced. The physico-chemical properties of the systems have been
investigated using a wide range of analytical methods. The systems are effective in
softening surface grime and aged varnish, allowing the conservator to remove the
bulk of the unwanted material through subsequent use of gentle swabbing with mild
solvents. To investigate if polymeric residues were present after treatment, a fluorescent tag was covalently attached to the polymers. The contents of gels used in a
treatment have been analyzed via gas chromatography mass spectrometry, nuclear
magnetic resonance, spectrofluorimetry, and Fourier transform infrared and compared to the material removed by swabs used alone as well as following application
of a gel. The systems show great potential as tools for art conservation.
248
Novel Methods for Accurate Measurement of One-Bond and LongRange Proton-Carbon Residual Dipolar Couplings (RDCs) to
Improve Structure Discrimination and to Assign Diastereotopic
Protons in Small Molecules
Roberto Gil, Carnegie Mellon University, Department of Chemistry, 4400
Fifth Ave., Pittsburgh, PA 15212
Nearly all of the work published thus far on the application of RDCs to the structural
analysis of small molecules involved the use of only one-bond H-C RDCs measured
from the cross-sections of F2 1H-coupled hetero-nuclear single quantum coherence (HSQC). These studies have shown that one-bond RDCs can suffice to solve
configurational problems in small molecules. However, there are many situations
in which it is desirable or essential to have more independent RDCs. A new pulse
program to accurately measure long-range (LR) H-C RDCs, the Selective J Scaled
(SJS) HSQC, was developed. We have shown that addition of LR-RDCs to the
analysis significantly improves the structure discrimination power. We have also
shown that it is possible to accurately measure one-bond H-C RDCs using J scaled
F1 1H-coupled HSQC without paying significant penalties in the experimental times.
On the other side, when the molecule contains diastereotopic methylene protons it
is necessary to previously assign them in order to perform structure discrimination
calculations. We have demonstrated that it is possible to discriminate structures
using the sum of the coupling of each CH bond of the CH2 group during the calculations and treating the CH2 group as a single structural feature. Hence, no previous
assignment is needed to determine the configuration. Once the correct configuration
is found, each CH2 diastereotopic proton is straightforwardly assigned by predicting their RDCs values using the calculated alignment tensor and comparing these
values with RDCs calculated either from F2 coupled HSQC or from J Scaled BIRD
HMQC/HSQC experiments.
[1] Carretti, E.; Grassi, S.; Cossalter, M.; Natali, I.; Caminati, G.; Weiss, R. G.; Baglioni, P.; Dei, L. Langmuir 2009, 25, 8656-8662.
[2] Angelova, L. V.; Natali, I.; Carretti, E.; Terech, P.; Dei, L.; Weiss, R. G. Langmuir
2011, 27, 11671-11682.
[3] Natali, I.; Carretti, E.; Angelova, L. V.; Baglioni, P.; Weiss, R. G.; Dei, L. Langmuir
2011, 27, 13226-13235.
252
Chemometrics Light: Using a FileMaker Pro Database to Model
Cleaning System Formulations for Art Conservation
Chris Stavroudis, Paintings Conservator, 1272 N. Flores St., West
Hollywood, CA 90069
The Modular Cleaning Program is a FileMaker Pro database that models the building of aqueous, solvent, solvent gel, and limited emulsion cleaning systems for
works of art. Built on the work and research of Richard C. Wolbers, the database
models the ionic equilibria calculations for aqueous cleaning systems and assists
the conservator in building complex cleaning systems from concentrated stock solutions. Solvent-solute interactions are modeled in Hansen solubility space and are
presented in a novel two-dimensional schema. Carbopol-based solvent gels are
modeled on empirically derived relationships and are presented on the same 2-dimensional solubility diagram. Emulsions based on Pemulen, a polymeric emulsion
stabilizer, and Gransurf 90, a silicone-based surfactant, are managed by tabulating
the properties of the aqueous phase and the solvent phase. The presentation shows
how a relational database that allows complex calculation fields and script-based
interactions can model the chemistry of these cleaning systems. The visual design
component of FileMaker Pro allows for the information to be presented in an interactive and appealing way.
249
Application of Carbon Detected NMR Methods to Biomolecules in
Solution
Scott A. Showalter, The Pennsylvania State University, Department of
Chemistry, University Park, PA 19502
13
C-detected NMR spectroscopy of proteins in solution has seen a recent resurgence, driven by improvement in cryogenic probe technology. For proteins with poor
1H amide chemical shift dispersion or relaxation properties, advances in carbon
detected NMR spectroscopy made in our laboratory and elsewhere allow comprehensive structural biology to be realized. While our analysis has focused on the
problem of defining intrinsically disordered protein (IDP) conformational ensembles
and dynamics, carbon-detected methodologies are also viable alternatives to existing protocols for studying large or paramagnetic proteins. In our group, we aim
to implement a new nuclear magnetic resonance (NMR)-based methodology for
assigning chemical shifts, establishing structural ensembles, and quantifying the
dynamics of IDPs, with the same level of rigor currently attainable for co-operatively
folded proteins. Here I present NMR methodological advances made in our laboratory, using our studies of the disordered C-terminal domain from human FCP1 as
an example system. I summarize or development of a fully triple resonance (i.e., 1H,
13
C, 15N) approach to chemical shift assignment of proteins with carbon direct detection that complements previous protonless approaches. Recently developed pulse
programs for paramagnetic relaxation enhancement measurements, coupled with
measurement of residual dipolar couplings, allow the generation of experimentally
constrained structure ensembles for FCP1. Finally, in order to more directly quantify
the conformational dynamics of IDPs in solution, we have developed experiments
for carbonyl 13C-detected acquisition of backbone 15N T1 and T2 relaxation rates.
Generalization of carbon-detected solution NMR methods for the study of intrinsically disordered and paramagnetic proteins is discussed.
253
Experimental and Innovative: Matisse Paintings from the Wertheim
Collection
Dan Kirby, Harvard Art Museums, Conservation and Technical Studies,
Cambridge, MA 02138, Gabriel Dunn
Henri Matisse’s body of work from 1913-17, has been referred to as his most experimental and innovative due to his altered palette and techniques. Two paintings
from the Fogg Museum’s Wertheim Collection, Geraniums, 1915 and Still-Life with
Apples, 1916, were subject to a technical analysis to understand the development
and the structure of these significant works. Observing slight variations in the under
drawing with the aid of infrared digital photography (IRDP), and X-radiography, and
identifying a diverse, bright palette through cross-sectional analysis with reflected
light microscopy (RLM), Fourier transform infrared (FTIR) micro-spectroscopy, Raman spectroscopy, and scanning electron microscopy (SEM), it was concluded, with
support from archival information that Geraniums is from an earlier period. Characteristics exhibited by Still-Life with Apples included a subdued, limited palette,
identified with cross-sectional analysis with RLM, FTIR, Raman, and SEM, and
compositional revisions seen in the X-radiograph, accurately match the 1913-17
period. Within the paint stratification of both paintings, striking color changes were
visible throughout. These color changes were replicated through digital image processing in order to view the paintings in various stages, as Matisse may have created them. Both paintings had non-original synthetic varnishes identified with FTIR,
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2012 EAS Abstracts
November 2012
257
cross-sectional analysis with RLM, and supported by conservation records. It was
proposed to remove the varnishes due to their poor aging, which had altered the
original aesthetics of the paintings. Geraniums has been cleaned, and while varnish
solubility testing has been performed on Still-Life with Apples, further discussions
with curators are still necessary to decide the course of treatment.
Analysis of Mineral Oils in Recycled Paperboard Food Packaging
Richard Castino, Sun Chemical, 631 Central Ave., Carlstadt, NJ 07072,
Chris C. Sommer, Jesus A. Ramirez
Recently, the potential migration of various substances inherently present in recycled paperboard used for food packaging has come under scrutiny. In particular, the content and the possibility for lower molecular weight mineral oil saturated
hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) to migrate
into food products has become of concern. In order to reduce migration of these
recycled paperboard mineral oils into food, functional barrier coatings are being
developed as an alternative to virgin paperboard substrates. We describe a gas
chromatography mass spectrometry analysis protocol which establishes the nature
of potential compounds of interest in recycled paperboard, provides for quantitation
of these compounds via extraction by a solid-phase food stimulant, and allows us
to show the effectiveness for potential barrier coatings for reducing the migration.
Both MOSH and MOAH have been monitored down to a total of 50 ppm in recycled
paperboard (and low ppb levels in food simulants) via this method.
NIR, a Rapid Screening Technique for Determining Procyanidin
Levels in Cinnamon
Walter A. Roy, McCormick and Company, Technical Innovation Center,
204 Wight Ave., Hunt Valley, MD 21031, Jonna Pratt, Roman Grypa
Spices, in addition to flavoring foods, are also sources of natural antioxidant compounds known as poly phenolics. Cinnamon is one of the spices rich in these poly
phenolic compounds, especially procyanidins. Preclinical trials have suggested
these poly phenols may decrease blood glucose levels and may have anti-inflammatory properties. Near- infrared (NIR) would be a useful technique for the
screening of procyanidin levels. In testing this hypothesis, thirty cinnamon samples
were scanned on a FOSS, model 5000, NIR with the spectra being collected from
1100 - 2500 nm at a 10 nm resolution. The procyanidin concentrations for these
samples were determined by a validated liquid chromatography fluorescence detection (LC-FLD) method using Catechin as the reference standard. The procyanidin
levels were calculated as mg/g catechin equivalent. The 30 samples ranged from
4 to 14 mg/g catechin equivalents and were used as the calibration model for the
NIR method. This calibration demonstrated an R-squared of 0.9. The results of this
experiment indicate that NIR could be a useful screening method for estimating
procyanidin levels in cinnamon samples. We are in the process of testing this model
against a set of unknown samples and having them confirmed by LC-FLD testing.
The LC-FLD method has demonstrated a relative standard deviation of 7% and it is
expected that this NIR method is close to that variation.
255
258
254
Detecting What Should and Should Not Be There: Rapid Screening
of Packaging and Finished Products for Contaminants by Using
Direct Analysis in Real-Time (DART)-Mass Spectrometry
Brian Musselman, IonSense, 999 Broadway, Suite 404, Saugus, MA
01906, Michael Festa, Joseph Lapointe
Direct analysis in real-time mass spectrometry (DART-MS) offers a rapid means to
scan objects including: plastic containers, sheets, fabrics, even finished goods for
their identity, homogeneity, and presence of contaminant in seconds per sample.
The analysis of these materials is facilitated by using a flatbed scanner under digital
control to scan the surface in a controlled manner. Methods for detection of contaminants in manufactured raw materials such as bulk chemicals are used every day in
analytical laboratories, however for consumer goods including packaging, plastics,
printed materials, and fabrics analysis is more challenging. Using DART allows for
rapid accumulation of reference spectra and the use of that data to compare with the
results obtained from analysis of these materials could provide a means to detect
contamination enters the production or packaging line. We describe a means to detect non-specific contaminants as well as targeting of known contaminants by using
rapid DART analysis of surfaces by comparing a library of results from past analyses with new quality control data processed automatically using statistical analysis.
Simple protocols for creating analytical methods to collect significant numbers of
results to populate the database are shown. Finally, detection of contaminants in
very complex samples in an automated means is described.
Carbohydrates in Coffee: The AOAC Official Method 995.13 vs. A
Fast Method
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, Lipika Basumallick, Deepali Mohindra, Jeff Rohrer, Art Fitchett
Coffee, a brewed drink from the roasted beans of the coffee plant, has emerged
as one of the most consumed beverages in the world. Globally, the coffee industry
ranks second only to petroleum in terms of dollars traded and green coffee (unroasted beans) is one of the most traded agricultural commodities. Coffee carbohydrates constitute at least 50% of a raw coffee bean’s dry weight. Carbohydrates act
as aroma binders and contribute to the flavor, the viscosity, and stabilize the foam in
the beverage. They are also very good tracers for authenticity assessment of instant
coffee. Commonly, the Association of Official Analytical Chemists (AOAC) Official
Method 995.13, which is based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD), is used for determining
carbohydrates in instant coffee. This method is also used by the British Standards
Institution. This presentation compares two HPAE-PAD methods for the determination of carbohydrates in instant coffee and green coffee: 1) the AOAC official
method 995.13, and 2) a fast method. For the official method, minor modifications
have been proposed to help resolve some of the sugars (rhamnose from arabinose,
and sucrose from xylose and mannose) that might otherwise be difficult to achieve.
259
Determination of Anions in Distillers Dried Grains with Solubles
(DDGS) by Liquid Ion Chromatography
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale,
CA 94085, Kasandra I. Oates, Jeffrey S. Rohrer, Brian De Borba, Kirk
Chassaniol
Due to a growing interest in alternative fuels, the production and use of distillers
dried grains with solubles (DDGS) is expected to increase significantly within the
next few years. Dried distiller grains are a co-product of the dry mill fuel ethanol
process that provides three times the amount of nutrients available from grain alone
when used as an alternative feed in ruminant diets. Because the composition of
DDGS can vary from the ethanol production process, it is of significant interest to
the distillery and feed industries to determine its mineral composition to formulate
a nutritionally balanced ruminant diet. Here we show the determination of chloride,
sulfate, and phosphate in DDGS using ion chromatography followed by suppressed
conductivity detection in less than 10 min. The method demonstrates excellent peak
shape, linearity, accuracy, precision (peak area relative standard deviations <1%),
and detection limits in the low µg/L range (LODs 1.7-5.7 µg/L and LOQs 5.7-18.8
µg/L). In addition, the simple sample preparation and the electrolytic eluent generator enhance the ease of use, reproducibility, and automation of the method.
256
Analysis of the Odor Profile of Food Products Using a Micro
Chamber Thermal Extraction System and Thermal Desorption GCTOF-MS Detection
Gareth M. Roberts, Markes International, Gwaun Elai Medical Science
Campus, RCT Llantrisant, CF72 8XL United Kingdom, Kurt Thaxton,
Paul Morris, Stephen Smith
The ability to identify the odor profile from food products is commercially important
for several reasons. These include product quality/consistency, consumer attraction
and off-odor analysis as an indicator of decay or contamination. To study this profile,
a micro chamber/thermal extraction system (µ-CTE) is described which enables the
volatile and semi-volatile organic chemicals (VOC/SVOC’s) to be monitored from
a variety of food products. This includes the odor profile of pork meat and several cheeses for both fresh and aged samples. Sample preparation, analysis and
detection employs a multi hyphenated approach combining dynamic headspace
extraction (µ-CTE), thermal desorption (TD) pre-concentration and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) analysis. The µ-CTE can
accommodate bulk (grams) samples, which are placed into a series of chambers,
heated (optional) and purged with an inert gas. The effluent from each chamber
is subsequently trapped by a thermal desorption sample tube containing selective
sorbents allowing the whole VOC/SVOC profile to be monitored, including sulpher
compounds. The ability of the TD system to quantitatively recollect a (split) proportion of the sample after desorption from the TD tube and/or cold trap is described
which enables re-analysis of the same sample. This provides a technique to look
at both the high concentration components, followed by a low split method for trace
level analysis. Detection using a high-performance GC-TOF-MS system provided
the optimum screening detector for both target and unknown compounds and the
classical EI spectra derived from the TOF enables identification using commercially
available libraries e.g., NIST, Wiley.
260
The Use of Ultraviolet/Visible/Near-Infrared Total Reflectance
Spectroscopy to Characterize Human Skin Chemistry
Jillian Dlugos, Glenelg High School, 940 Winter St., Glenelg, MD 21771,
Jeffrey Taylor
Reflected light from human skin carries chemical information from all three (stratum
corneum, epidermis and dermis) skin layers. Each skin layer has its own unique
chemistry that is apparent in the total reflection spectra. This paper evaluates reflectance spectra from a wide range of human ethnic skin types. The data are measured
with a Lambda 1050 UV/Vis/NIR spectrophotometer fitted with a 150 mm, InGaAs
detector equipped, Spectralon integrating sphere. New instrument and detector
technology can now measure high fixed resolution reflectance spectra in the nearinfrared region. The chemistry of all three skin layers is discussed, with particular
41
2012 EAS Abstracts
November 2012
attention on the spectroscopy of melanin. Melanin is a useful component of human
skin since it protects the skin from damaging ultraviolet radiation, which can cause
cancer or other related health problems. The quantitative and qualitative characterization of changes in melanin chemistry is critical for many areas of skin care and
medical research. This study demonstrates how skin thickness and data processing
procedures (Kubelka-Munk and Log 1/R) influence reflectance spectroscopy data.
Total reflection spectroscopy offers a non-destructive and non-invasive analytical
process to study skin chemistry that would be of interest to the medical (cancer) and
skin care industries (cosmetics and sunscreen).
investigations into the differences between the polymer grades and their impact on
chemical degradation were also conducted. This involved looking at the chemical
substitution of the polymers and the direct and indirect effects this change can have
on the polymer properties (micro-environment pH, H-bond acidity, hygroscopicity).
This presentation provides an evaluation of the tools used to interrogate the stability
data, information on the effectiveness of the DoE to determine the reaction kinetics
for the individual and combined parameters and discusses why the differences in
the polymer grades are thought to result in the different rates of chemical degradation.
261
264
Sample Preparation and Examination of Imported Children’s Toys
for Phthalates and Bisphenol A
Patricia L. Atkins, SPEX CertiPrep, 203 Norcross Rd., Metuchen, NJ
08882, Thomas Mancuso, Ralph Obenauf, Richard Bostwick
The debate over potentially toxic compounds used in plastic production has created
new testing methods and regulations controlling or banning plasticizers such as
phthalates and Bisphenol A. Children’s products are often targeted for testing and
regulation due to their contact and use by small children where the potential for toxic
exposure is greater than for adults. The United States has started to limit levels of
some phthalates for use in children’s’ products including DEP, DEHP, DBP, BBP.
The Consumer Product Safety Commission (CPSC) has published testing methods
for these regulated phthalates. The regulation of Bisphenol A (BPA) remains under
debate. This study examined the levels of phthalates and BPA in small inexpensive imported children’s toys purchased at discount stores. New cryogenic grinding
methods were employed to process the different types of plastic toys examined.
Microwave extraction methods were created and optimized against newly developed certified solid reference materials to compare levels of phthalates and BPA
found in the toys. The samples were examined using gas chromatography mass
spectrometry. High levels of phthalates and BPA were detected in the majority of the
PVC toys. In many samples the concentration of phthalates far exceeded the limits
set by the CPSC.
Content Uniformity by Transmission Raman Spectroscopy of
Intact Tablets using only Powder Blend Calibration
Andrew Owen, Cobalt Light, The Electron Building, Fermi Ave., Harwell
Oxford, Oxfordshire, OX11 0QR United Kingdom, Matthew Bloomfield,
Darren Andrews, Hilary Jeffrys, Steven Brown, Pavel Matousek
Transmission Raman spectroscopy (TRS) is a technique that enables content uniformity of intact tablets and capsules without complicated calibration models. In contrast with other vibrational spectroscopy techniques TRS is able to analyze through
thick tablets with little or no sensitivity to changes in particle scattering effects arising
from tablet compaction variances, source material changes and other processing
effects. Ultimately, this means that it may be possible to build calibration models using simply blended powders and apply those to compressed tablets. Examples are
shown of the insensitivity to particle scattering effects and the building of models for
tablets using powder blends.
265
Can HPLC be a PAT Tool for In-Process Testing During
Manufacturing of Active Pharmaceutical Ingredient?
Tseng-En Hu, Merck, 126 East Lincoln Ave., PO Box 2000, Rahway,
NJ 07065, Elie Chaaya, Zhihao Lin, Jonathan P. McMullen, Frank
Bernardoni, Joseph Hinksmon, John P. Higgins, Charles J. Orella, Keith
E. O’Neill, Ike Diribe, John H. Roosa, Dale A. Robinson
High-pressure liquid chromatography (HPLC) is ubiquitous in pharmaceutical development, commercialization and supply, yet its use by process analytical technology
(PAT) on shop floors is rarely reported, partly due to equipment reliability, particularly
sampling system reliability. In the past two years, we have embarked on a journey
to design and implement a sampling system so as to be able to leverage a Dionex
Process HPLC unit. The system has received systematic design review and testing
in Merck’s Rahway Pilot Plants. In this presentation, I’ll review the principles behinds
the design of the sampling system, share testing results from our laboratory and
examples of on-line analysis from our Pilot Plants.
262
Application of iChemExplorer for Semi-Automatic Solution Stress
Testing
Fenghe Qiu, Boehringer Ingelheim, 900 Ridgebury Rd., Ridgefield CT
06877, Lily Du, Ashish Soman, Chan Li
Solution stress testing is an integral part of pharmaceutical stress testing for the
purposes of identifying the likely degradation products, establishing the degradation
pathways and the intrinsic stability of the drug molecule, and validating the stability
indicating power of the analytical procedures. Solution stress testing involves multiple conditions and multiple pulling, therefore, can be very tedious and labor intensive if done manually. Although complete or semi-automation (e.g., ReactArray) is
possible, due to the complexity of the hardware and software, it requires significant
capital investment and often needs dedicated personnel. iChemExplorer is a much
simpler solution to semi-automate solution stress testing. It is a simple add-on to
any Agilent HPLC to provide programmable temperature control and stir. The data
can either be processed with the high-pressure liquid chromatography (HPLC) software (ChemStation or Empower) or the iChem software. The latter can also provide
graphical display of the degradation and calculations on the degradation kinetics,
e.g., activation energy. Boehringer Ingelheim has evaluated these techniques for
two years and now has fully implemented it in routine drug substance and product
solution stress testing. For drug product application, we combined iChemExplorer
with filter vials to avoid manual filtering of potential precipitation of formulation components. This presentation provides the evaluation results and some case studies. A
cost benefit comparison for the use of iChemExlorer vs. the conventional approach
is also discussed.
266
pH Stability Prediction of a Pharmaceutical Formulation Using a
Chemometric Hard Modeling Approach
Husheng Yang, AstraZeneca, 587 Old Baltimore Pike, Wilmington, DE
19702, Kirk A. Lewis, Susan E. Pegg
In a chemometric hard modeling approach, experimental data are fitted to known
mathematical equations to obtain a prediction model. In this work, we applied this
approach to predict the pH stability of a liquid pharmaceutical formulation whose
shelf-life was dependent on this critical quality attribute. A graphical user interface
was also developed to allow the model to be used routinely as a general software
package. The prediction model is based on the acid-base equilibrium which is
well understood in chemistry. There was reasonable understanding about the degradants of the active pharmaceutical ingredient in the formulation. However, not all
the parameters and degradants were known for the formulation and the available
experimental data covered only 10% of the desired shelf-life. A general model that
incorporated multiple poly protic acids and multiple acidic degradants was first developed. Unknown parameters were then determined through simulation using the
general model. Chemistry knowledge was used to guide the simulation process.
The final model was applied to predict the pH stability of proposed commercial formulations before experimental data became available.
263
Investigation into the Role of Polymer Chemistry, Temperature and
Humidity on the Degradation of an HPMC-AS Spray Dried
Dispersion
Adele L. Patterson, Bristol-Myers Squibb, Reeds Ln., Moreton, CH46
1QW United Kingdom, Elizabeth Banks
The increasing use of enabling technologies, i.e., spray dried dispersions, hot melt
extrudates and co-processed products, to deliver drugs with challenging physicochemical properties, e.g., poor aqueous solubility, has resulted in a number of
new challenges for analysts. One example is the increase in chemical degradation seen for a generally stable active pharmaceutical ingredient (API) when spray
dried with the polymer hydroxypropylmethylcellulose acetate succinate (HPMC-AS).
The spray dried dispersion showed increased chemical degradation relative to the
API, at elevated temperatures and humidity and perhaps more interestingly different
grades of this polymer showed differing degrees of degradation. A design of experiment (DOE) study was conducted to evaluate the effects of a range of temperature
and humidity combinations and the data generated was analyzed in a number of
ways to gain understanding of the degradation kinetics with respect to the individual
storage parameters as well as their combined impact. Tools used include the Arrhenius equation, amended Arrhenius equation and multi-linear regression. Concurrent
267
The Characterization of Bulk Powder Properties for Pharmaceutical
Manufacture: The Need for a Multivariate Approach
Tim C. Freeman, Freeman Technology, 1 Miller Court, Severn Dr.,
Tewkesbury, GL20 8DN United Kingdom, Mike Delancy, John Yin, Brian
Armstrong
As oral drug delivery is still the preferred vehicle for administration of active pharmaceutical ingredients, either as tablet/capsule or in the form of an inhalable powder,
the development of quality-by-design strategies for powder processing must be a
priority. Production of any of these dosage forms relies on the flow properties of the
powder formulations – they must be well-matched to their processing environment
and their delivery system. However, such a requirement demands a thorough understanding of the nature and behavior of the powders themselves. It is entirely inadequate to suggest that a powder’s characteristics can be represented by a single
number from a single test. Therefore knowing how a powdered material behaves
over a range of stress conditions – when stationary, in motion or about to move – is
42
2012 EAS Abstracts
November 2012
vital for designing and running pharmaceutical manufacturing processes, especially
in the light of the published guidance. This presentation demonstrates how recent
advances in powder characterization allow the measurement of a range of powder
properties which can predict material behavior during processing and allow optimal
equipment settings for powder, process, and delivery system compatibility. This is
illustrated by case studies where knowledge of powder characteristics can assist
with the understanding of process behavior in mixing, hopper flow, and die filling
applications. Furthermore, it also demonstrates how this information can then be
fed back in at the development stage to provide process ability specifications which
generate valid quality-by-design.
according to charge, mass and hydrophobicity, respectively. Future plans involve
pushing this technology to new heights and with the founding of a new consortium
for top-down proteomics, we anticipate significant acceleration in pace of development for mass spectrometry-based proteomics using intact protein isoforms (or
“proteoforms”) as the primary unit of measurement.
271
Nanospray Enabled Mass Spectrometry: A Journey from Ultra-Low
Flow to Ultra-High Throughput
Gary Valaskovic, New Objective, 2 Constitution Way, Woburn, MA 01801
Nanospray technology has historically existed in two very distinct, almost unrelated,
forms. Off-line, or static nanospray, is characterized by ultra-low flow rates (< 20 nL/
min). There is typically no active pumping other than electrostatic attraction between
source and emitter. Actual flow rates are often a best-guess estimate. Unfortunately
much of the significant analytical benefit of static nanospray (efficiency, ionization
response, matrix effect reduction) is lost even at “modest” low flow rates of 100-200
nL/min. Sample throughput is often very poor and flow control non-existent. The
second implementation of nanospray, on-line nanospray, is combined with nanobore liquid chromatography (LC). It is characterized by controlled and pumped flow
rates of 200-1000 nL/min. On-line nanospray enables highly complex mixture analysis, and reasonable throughput through a traditional, though limited, LC approach.
Unfortunately, due to the higher flow rates, it offers a small fraction of the ionization
benefit of static nanospray. Analytically, the analyst would benefit greatly from a new
methodology for nanospray combining these two worlds. Examples for combination
of these two methods through flow control, spray control, and segmented flows are
presented. This enables a truly high throughput, ultra-low flow nanospray workflow
suitable for many areas of chemical analysis by mass spectrometry.
268
Chromatographic Design of Experiments
Jeffry R. Trevenen, Teva Pharmaceuticals, 223 Quaker Rd., Pomona,
NY 10970
Quality-by-design has become an increasingly important aspect to both manufacturing of pharmaceuticals and development of analytical methods. Design of experiments, along subsequent statistical analysis, are used together as tools to determine the overall robustness and variability of an analytical test method. Recently,
a case study was performed on a raw material to evaluate the influence of method
factors on the amount of residual solvents present in the raw material. Since multiple
critical method factors were determined to be significant, a Placket Burnam design
of experiments was utilized and the method was broken down into two separate
experimental designs; one for the headspace parameters and one for the chromatographic conditions. A failure mode effect analysis (FMEA) was performed on
the analytical method and multiple critical method factors were determined. The
effects of slight changes in vial equilibration time, oven temperature, vial pressure,
loop temperature, and transfer line temperature were studied in a statistical design
and the impact on the critical method attributes (responses) were determined for
the headspace parameters. The effects of slight changes in inlet temperature, oven
temperature, flow rate, and gradient slope were determined for the chromatographic
parameters. Jump software was used to evaluate the robustness and establish a
working design space to determine the optimal conditions of the analytical method.
272
Mass Spectrometry, Growing Up in Pharmaceuticals and
Biotechnology
Edward Chait, Life Sciences Consulting, 22015 Turner, Chapel Hill, NC
27517
As a pioneer in organic mass spectrometry, Fred McLafferty had a keen vision of
what mass spectrometry would be like and its influence on analytical chemistry
almost 50 years hence. He imparted that vision to his students. That vision has
now been realized in both exotic and routine applications of mass spectrometry in
pharmaceuticals and biotechnology. These analytical applications are discussed in
fields as diverse as pharmacology applied to clinical trials and tissue imaging with
the promise of mass spectrometry in the surgical suite. Overall these examples are
interpreted in light of the contributions Professor McLafferty has made to the progress and future of mass spectrometry.
269
Advances in Automating Solid-Phase Extraction for UCMR3:
Hormones and 1, 4-Dioxane
Michael Ebitson, Horizon Technology, 45 Northwestern Dr., Salem, NH
03079
Currently the United States Environmental Protection Agency (EPA) is proposing
monitoring for the third Unregulated Contaminant Monitoring Regulation (UCMR3).
This action, taken every five years, establishes a program for no more than 30 contaminants in drinking water. Laboratories that have been approved for this program
by the EPA by passing their proficiency testing can take place in this program from
2013-2015. The objective of this project is to create two automated solid-phase
extraction (SPE) procedures that are fast and provide consistent recoveries within their respective recovery acceptance criteria’s. This research project focuses
on seven hormones (17-estradiol, 17-ethynylestradiol (ethinyl estradiol), 16-hydroxyestradiol (estriol), equilin, estrone, testosterone and androstene-3,17-dione)
based off of method 539 and one compound (1,4-dioxane) from method 522. Both
methods have different SPE and analysis procedures. Method 539 is based off of 47
mm SPE disks and requires analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and 522 is based off or cartridges
and requires analysis by gas chromatography by mass spectrometry with selected
ion monitoring (GC-MS by SIM). This paper explains new developments and procedures in automated SPE technology, methodology and chemistry. These new developments not only provide a solution for processing drinking water samples with two
different automated SPE extractors, but in fact provide an increase in throughput,
recoveries and consistency over manual SPE procedures.
273
Protein Conformational Evolution from Solution to Gas Phase:
Unfolding and New Folding Pathways in Native Ubiquitin after
Electrospray Ionization
Fred W. McLafferty, Cornell University, Baker Lab, Ithaca, NY 14853,
Owen S. Skinner, Kathrin Breuker
Electrospray ionization (ESI) of a singular protein tertiary structure in solution can
produce ions of many charges, each of which can exist as multiple conformers. In
contrast to accepted knowledge on solution behavior, however, there is no overall
picture, even few structural details, on how gas phase unfolding and folding of the
native conformer can produce these modifications. For transfer into the gas phase
of the extensively-studied native ubiquitin ions, we can now report substantial details of near-complete conformational evolution using a combination of techniques
such as electron capture dissociation (ECD), infrared (IR) or collisional activation
(CA), and Hydrogen–deuterium (H/D) exchange (HDX). ESI of native ubiquitin
yields conformationally unstable 7+ ions; pioneering studies with ion mobility spectroscopy by the Clemmer group have shown that possibly a dozen conformers are
produced that have up to 65% increases in cross section. Our ECD spectra 0.2
and 2 s after ESI are consistent with unfolding in two major processes followed by
immediate formation of two new overlapping salt bridges, Asp-21-Arg-54 and Arg42HOOCGly76. Additionally, both terminal regions become α-helical, consistent with
conclusions of our 2002 ECD studies. CA dissociation of the salt bridges provides
confirmatory evidence of their location; the postulated double overlap region of residues Arg42-Arg54 is supported by the largest increases in H/D exchange after CA.
After IR de-naturation of “mature” ions, refolding is characterized kinetically by ECD
for 0.2 s to 10 min; surprisingly, the earliest (1 s) first-order folding is consistent with
formation of the same two salt bridges observed for the native state 0.2 s after ESI.
270
On the Impact of Top-Down Analytical Strategies in Measurement
Science
Neil L. Kelleher, Northwestern University, Department of Chemistry,
Evanston, IL 60208
Although the traditional proteomics strategy has been peptide-based, unambiguous
characterization of proteins in-vivo is best achieved through the analysis of intact
proteins. While I served as a graduate student under Prof. Fred McLafferty, this
new paradigm for intact protein analysis was born. In my independent career, my
lab has championed the use of top-down proteomics to study complex protein mixtures. Early incarnations of top-down proteomics suffered from low throughput and
incomplete protein characterization. This was attributed to an underdevelopment of
intact protein separations, mass spectrometry instrumentation and bioinformatics
tools. Recently, our lab has developed a comprehensive platform for the large-scale
use of multiplexed two-dimensional separations coupled to capillary liquid chromatography Fourier transform infrared mass spectrometry to achieve unprecedented
proteome coverage. To deal with the complexity of the human proteome, our separation platform implements the techniques of solution information exchange forum,
GELFREE, and capillary- reversed-phase liquid chromatography; which fractionate
274
Super Fluid Chromatography (SFC) for Chiral Method Development
Screening and Analysis
Gregory K. Webster, Abbott Laboratories, 200 Abbott Park Rd., Abbott
Park, IL 60064
With many of the new chemical entities under development in the pharmaceutical industry being chiral, the specific stereochemistry of these substances effects
their biological activity, and commercial viability of the potential new drug. Thus,
enantio-selective separation techniques play a vital role in the development of these
43
2012 EAS Abstracts
November 2012
entities into commercial products. In an attempt to improve upon the efficiency of
chiral method development, column manufacturers and industry scientists have
developed screening procedures to efficiently evaluate various chiral separation
conditions in an unattended mode. This presentation focuses of current method
development and preparative scale strategies in use with SFC. In addition, SFC is
no longer limited solely to chiral purity analysis. With the dynamic improvement in
sensitivity and precision of commercial SFC instrumentation, this presentation also
illustrates how SFC is meeting compendial method validation requirements used for
good manufacturing practice active pharmaceutical ingredient release and stability
testing.
demands of capacity and capability of the forensic laboratory. New technologies and
analytical methods are routinely evaluated for implementation in the laboratory or
the field. The overall counterfeit pharmaceutical threat is discussed with emphasis
given to Merck’s anti-counterfeiting strategy and analytical detection capabilities.
279
Marker Technology used in the Fuel Industry
Duncan McCallien, Tracerco, Pavilion 10, Belasis Hall Technology Park,
Billingham, TS23 4AZ, United Kingdom
Counterfeit and illegally altered fuels present a global problem. Not only do they
lead to a large loss of revenue ($100’s billion) for governments but they contribute to
poorer air quality and fund criminal activity. The problem can be tackled by adding a
tracer molecule to either the genuine fuel or to the most likely adulterant. A number
of analytical technologies can be used to detect the tracer, each with its own pros
and cons. Short sequences of DNA have the potential to be used as fuel tracers as
they offer the possibility of unlimited permutations, parts per trillion addition levels,
unambiguous detection and security from being copied. An objective assessment
of DNA as a fuel tracer was therefore carried out. A selection of non-natural, single
and double stranded, linear and plasmid, DNA sequences was synthesized. They
were tested in a range of fuels and other hydrocarbon liquids over a period of time.
Properties such as plurality, covertness and addition levels were assessed as well
as other critical attributes such as resistance to unauthorized removal (laundering),
long term stability and precision of quantification. Overall the assessment revealed
several critical deficiencies in the use of DNA as a tracer for fuels. These are discussed and comparisons made with more robust technologies.
275
An Overview of Supercritical Fluid Chromatography Mass
Spectrometry (SFC-MS) in the Pharmaceutical Industry
Laila Kott, Millennium Pharmaceuticals, 40 Landsdowne St., Cambridge
MA 02139
Although SFC-MS has been around since the late 1980’s, it has not become as
prevalent as liquid chromatography mass spectrometry (LC-MS) in the pharmaceutical industry because, until recently, most of the work was in the form of preparation SFC for isolating compounds (usually chiral). However, with more sensitive
instruments coming online, the analytical interest in SFC has reawakened and along
with that has come an increase in SFC-MS activities. This talk describes the progression of SFC-MS technology, the types of interfaces, back pressure regulators
and analyzers that have been coupled to SFC. Some instrumental parameters are
discussed, such as what interface or backpressure regulator to use depending on
the analysis. An overview of chiral SFC-MS and prep SFC-MS in the pharmaceutical
industry is presented.
280
Molecular Tracking: Using Naturally Occurring Stable Isotopes to
Flag Counterfeits
Iain Green, Picarro, 3105 Patrick Henry Dr., Santa Clara, CA 95054,
Nabil Saad
Stable isotopes provide important information about the authenticity, origin and contamination of the raw materials and final products in the chemical, food and pharmaceutical industries. The amount of isotopes such as carbon-13, hydrogen-2 (deuterium) and oxygen-18 in any plant, compared to their more common alternatives
(carbon-12, hydrogen-1 and oxygen-16) is related to plant species (and variety),
growing region and growing conditions. This chemical record constitutes Nature’s
Barcode and can be used as a profile of any chemical derived from a plant, including those derived from eons-old vegetation long since converted to oil, coal or gas.
Chemical processes can change the signature from the raw plant, but the chemicals
used carry their own signatures and the end product still has a distinct isotopic profile. These profiles can be used to distinguish fraudulent activities such as economic
adulteration, transshipment, dilution and other supply chain integrity problems. Cavity ring-down spectroscopy (CRDS) is a high precision, high sensitivity technique
that enables bench-top, fast and easy stable isotope measurements. The key to this
optical technique is that it is time-based; which provides the raw performance characteristics for demanding stable isotope measurements. The availability of CRDS is
revolutionizing stable isotope analyses; it is an instrument that can be used by any
analytical lab in the world. Samples can be run without any chemical processing in
as little as 10-15 minutes. In this paper we describe the technology and show data
describing the breadth of potential applications.
276
Design, Development, and Analysis of a Novel Series of Stationary
Phases for SFC
Jeffrey W. Caldwell, Princeton Chromatography, 1206 Cranbury South
River Rd., Cranbury, NJ 08152, Walton B. Caldwell
Success in achiral SFC is highly dependent on stationary phase choice for a particular application or class of compounds. Often very subtle changes in the structure
of the stationary phase are reflected in the resulting chromatography. We have created a series of closely related novel stationary phases for use in achiral SFC. We
examine closely the relationship of these subtle phase changes to selectivity and
retention for several groups of compounds.
277
From Drug Discovery to Drug Development and Commercialization:
The Evolving Role of SFC in Pharmaceutical Analysis
Zhenyu Wang, Merck, 556 Morris Ave., Summit, NJ 07901, Honggen
Zhang, Oscar Liu, Brent Donovan
Supercritical fluid chromatography (SFC) has to date been very successful in pharmaceutical industries. So much so that it dominates chiral separation and purification. It provides several advantages over high-pressure liquid chromatography
(HPLC) in terms of resolution, throughput, and reduced consumption of hazardous/expensive solvents. Despite its increasing popularity, most implementations of
SFC are still in drug discovery, where the applications are generally qualitative and
semi-quantitative. SFC is seldom seen in highly regulated environments, such as
drug development and quality control (QC) labs where the analytical activities and
requirements are quite different. Recent efforts have been made by both instrument
manufacturers and users to set the stage for implementation of SFC into these regulated environments. In this study, newly developed SFC method for the separation
and quantitation of mometasone furoate and its trace impurities demonstrate the
potential and even advantage of SFC in impurity analysis, one of the most common
and difficult analytical tasks in drug development and QC labs. The SFC method
provides an orthogonal selectivity complementary to the reversed phase HPLC (RPHPLC) method. All of the impurities and the active were baseline separated using
less than one third of the RP-HPLC method run time. This study demonstrated that
the SFC method, with improved sensitivity, can be a valuable chromatographic tool
in drug development and QC labs.
281
Big & Small; Inside & Out; Long & Short Sample Analyses for
Forensic and Anti-Counterfeit Identification
Frank Weston, Agilent Technologies, 2850 Centerville Rd., Wilmington,
DE 19808
Big & small; inside & out; long & short sample analyses presents the use of modern
interferometer based spectroscopy techniques for forensic & anti-counterfeit sample identification. This presentation demonstrates, using real-world examples, how
state-of-the-art Fourier transform infrared (FTIR) hardware and a variety of sampling
accessories are used for modern day identification of potential counterfeit species
ranging from chemical and biological to pharmaceutical in origin. Techniques discussed include macro and micro samples, surface and depth profiling, UV-visible
through Far-IR regions of the spectrum, and cover both in-lab and out-of-lab FTIR
spectrometers keeping the lab bench high-end researcher to the field technician
users in mind.
278
Pharmaceutical Anti-Counterfeiting Strategies and Technologies –
An Overview
Richard Steinbeiser II, Merck, 770 Sumneytown Pike, West Point, PA
19486, Anthony Zook
Counterfeit pharmaceutical products continue to be a global threat to public health.
Merck combats counterfeit pharmaceuticals through ever evolving strategies and
uses of advanced and emerging technologies. Merck strives to provide a best-inclass strategy to protect our patients from the threat of counterfeit medicines. Our
anti-counterfeiting strategy is intelligence-based and very proactive. We actively
seek the primary offenders who are having the greatest negative impact on the safety of our patients. We are positioned to provide investigative and analytical support
whenever requested. Successful anti-counterfeiting strategies require conclusive
laboratory analyses of the suspected counterfeit products. The number and complexity of suspect counterfeit products continuously increases, placing increased
282
Skip Palenik – Consummate Microscopist
Peter R. De Forest, Forensic Consultants, PO BOX 141, Ardsley, NY
10502
I first met Skip Palenik in 1977 when we were both asked to co-teach a course on
polarized light microscopy for the New York Microscopical Society (NYMS). This
was at the time of the NYMS Centennial. Although I had never met Skip before, I
knew him by reputation. I welcomed the opportunity to teach with him. Following our
experience teaching together, we became very good friends. It seems like a long
time ago. Then, and over the years, I observed that Skip is a great teacher. In addition to effectively conveying a wealth of knowledge, he imbues his students with
enthusiasm for the subject. Skip worked for McCrone Associates for many years
44
2012 EAS Abstracts
November 2012
doing microscopy with industrial and forensic casework samples. He then struck out
on his own and established Microtrace. This has been a great success from several
perspectives. Skip has hired and trained two of my former graduate students. They
have learned a great deal and enjoyed working for him. Skip brings three things
to his casework. The first is an encyclopedic knowledge of microscopic objects or
microscopic particles resulting from the division of larger objects. Over the course
of his career he has amassed a huge collection of reference materials. His study
of these has contributed to the encyclopedic knowledge he possesses. The second
factor is his broad knowledge and skill with an impressive array of microscopical
techniques. Third, he is very adept at scientific reasoning and problem-solving. This
presentation attempts to capture and illustrate something of the essence of this
consummate scientist.
the room, hits a wall, turns and takes off into another direction. Hitting a wall is just
a way of defining a boundary, change direction but never give up. It is resilient. It
relentlessly crisscrosses the room in an unpredictable pattern until every piece of
dirt and dust are picked up. The job is accomplished, but success is not a straight
line. I can press the button and leave for work, knowing the work will be done when
I return, because it does the right thing, even when nobody is watching. It returns to
its docking station when its batteries are running low, always making it back before
the batteries run out. It manages its energy, not its time. But most interesting, is what
we can learn about developing and enhancing our own creativity by watching and
learning from a robotic vacuum cleaner.
287
Using Your Emotional Intelligence at Work
Mimi Sherlock, Sherlock Creative Thinking, 414 Corlies Ave., Allenhurst,
NJ 07711
IQ only gets you so far. Emotional intelligence, commonly referred to as “EQ” is the
other kind of smart... the one most important predictor of personal and professional
success for individuals and the organizations they serve. A wide body of independent research has proven it. Emotionally intelligent people communicate effectively,
form healthy relationships, and create powerful coping strategies; emotionally unintelligent people don’t - no matter how high their IQs. As in many demanding jobs,
we can encounter a stressful work environment with many high-stake challenges,
a fast pace, and complex relationships. In the face of these pressures, professionals must carefully manage their reactions and interactions (within themselves, with
each other, and with customers) to achieve optimal work satisfaction and outcomes.
Emotional intelligence is a key contributor to this dance of managing competing
pressures, and an invaluable asset for professionals. Research shows that emotional intelligence is positively related to the ability to be innovative and creative in the
workplace. Strategies are available which can impact positively on creativity and innovation. EQ can be substantially strengthened and developed. With simple awareness and training, your EQ can improve considerably. Those who lack it can acquire
it. Those who have it can enhance it. The benefits of developing an increased level
of emotional awareness and understanding will pay off in all aspects of our lives.
You can perform better, enjoy less stress, work in a more effective and creative
environment, be happier and more successful and so will the people around you.
283
Rapid Characterization of Forensic Paint Samples by Raman
Microspectroscopy
Christopher S. Palenik, Microtrace, 790 Fletcher Dr., Suite 106, Elgin,
IL 60123
Raman microspectroscopy is the first analytical technique that provides a practical means to definitively identify a large number of pigments, in-situ, in a relatively
short amount of time. This presentation provides highlights of the past six years of
research conducted at our laboratory in this area and how Raman spectroscopy can
be effectively utilized for: a) quick, quantitative screening of forensic paint samples,
b) to improve the value of a traditional paint examination through the identification
of major and minor pigments in a sample, and c) to provide investigative leads by
specifically identifying pigments which can be related to a particular manufacturer.
Applications to automotive, architectural and artistic paint samples are discussed.
284
Botany and Mycology with the Light Microscope: Exciting Weapons
in the Forensic Armoury
Patricia E.J. Wiltshire, University of Aberdeen, Department of Geography
and Environment, Elphistone Rd., Aberdeen, AB24 3UF United Kingdom
The light microscope is pivotal in helping police investigations into criminal activities ranging from rape and abduction to mass murder. It is one of the most valuable tools available to the forensic scientist. The ubiquity, but specificity in habitat
requirements of organisms, means that the patterning of distribution of fragmented
structures, pollen grains, or spores, will vary from location to location. Thus, it is
possible to differentiate one place from another by analysis of the assemblages and
profiles of proxy indicators of plants, animals, fungi, and even, on occasion, certain
bacteria. This makes them invaluable forensic indicators. However, for them to be
useful in criminal investigation, the practitioner must have a great deal of knowledge
and experience of the taphonomic factors affecting their distribution throughout the
environment. Such taphonomic factors are complex, and variable according to circumstances, so any anyone involved in this field requires substantial field as well as
laboratory experience. Animals, plants, fungi and other microbes have all helped in:
establishing post mortem interval and cause of death; differentiating kill sites from
deposition sites; demonstrating contact between offenders and locations; eliminating irrelevant locations; locating clandestine burial or deposition; establishing the
truth of testimony; and predicting the movement and origins of drugs, people, and
objects. Examples are presented from recent criminal cases. The light microscope
is an invaluable tool in the forensic toolbox.
288
Innovative Approaches to Staffing the Lab in Challenging Times
Mary Schwans, Astrix Technology Group, 123 S. Broad St., Suite 1945,
Philadelphia, PA 19109
Our economy is moving quickly and we are continually facing challenges in staffing
laboratories between workforce reductions, aging staff, and leaner budgets. Lab
Managers and Leaders are learning to be more than just innovators in the lab, but
also in their staffing needs. In this presentation, the author focuses on four approaches to staffing those needs: staff augmentation, subject matter experts, outsourcing and empowerment of existing staff.
289
The Importance of Support Labs in Innovation and Intellectual
Property Activities
Richard R. Durand, Jr., Sun Chemical Corporation, 631 Central Ave.,
Carlstadt, NJ 07072
There are many ways that support laboratories can contribute to intellectual property and innovation. However, support teams are frequently asked only to provide
data to development teams which will be used in framing potential patent protection
for new products. For many of these situations, the analyst may not be an intimate
part of the inventive process. There are several other scenarios where the support
lab may be in the position to lead the quest to define and protect innovative concepts
through various measurements that can be made within their laboratories. Three different examples in which the support team was the key to developing the inventive
concept are outlined. These scenarios illustrate opportunities which may present
themselves for integrated participation in the innovation process.
285
Microscopic Trace Evidence – The Overlooked Clue
Skip Palenik, Microtrace, 790 Fletcher Dr., Suite 106, Elgin, IL 60123
Because microscopic traces are too small to see and too difficult to guard against,
they have provided valuable evidence in criminal investigations since they were first
exploited by pioneering forensic scientists in the first half of the Nineteenth Century.
The key to the study and understanding of microscopic evidence is the microscope
and the author has been fortunate in being able to apply the microscope and microscopical methods to the investigation of a wide variety of crimes over nearly 40
years. The influences of Edmond Locard, Walter McCrone and Max Frei-Sulzer and
well as the fictional scientific sleuth John Thorndyke is recognized as the audience
is taken through a series of cases from the presenter’s casebook that includes the
Atlanta Child Murders, the Narita airport bombings, Jon Benet Ramsey case, Green
River Murders and others. The emphasis is on the microscope and the clues it revealed in solving (or in some cases attempting to solve) these mysteries.
290
Analysis of Bisbenzylisoquinoline Alkaloids by LC-SPE-NMR
Robert Krull, Bruker-BioSpin, 15 Fortune Dr., Billerica, MA 01821,
Kenneth Johansen
In recent years, the use of liquid chromatography solid-phase extraction nuclear
magnetic resonance (LC-SPE-NMR) has become an increasingly popular technique
for the isolation of pharmaceutical compounds and natural products. Alkaloids are
an interesting class of compounds in both of these areas. Due to the inherently basic nature of alkaloids, often times the “traditional” methods of compound trapping
by LC-SPE-NMR are less than optimal. Improved methodology for the trapping of
these types of compounds is presented.
286
Everything I Learned About Leadership, I Learned From My
Vacuum Cleaner
Stephanie Olexa, Lead to the Future, 2425 Butter Creek Rd.,
Quakertown, PA 18951
A few years ago, I bought a robotic vacuum cleaner. It has worked tirelessly to keep
the cat fur contained and minimize the sneezing from my allergy prone husband.
But, I’m also amazed at how much it knows about success in business and in life. It
sits patiently in a corner, linked to its docking station ready to jump into action. It is
always ready to take on a new challenge. With the press of a button, it zooms across
291
Metabolite ID: A Synergistic Journey to Structure Elucidation
Chad Hadden, Eli Lilly, Lilly Corporate Center, Indianapolis, IN 46285
The modern mass spectrometer offers superior sensitivity and accurate mass data
with impeccable precision that have afforded it the primary method for metabolite
45
2012 EAS Abstracts
November 2012
identification. When coupled with liquid chromatography, the acquisition of fragmentation data, and some mechanistic insight, it has proven to be a very powerful technique for the understanding of major circulating entities and metabolic pathways.
Unfortunately, metabolites don’t always fragment in ways which provide unambiguous structures. This approach can often fail to provide definitive structures, and assigned structures may be at a confidence limit far less than 100%. Nuclear magnetic
resonance (NMR) spectroscopy can afford a more definitive structural assignment,
but it requires considerably more material and greater purity. In the course of our
work we have encountered a variety of compounds that yielded unexpected metabolites, or were inconsistent with the initially mass spectrometry (MS)-proposed
structure. Several examples are discussed that highlight the value of utilizing NMR
in conjunction with MS data for metabolite structure identification.
The specific vapor is an indicator of a leak from a buried pipeline transporting liquid
hydrocarbons such as gasoline. Previously, we developed a handheld batterypowered near-infrared (NIR) standoff tunable diode laser absorption spectrometer
(s-TDLAS) platform based on telecom-style NIR InGaAs DFB lasers. A commercial
methane detector, commonly utilized for natural gas pipeline leak surveying, utilizes
this platform. We are now adapting a first-of-its-kind room temperature DFB-ICL to a
similar platform. The ICL produces several tens of milliwatts at about 3.4-µm. Using
the wavelength modulation spectroscopy (WMS) technique, the s-TDLAS/ICL platform detects gasoline constituent vapor plumes having path-integrated concentrations ~10 ppm-m from 30 m standoff distance.
296
High-Resolution Infrared Spectroscopic Imaging: Theory and
Applications
Rohit Bhargava, University of Illinois-Urbana-Champaign, 405
N. Mathews Ave., Urbana, IL 61801, Paul Carney, Rohith Reddy,
Matthew Gelber, Kevin Yeh, Thomas van Dijk, Matthew Kole, Matthew
Schulmerich
Infrared (IR) spectroscopic imaging was widely believed to be a simple combination
of spectroscopy and optical microscopy. Our recent theoretical work has demonstrated that this is not the case. There is a non-trivial coupling of the structure and
spectral content of a sample. Understanding the data obtained in IR spectroscopic
imaging is considerably more complicated than previously believed and presents a
rich area of investigation. Here, we report on the extension of a scalar wave theory
model for IR imaging to understand complex samples in a variety of microscopy
configurations. We predict the performance of microscopy systems and resulting
images from first principles, resulting in several new insights. Microscope configurations based on these predictions were set up and performance was found to agree
well with predicted response. We then extended the work to a variety of samples,
optical configurations and sources, leading to the development of instruments with
new capability. Specifically, we show how unprecedented spatial resolution can be
obtained and describe the fundamental limits of information extraction based on
rigorous theory. Finally, we demonstrate the experimental realization of the theory
and simulation to demonstrate the recording and subsequent reconstruction of accurate spectral data.
292
LC-NMR: Applications, Permutations, and Methodology
Paul A. Keifer, Agilent Technologies, 5301 Stevens Creek Blvd., Santa
Clara, CA 95051
Liquid chromatography nuclear magnetic resonance (LC-NMR) and LC-NMR-mass
spectrometry are very powerful analytical techniques for resolving complex mixtures. We discuss the techniques that are available, the applications for which they
can be used, and the limitations of the methodology.
293
Investigation of Drug Metabolism Using a Multinuclear NMR
Approach
Igor Goljer, GlaxoSmithKline, 709 Swedeland Rd., PO Box 1539, King
of Prussia, PA 19406
An essential part of the drug development process is to gather information about a
drug candidate’s absorption, distribution, metabolism and excretion properties. This
information includes the identification and quantification of drug related material
(DRM) in circulation, tissues and excreta typically via the use of radio labeled material. However, given the high attrition rates of drug candidates, the choice to delay
the radiolabel synthesis until later in drug development process represents a viable
cost savings. This talk describes how 1H and 19F NMR were used to identify and
quantify DRM in biological samples using non-radiolabeled drug candidates at sub
microgram levels using cryogenically cooled nuclear magnetic resonance (NMR)
probes. Furthermore, this talk describes various multinuclear NMR techniques (e.g.,
19
F, 13C and 15N) which yield the definitive structure of DRM components on a microgram scale. This structural information is critically important to the identification
of a drug candidate’s metabolic fate and determination of nonclinical toxicological
coverage.
297
Developments in In-Situ Industrial Raman Analysis
Ian R. Lewis, Kaiser Optical Systems, 371 Parkland Plaza, Ann Arbor,
MI 48103, Kevin L. Davis, David J. Strachan, Sean J. Gilliam, Maryann
Cuellar, Pat Wiegand, Joseph B. Slater
Over the last 20 years developments in instrumentation for practical Raman spectroscopy have led to successful industrial applications of Raman spectroscopy. The
text-book benefits offered by Raman spectroscopy include the specificity of Raman
measurements the non-destructive nature of the measurement, the quantitative capability from sub-percent levels to 100%, the low-Raman cross-section of water, the
ability to measure samples remotely and in-situ, the ability to measure spectra from
materials in different physical states, etc. While the demonstration of new Raman
applications remains important to the understanding of the wider applicability of Raman spectroscopy for industrial analysis a critical element is often overlooked – appropriate sampling. Burdensome sampling approaches requiring either extensive
sample work-up or extractive sampling can render the most promising laboratory
analytical methods inappropriate for real-time industrial process sampling. Each
sample’s chemical phase presents its own unique challenges and thus different
approaches are required to optimized in-situ instrumentation to ensure robust Raman spectral analysis. In this presentation new development in probe technology,
micro-sampling, and alterative excitation wavelengths are given. The presentation
includes a selection of applications, the challenges associated with those applications, and the solutions developed for several different industrial market segments.
294
Identifying Pathological Minerals by Raman Spectroscopy as an
Early Sign of Bone Infections
Karen Esmonde-White, University of Michigan, Chemistry Department,
930 N. University, Ann Arbor, MI 48109, Mekhala Raghavan, Francis
Esmonde-White, Crystal Holmes, Michael Morris, Blake Roessler
We present research in Raman spectroscopy for diagnosing bone infections in
non-healing, infected diabetic foot ulcers (also called diabetic osteomyelitis). Bone
specimens were collected from patients with diabetic osteomyelitis requiring either a
biopsy or amputation. Spectra were collected across the surface of bone fragments
with a near-infrared Raman microspectroscopy instrument. Environmental scanning
electron microscopy (ESEM) of the bone samples was performed to visualize biofilm
formation and re-precipitated mineral crystals. Energy dispersive X-ray (EDX) spectroscopy provided elemental analysis of mineral crystals. An unexpected finding was
Raman spectral patterns corresponding to brushite, an acidic calcium phosphate
mineral. This pathological mineral was found in some osteomyelitic specimens, but
not in the controls. ESEM identified bacterial biofilms and recrystallized mineral in
bone specimens where Raman identified brushite. EDX spectroscopy of recrystallized mineral confirmed the presence of brushite and also identified a hypercalcified mineral. Formation of brushite was coincident with bacterial biofilm formation.
Our work suggests that bacteria directly form bio films on bone and creates an
acidic environment favorable to the formation of brushite. Because this mechanism
of bone destruction is unlike the mechanism observed in osteoporosis or skeletal
cancer metastasis, we propose brushite as a spectroscopic marker unique to bone
infections. We also present preliminary results of bone fragments examined by a
prototype clinical fiber-optic probe.
298
Characterization of Particle Size Standard NIST 1019b with
Synchrotron X-Ray Microtomography and Digital Data Extraction
Jon M. Friedrich, Fordham University, Department of Chemistry, 441
East Fordham Rd., Bronx, NY 10458, Mark Rivers, Michael Perlowitz,
Zachary Meinhart, Vanessa Ramirez
We show that synchrotron X-ray microtomography (µCT) followed by digital data
extraction can be used to examine the size distribution and particle morphologies
of the polydisperse (750 to 2450-µm diameter) particle size standard NIST 1019b.
Our size distribution results are within errors of certified values with data collected
at 19.5-µm/voxel. One of the advantages of using µCT to investigate the particles
examined here is that the morphology of the glass beads can be directly examined.
We use the shape metrics aspect ratio and sphericity to examine of individual standard beads morphologies as a function of spherical equivalent diameters. We find
that the majority of standard beads possess near-spherical aspect ratios and sphericities, but deviations are present at the lower end of the size range. The majority
(>98%) of particles also possess an equant form when examined using a common measure of equi-dimensionality. Although the NIST 1019b standard consists
295
Mid-IR TDLAS for Standoff Detection of Leaks from Liquid
Hydrocarbon Pipelines
Mark A. Druy, Physical Sciences Inc., 20 New England Business Center,
Andover, MA 01810, Richard T. Wannier, Mark G. Allen, Mickey B. Frish,
Joel M. Hensley
The recent advent of distributed feedback inter band cascade lasers (DFB-ICL)
providing wavelengths in the mid-infrared hydrocarbon fingerprint spectral region
(3-5-µm) and operating at or above room temperature enables novel compact
spectrometers for trace gas sensing applications. In this paper, we describe the
application of ICL technology to standoff detection of a specific hydrocarbon vapor.
46
2012 EAS Abstracts
November 2012
302
of loose particles, we point out that an advantage of µCT is that coherent materials
comprised of particles can be examined without disaggregation.
Unique
NMR-Based
Approach
to
the
Analysis
of
Radiopharmaceuticals
David J. Schenk, Merck Sharp and Dohme Corp, 126 E Lincoln Ave.,
Rahway, NJ 07065, Mary M. Senior, Peter G. Dormer, Robert A.
Reamer, David Hesk, Roy Helmy
Labeled compounds, such as radiotracers containing tritium, carbon-14 or sulfur-35
and stable isotopically labeled compounds containing deuterium, carbon-13 and
nitrogen-15, are used across the stages of drug development to identify biological
targets, understand preclinical species and human metabolism (PK/PD), determine
environmental impact, and to calibrate parent concentration in clinical samples. At
Merck, tritium labeling represents ~60% of the radiolabeled compounds (~100/yr)
provided due to the direct synthetic approach afforded by tritium dehalogenation,
tritium reductions and catalytic tritium exchange reactions. Currently, radiotracer
analysis is completed on four major platforms: radio high-pressure liquid chromatography (HPLC) for radiochemical purity, automated dilution and scintillation counting
for the measurement of radioactivity per volume (mCi/mL), LC mass spectrometry
analysis for specific activity (the distribution of labeled compound species and nonradiolabeled species), and tritium nuclear magnetic resonance (NMR) for tritium
atom location. Each of these assays is well developed, reliable and efficient. Nevertheless, some compounds do not readily fall within a standard assay approach,
due to inherent chemical or physical properties. Examples of platform issues are
where compounds are not stable or readily soluble in the platform diluent, or are not
ionized under standard MS conditions. Presented here is a new approach to provide all typical tritium-compound analytical data on a single instrument. This method
relies on the accurate calibration of the proton and tritium NMR channel intensities
and provides radiochemical purity, radioactive concentration, specific activity, tritium
atom location and chemical concentration. This method may be used alone or as a
confirming assay.
299
Showing His True Colors: Höroldt’s Enamel Recipes vs. Those of
18th and 19th Century Independent Decorators as Studied by
X-Ray Fluorescence
Nicholas Zumbulyadis, 2 Seneca Pkwy., Rochester, NY 14613, Jennifer
L. Mass, Aniko Bezur, Franesca Casadio, Kelly Domoney, Andrew
Shortland, Katherine Eremin, Lynn Lee
In the years after his arrival at the Meissen Porcelain Manufactory in 1720, Johann
Gregorius Höroldt developed a palette of brilliant colors for the decoration of Europe’s first hard paste porcelain. His detailed recipes, however, were not recorded
until the end of 1731 in a hand-written parchment volume titled “True and correct description of the enamel colorants I have invented with the help of God that are in use
in the Royal Porcelain Manufactory similarly the Gold and Silver and how it should
be treated.” This document serves as the main literary source for the elemental
and chemical composition of polychrome decoration on early Meissen porcelain.
At about the same time independent decorators, known as Hausmaler, began developing their own palettes and styles. While the biographies of most Hausmaler
are known in considerable detail, very little is known about F. J. Ferner. He and his
workshop appear to have been active ca. 1730-1770 and quite prolific. The Ferner
school is characterized by a very distinct style of painting over glaze motifs totally
disjointed from the under glaze blue decoration that until now was considered sufficient evidence for a high confidence attribution of authorship. In this paper we
report X-ray fluorescence on eight objects attributed to Ferner to gain insights into
the enameling techniques of this enigmatic Hausmaler. Our studies reveal subtle differences between Höroldt and Ferner in reproducing complex colors like black and
brown. We demonstrate for the first time that Ferner’s idiosyncratic compositions
have found 19th century imitators.
303
Terahertz Spectrometry: A New Frontier for Nano- and Pico-Scale
Investigations
Anis Rahman, Applied Research & Photonics, 470 Friendship Rd., Suite
10, Harrisburg, PA 17111, Aunik Rahman
Terahertz scanning reflectometry offers an opportunity to investigate both the surface and the sub-surface of biological tissues (e.g., skin) in a non-invasive fashion.
The non-ionizing nature of T-ray eliminates radiation damage of sensitive tissues
while be able to probe disease conditions in the deeper layers leading to an effective real-time diagnostic tool. For example, measurement of permeation kinetics and
concentration gradient in a non-invasive mode is important in several areas such
as in the study penetration behavior of an active ingredient through human skin
or other tissue. Two critical factors are: the concentration gradient of permeating
ingredient across the depth of skin and the kinetics (or rate) of such permeation.
These factors are essential for quantitative analysis, for example, via Fick’s laws
of diffusion. In this study, a terahertz scanning reflectometer (TeraScanR) has been
used for direct measurement of both concentration gradient across the thickness of
stratum corneum and permeation kinetics of an active ingredients. Two analytes,
hydrocortisone and caffeine were investigated. Some details are discussed in terms
of experimental data.
300
A Closer Look at Egg-Oil Paint Systems Using Imaging TOF-SIMS
and XPS
Zachary E. Voras, University of Delaware, Department of Chemistry,
Newark, DE 19716, Kristin De Ghetaldi, Thomas P. Beebe, Jr., Jennifer
Mass
The early adoption of oil-painting techniques in Quattrocento, Italy artwork has been
revealed by both research and scientific analysis, and combinations of egg and oil
have been discovered in easel paintings by several Northern Italian painters of the
15th century. Further scientific investigation into the use of binding media employed
by Quattrocento painters will provide scientists, curators, and conservators with
more information that may shed light on workshop practices, attributions, and other
topics associated with provenance. In this study X-ray photoelectron spectroscopy
(XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used
to analyze paint cross-sections collected from historically accurate paint samples
that were naturally aged for four years. These samples were made with various
pigments and also various binding medium per pigment. XPS was employed to
provide pigment confirmation and spatial positioning in relation to binding medium.
TOF-SIMS provided an estimate of the ratio of mixed-binding-medium composition
where appropriate (e.g., tempera grassa). The results obtained from these reference samples are summarized and a detailed account of the sample preparation
is included. The creation of a comprehensive chemical database of pigments and
binding media, a long-term goal of this research team, allows art historians to better
understand an artist’s medium selection, and conservators to better preserve these
works of art for generations to follow.
304
Leveraging UV Fiber Optic Dissolution to Increase Scientific
Knowledge and Operational Efficiency
Limin Zhang, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ
08903, Yueer Shi, Brent Kleintop
Fiber optic based UV (UVFO) dissolution methods have gain momentum due to
their unique advantage of in-situ dissolution measurement. This technology provides both technical and operational benefits, such as providing resource and cost
savings by eliminating manual dissolution sampling, filtering and offline high-pressure liquid chromatography (HPLC) or UV spectrometer setup. Use of UVFO also
provides real-time data generation (typically every 5 seconds) that enables a better
understanding of the dissolution release at early time-points for fast release compounds and provides enhanced dissolution profile for method discrimination evaluation. Real-time data generation may help to expedite formulation development when
generating the dissolution data is the rate limiting step. Recently we applied this
technology in two case studies where the advantage of frequent sampling provided
valuable scientific insights. In one study UVFO dissolution was used to understand
the in-vitro dissolution profile of citric acid (drug-in-capsule) in the pH range of 1 to
4 to help design an effective clinical study where co-administration of citric acid capsules is needed to mitigate a pH effect on the drug under investigation. Citric acid
is highly soluble in aqueous media in the pH range of 1 to 4 such that dissolution is
rapid and frequent sampling is required to understand when dissolution reaches the
plateau. We also present results from a second case study investigating the dissolution variation caused by hydroxypropyl methylcellulose capsule shell. Monitoring
the release of the drug from the granulation and disintegration of capsules by UVFO
dissolution provided useful information in identifying the root cause.
301
Insights on the Structure of Lead Carboxylates (Lead Soaps)
Found in Traditional Oil Paintings by 207Pb Solid State NMR
Jaclyn I. Catalano, Metropolitan Museum of Art, 1000 Fifth Ave., New
York, NY 10028, Yao Yao, Anna Murphy, Nicholas Zumbulyadis, Silvia
Centeno, Cecil Dybowski
Lead carboxylates have been detected in hundreds of oil paintings dating from the
15th to 20th centuries. Soap formation is produced when lead salts, such as lead
white (2Pb(CO3)2-Pb(OH)2) or lead tin yellow type I (Pb2SnO4) react with fatty
acids in the oil binding medium. The mechanism and factors that trigger soap formation are not well known. Knowledge of this process is essential, since soaps can affect the integrity of artwork by forming protrusions and/or by increasing the transparency of the paint films. Understanding the mechanism of these reactions requires
chemical information, which can be readily obtained with solid-state 207Pb and 13C
NMR. We have examined the spectroscopy of materials known to be involved in
soap formation, such as lead palmitate, stearate, and azelate, and interpreted NMR
parameters in terms of local structure. The information gained in these experiments
provides new information on the structure of lead carboxylates and serves as a
basis for analyzing mixed-phase materials, such model paint samples.
47
2012 EAS Abstracts
November 2012
305
308
Validation and Application of QuEChERS to Flavor and Fragrance
R&D
Nicole L. Harmuth, International Flavors and Fragrances, Union Beach,
NJ 07735, Fang Fang, George Reiner
This presentation highlights the initial validation process and application of QuEChERS (quick, easy, cheap, effective, rugged, and safe) to suit research and development (R&D) needs in the flavor and fragrance industry. It discusses the general
theory behind QuEChERS, how this theory can be applied to flavors and fragrances,
and the overall benefits the technology provides to R&D. Many R&D inquiries require the quantification of flavor and fragrance components in a sample. This is
generally done by the use of external standards and requires the use of harmful solvents such as methylene chloride and toluene. In addition, the liquid-liquid extraction generally takes over an hour. Through the use of QuEChERS, we have been
able to eliminate some of the more harmful solvents, decrease the loss of highly
volatile compounds, and significantly reduce extraction times. Furthermore, many
samples have complex matrices that require multi-step extractions with varying solvents. This has generated a need for variations in methods with detailed tables
specifying the appropriate reagents to use. In a setting where sample matrices are
constantly evolving and changing, this approach can only allow the chemist to go so
far, leading to a constant need for method development. QuEChERS has provided
a universal method that addresses diverse sample types. The QuEChERS method
was modified to suit our needs, and parity was established to ensure accurate and
reproducible results. Overall, employing QuEChERS has reduced resources and
provided a faster method of analysis, allowing us to increase sample throughput and
decrease turn-around-times… all great benefits in today’s laboratory environment.
Investigation of Bio-SPME Technology for the Enrichment of Illicit
Phenethylamine and Cathinone Compounds from Biological
Samples
Craig R. Aurand, Supelco/Sigma Aldrich, 595 North Harrison Rd.,
Bellefonte, PA 16823, Robert Shirey, Yong Chen, David S. Bell, Janusz
Pawliszyn
Recent advances in solid-phase microextraction (SPME) devices, termed bioSPME, are physically stable toward polar media. Bio-SPME devices, when coupled
with liquid chromatography mass spectrometry (LC-MS), provide analysts with an
interesting alternative set of tools for bio analyses. Some potential advantages of
Bio-SPME over traditional sample preparation may include improved selectivity toward target analytes, micro sampling, in-vivo sampling, and high-throughput analyses through automation. Bio-SPME fibers prepared in a 96 tip array were utilized
for the enrichment of nine illicit “bath salts” from urine and plasma samples. BioSPME fibers functionalized with hydrophobic and cation exchange particles were
employed. Following enrichment, the bio-SPME fibers were directly desorbed in
a 96 well plate for LC-MS time-of-flight analysis. Key parameters that impact analyte enrichment efficiency, such as sample pH, and extraction time were explored
along with the impact of biological sample matrix. In addition to extracted analyte,
overall sample matrix interferences were monitored and compared with typical dilution/precipitation techniques. Extraction studies demonstrated the capability of
analyzing sub-10 ng/mL concentration levels in both plasma and urine samples.
Unlike traditional SPE approaches, the unique utility of bio-SPME enables the direct
enrichment of targeted small molecules without the need for sample dilution or precipitation. Plasma samples prepared using the bio-SPME technique exhibited a five
time reduction in detected endogenous matrix as compared to standard precipitation techniques, while demonstrating increased analyte response. The capability to
configure the fiber tips in a 96 tip array allows for handling high numbers of samples
comparable to that of 96 well plate formats.
306
Automated Solid-Phase Extraction for Emerging Contaminants in
Drinking Water
William R. Jones, Horizon Technology, 45 Northwestern Dr., Salem, NH
03079, Julie McGettrick
As analytical technology gets more sophisticated and detection limits get lower,
chemicals that were previously undetected are being found in drinking water. Pharmaceuticals, caffeine, and artificial sweeteners are passing through wastewater
treatment plants and into water supplies around the world. Artificial sweeteners have
been found in lakes and rivers in Europe; and pharmaceuticals have been detected
in municipalities across the United States. Although these chemicals are safe at
their recommended doses the effect of long term, low dose exposure is unknown.
New extraction procedures are necessary to monitor these compounds. Last year
we presented the development of a solid-phase extraction (SPE) disk for use in the
extraction of pharmaceuticals and personal care products. The greater surface area
of disks is advantageous when samples are dirty and particulate laden, but this is
not necessary with drinking water. We have adapted the multi-modal chemistries
used in our SPE disks to a cartridge format. The slower flow rate through a cartridge improves retention for some hydrophilic compounds that can break through
a disk. A cartridge also retains less water than a glass fiber disk, leading to faster
evaporation times during concentration. In this paper we discuss the development of
an automated SPE method for the extraction of emerging contaminants in drinking
water. Commercially available and custom made SPE cartridges were used with a
new automated SPE cartridge extraction system. Recovery and precision data for
analytes including caffeine, carbamazepine, and sucralose are presented.
309
Determination of Phthalates in Infant Shampoo/Bodywash
Karyn M. Usher, West Chester University, 750 South Church St., West
Chester, PA 19383, Devon C. Zimmerman, Henry F. Rossi III, Jacqueline
Rizzo
Phthalates are a family of compounds that are often used as additives in flexible
vinyl products, or as solvents or fixatives in cosmetics. The Environmental Protection Agency is concerned about phthalates because of their toxicity and because it
is common for humans to have a high daily level of exposure to phthalates. Previous
studies have shown that some of the products with the highest levels of phthalates
are highly scented infant products. The extraction of four phthalates (diethyl phthalate, dipropyl phthalate, benzylbutyl phthalate and dicyclohexyl phthalate) from infant
shampoo/body wash was studied using Agilent Chem Elut 5 mL unbuffered solid
supported liquid-liquid extraction (SLE) cartridges. An Agilent 1200 HPLC system
with diode array detection was used for the separation and determination of the
extracted phthalates. Gradient elution was used to efficiently separate the analytes
of interest. Recoveries for these four phthalates from the infant shampoo/body wash
were higher than 91%.
310
Fourier Transform Infrared (FTIR) for Measurement of HCl on
Various Emission Source Applications
Kevin Ramazon, California Analytical Instruments, 1312 W Grove Ave.,
Orange, CA 92865
With the recent introduction of the new Portland Cement MACT and Utility Boiler
MACT, the measurement of acid gases such as hydrogen chloride (HCl) and hydrogen fluoride (HF) have been brought to the forefront as a surrogate measurement
for various hazardous air pollutants (HAPS). FTIR has been listed as the technology
required to measure HCl on a continuous basis according to these new rules. This
paper looks at the current state of both the Portland Cement MACT and the Utility
MACT regulations and the current requirements for the measurement of HCl as a
surrogate HAPS. In addition to the regulatory requirements, the paper looks at other
industries already using FTIR in complete emission monitoring system (CEMS) as
well as the European marketplace which is currently using FTIR for many of the utility site using solid waste as a fuel source. Next, the paper examines data generated
from a cement kiln and municipal waste incinerator and what lessons we can take
forward into the utility industry for the measurement of HCl and other acid gases.
In addition to HCl measurement, this paper looks at the comparison of FTIR and
traditional CEMS instruments for the measurement of NOx, CO, SO2, and various
hydrocarbons. Finally, this paper summarizes what is required by the regulations,
how to implement the use of FTIR to meet these regulations, and show the limitations of this technology based on real world data from other similar applications.
307
Removal of Pharmaceutical Dosing Excipients from Plasma
Samples Using Mixed-Mode Solid-Phase Extraction (SPE)
Erin E. Chambers, Waters, 34 Maple St., Milford, MA 01757, Jonathan
Danaceau
Pharmaceutical vehicle excipients are often added to formulations to facilitate dissolution. These compounds can cause significant matrix effects, typically ion suppression, in liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses.
The use of fast LC gradients can result in co-elution of target analytes with these
compounds. Early pharmacokinetic time points, when the concentration of these
excipients is elevated, can be particularly troublesome as this co-elution has been
shown to be a major contributing factor to excipient related ion suppression. Several approaches have been investigated to attempt to minimize this problem. While
some of these strategies have been successful, they each have their limitations.
Using mixed-mode cation exchange solid-phase extraction (SPE), basic analytes
are bound to the sorbent by strong cation exchange, and non-ionic excipients, such
as PEG 400 or Triton X-100, are bound by reversed-phase and are selectively removed before analyte elution. PEG 400 and Triton X-100 were completely removed
from plasma samples using mixed-mode SPE and matrix factors (ion suppression)
associated with dosing excipients were eliminated. This work represents a simple,
quick and elegant method for the removal of high concentrations of non-ionic dosing
excipients from plasma samples, eliminating matrix effects (and thus ion suppression) often seen in early time points of pharmacokinetic studies. Furthermore, this
problem is solved without any additional method development or other time consuming or cumbersome procedures, which makes it suitable and broadly applicable
for a high-throughput environment.
48
2012 EAS Abstracts
November 2012
311
314
Purge-and-Trap GC Analysis of Water Samples Associated with
Hydraulic Fracturing and Natural Gas Extraction
Gary Engelhart, OI Analytical, PO Box 9010, College Station, TX 77842,
Phil Griffiths, Scott Hazard
Shale gas reservoirs are a growing source of natural gas in the United States. The
US Energy Information Administration estimates the total natural gas resource base
of the US to be 2,553 trillion cubic feet (Tcf). Shale gas represents greater than
20% of the current US supply an increase from 1% in 2000. Hydraulic fracturing
or “fracking” involves pumping water, sand and chemicals at extremely high pressure into deep underground wells to crack open hydrocarbon-rich shale formations
and extract natural gas. Chemicals used for hydraulic fracturing include potentially
toxic substances such as diesel fuel and disinfectants, which can contaminate underground sources of drinking water. In some cases, methane has been detected
in drinking water from wells. A study conducted in the Marcellus and Utica shale
formations found methane concentrations were 17-times higher on average in wells
from active drilling and extraction areas than in non-active areas. Methane is not
a regulated contaminant under US National Drinking Water Regulations. Consequently, there are no USEPA-approved testing methods for measuring methane in
drinking water and groundwater. This poster describes the use of a purge-and-trap
gas chromatography (GC) system to analyze methane, ethane, ethene, and propane hydrocarbons (C1-C3) in drinking water samples.
Large Volume Splitless Injection with an Unmodified Split/Splitless
GC Inlet to Lower Reporting Limits For 1,4-Dioxane in Drinking
Water Analyzed by EPA Method 522
Christopher M. Rattray, Restek, 110 Benner Circle, Bellefonte, PA
16823, Jack Cochran, Christopher English
The third Unregulated Contaminant Monitoring Regulation (UCMR 3) is scheduled
to begin next year and includes a requirement for 1,4-dioxane analysis using Environmental Protection Agency (EPA) Method 522. EPA Method 522 was originally
written to be performed using a standard gas chromatograph (GC) equipped with
a single quadrupole mass spectrometer (MS). However, a recent re-evaluation of
1,4-dioxane’s carcinogenic risk resulted in the EPA lowering the suggested minimum reporting limit from 0.3 to 0.07 µg/L, and even when operating in selected ion
monitoring (SIM) mode, most GC-MS instruments would be hard pressed to meet
this lower 0.07 µg/L limit. One way to lower detection limits is to inject more sample,
but large volume splitless injection for gas chromatography typically requires a special injection port such as a programmable temperature vaporizer. The technique
described here for the analysis of 1,4-dioxane in drinking water uses an unmodified
split/splitless inlet with very-large-scale integration to lower reporting limits without
concentrating extract. With an injection volume of 10 µL, we achieved a linear calibration range exceeding 3 orders of magnitude, with levels ranging from 5 to10,000
pg on column. We saw 89% recovery when 1 L of bottled water fortified at 0.005
µg/L was extracted using coconut charcoal solid-phase extraction tubes and analyzed according to EPA Method 522 with LVSI, matching the method performance
data published by the EPA. This LVSI technique requires absolutely no changes to
standard laboratory instrumentation.
315
Simultaneous Analysis of Available and Total Cyanide by Gas
Diffusion Amperometry Methods USEPA OIA-1677 and ASTM D
7511-09
William Lipps, OI Analytical, PO Box 9010, College Station, TX 77842,
Phil Griffiths
United States Environmental Protection Agency (USEPA) methods OIA-1677 and
American Society for Testing and Materials (ASTM) D 7511-09 have the advantage
of determining available cyanide and total cyanide respectively without the burden
of a preliminary distillation. Historically, these methods required separate determinations due to a manual ligand addition step required by OIA-1677 to release cyanide
from certain metal-cyanide complexes. Method OIA-1677 has been modified to automatically inject a diluted ligand reagent into the sample prior to determination. A
modification to the TA1 reagent of ASTM D 7511 improves recovery of total cyanide,
decreases interferences, and shares reagents with the available cyanide method.
This automatic ligand injection enables total and available cyanide to be determined
simultaneously from the same sample aliquot and using the same reagents.
312
Rapid Quantification of Dioctyl Sulfosuccinate Sodium Salt (DOSS)
in Birds Egg Tissue Utilizing QuEChERS Extraction Method and
UHPLC-MS/MS Multiple Reaction Monitoring (MRM)
Aliaksandr V. Yeudakimau, University of Connecticut, 3107 Horsebarn
Hill Rd., Building 4, Storrs, CT 06269, Anthony A. Provatas, Christopher
R. Perkins, James D. Stuart
With the Deep Water Horizon offshore rig explosion and subsequent sinking of a
platform on April 22, 2010, an estimated amount of 4.4 million gallons of crude oil
was released into the Gulf of Mexico. To contain it from spreading through vast
ocean surface areas, over 1.8 million barrels of oil dispersants were applied. Oil
dispersants are chemical mixtures of solvents, typically containing a high percentage of one or more uncharged or charged anionic surfactants of different solubility,
designed to mitigate the formation of oil slicks by increasing the solubility of crude
oil. Due to insufficient scientific data, it is uncertain whether dispersants are toxic at
present concentrations to the aquatic ecosystem, but it worth noting that they can
contribute to the enhancement of more toxic oil components to the habitat through
increased solubility. This project focused on sample preparation, analysis and
quantification of DOSS in egg matrix at concentrations as low as 500 ppt. Sample
preparation was carried out by QuEChERS (quick, easy, cheap, effective, rugged,
and safe) extraction method followed by weak anionic exchange cleanup. This technique proved to be a rapid, yet efficient extraction method that gave excellent quality
control recoveries. A ultra high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) system was utilized providing enhanced chromatographic separation combined with increased analyte sensitivity and short acquisition time.
316
Sample Preparation Effects on Data Quality in Total Organic
Carbon (TOC) Analysis of Bauxite Ore Samples
Garrett Slaton, OI Analytical, 151 Graham Rd., College Station, TX
77845, Jeffrey Lane, Gary Engelhart
Bauxite ore is the most important source of aluminum, which is recovered from the
ore using the Bayer extraction process. Organic carbon present in bauxite can interfere with the extraction process, and downstream processing of alumina. Inorganic
carbon in the ore also causes difficulties, so the ore is monitored using total carbon
(TC) and total organic carbon (TOC) measurements. We present a series of measurements of TC and TOC of bauxite samples determined using combustion with
the resultant CO2 analyzed by a non-dispersive infrared detector. Sample preparation details of combustion temperatures and times, sample sizes, and total inorganic
carbon removal methods for optimally accurate and precise data are presented.
313
Liquid-Liquid Extraction and SPE-Cleanup Followed by UPLC-UV
Analysis of Polycyclic Aromatic Hydrocarbons from Blood
Samples Taken from Birds Impacted by the Deepwater Horizon Oil
Spill
Anthony A. Provatas, University of Connecticut, 3107 Horsebarn Hill Rd.,
Building 4, Storrs, CT 06269, Aliaksandr V. Yeudakimau, Christopher R.
Perkins, James D. Stuart, Joseph E. Seiss
Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds consisting of two or more fused aromatic rings, which are found in significant amounts
in crude oil. Due to the recent Deepwater Horizon offshore rig explosion in the Gulf
of Mexico, the fate of PAHs have become a major environmental concern related to
potential effects on the surrounding ecosystem. Because of their carcinogenic and
mutagenic nature, the United States Environmental Protection Agency has classified 16 un-substituted PAHs as priority pollutants (EPA 610). We developed a method allowing quick and efficient sample preparation and analysis for PAHs in blood
samples from birds potentially impacted by the oil spill. Sample preparation was
carried out by hexane liquid-liquid extraction, followed by Alumina-N solid-phase
extraction cleanup. The analytes were eluted with methylene chloride, followed by
acetonitrile solvent exchange, and analyzed using ultra-pressure liquid chromatography (UPLC)-UV. Representative recoveries averaged in the range from 70-120%,
with detection limit of 1.5 ppb. The UPLC-UV analytical system provided enhanced
chromatographic separations combined with increased analytical sensitivity for the
16 PAHs of interest.
317
318
Withdrawn by the author.
Validation of New VPH GC-MS Method Using Multi-Matrix Purge
and Trap Sample Preparation System
Nathan Valentine, Teledyne Tekmar, 4736 Socialville Foster Rd., Mason,
OH 45040, Tom Hartlein, Holly Taylor
The Massachusetts Department of Environmental Protection (MassDEP) developed
the “Method for the Determination of Volatile Petroleum Hydrocarbons (VPH) by
Gas Chromatography/Mass Spectrometry” to measure the collective concentrations
of volatile aliphatic and aromatic petroleum hydrocarbons in aqueous and soil/sediment matrices. The previous method for VPH determination required both flame ionization and photoionization detectors, while this method utilizes mass spectrometry.
This method uses purge and trap technology, which allows for the determination of
volatile organic compounds (VOCs) at low concentration levels with precision and
accuracy. The Teledyne Tekmar Atomx automated VOC sample preparation system
is used to validate this method for both water and soil matrices. Calibration curves
and detection limits are established for the aliphatic and aromatic petroleum hydrocarbons listed in the MassDEP VPH method.
49
2012 EAS Abstracts
November 2012
319
324
Extraction of Sub-Nanogram Levels of Catecholamines from
Human Plasma Using Mixed-Mode Cation Exchange Resin Prior to
LC-MS/MS Analysis
Victor Vandell, Biotage, 10430 Harris Oaks Blvd., Charlotte, NC 28269,
Elena Gairlock, Ira Bellew
Catecholamines act as neurotransmitters in mammals and are classic biomarkers
for the detection of diseases like hypertention, pheochromocytoma and neuroblastoma. The catecholamine analytes evaluated in this study were Epinephrine, Dopamine and Norepinephrine. The detection of catecholamine analytes in plasma
and at sub-nanogram per milliliter levels is critical toward the quantitation and ultimately towards clinical diagnosis for diseases. This poster demonstrates a rapid
and reliable mixed-mode weak cation exchange solid-phase extraction assay for
the extraction of Epinephrine, Dopamine and Norepinephrine from plasma at subnanogram per milliliter concentration levels prior to liquid chromatography tandem
mass spectrometry (LC-MS/MS) analysis. All of target catecholamines were successfully extracted from spiked plasma samples at concentration levels of 100pg/
ml for Epinephrine, 200 pg/ml for Dopamine and 1000 pg/ml for Norepinephrine.
Sample recoveries at the limit of detection of 100 pg/ml were >85% for epinephrine
and dopamine with percent relative standard deviations less than 10%.
High Temperature Arylene Cyanopropylphenyl Stationary Phase
for Analysis of Volatile Organics
Christopher Rattray, Restek, 110 Benner Circle, State College, PA
16823, Michelle Misselwitz, Jason Thomas, Chris English
Typical non-arylene cyano (624-type) stationary phases for capillary chromatography provided an effective solution for the separation of many common volatile
organic compounds, such as those found in United States Environmental Protection
Agency (USEPA) Method 8260. However, along with this unique and versatile polymer came some drawbacks, these being primarily high bleed, which became particularly problematic for those analyses utilizing ion trap mass spectrometry detection,
where excess bleed significantly impairs detector sensitivity, as well as, increases
detector maintenance frequency. In this work we examined the effectiveness in utilizing a 624 type capillary column that incorporates a new highly inert deactivation
with an improved stationary phase that incorporates aryl groups into the backbone
of the polymer; for analyzing common volatile organic contaminants in the environment. This new stationary phase-significantly reduces column bleed.
320
Collaborative Study with USEPA for Optimization of Sample
Preservation for Hexavalent Chromium in Drinking Water
Jay Gandhi, MetrohmUSA, 4738 Ten Sleep Ln., Friendswood, TX
77546, Matthew Johnson, Carl Zhang
Recently published United States Environmental Protection Agency (USEPA) method 218.7 had a major facelift in terms of sample preservation, analytical and other
parameters to achieve ultra-trace level detection limits to meet State of California
health goals. This presentation highlights the collaborative study to update USEPA
method 218.6 to 218.7
325
Extraction of Methylmalonic Acid (MMA) from Human Serum Using
Supported Liquid Extraction in 96-Well Plate and Column Formats
Prior to LC-MS-MS Analysis
Victor Vandell, Biotage, 10430 Harris Oaks Blvd., Charlotte, NC 28269,
Elena Gairloch
The screening for elevated levels of methylmalonic acid in serum is commonly used
as a clinical diagnostic indicator of cobalamin (Vitamin B12) deficiency in mammals.
The significance of this screening is important for the detection of inborn metabolic
disorders that can lead to the build-up of MMA in human biological systems. The detection and quantification of MMA can be problematic due to the abundant presence
of the endogenous isobaric succinic acid (SA). The SA offers no clinical diagnostic
value. The development of reliable sample cleanup methods is necessary for the
accurate detection and quantification of MMA using mass spectrometry. Methylmalonic acid was extracted from serum using the ISOLUTE SLE+ cartridge (1 ml) and
96-fixed well formats. The target analyte was spiked into human un-pooled serum
at a concentration of 1 µg/ml. The sample was loaded at full capacity onto the well
plate or cartridge at a 1:1 ratio using a novel acidic buffer solution of 4.6 M formic
acid. Matrix interferences like proteins, salts, non-ionisable molecules and phospholipids were successfully absorbed onto the SLE+ sorbent and the targets analytes
were subsequently eluted using 100% methyl tert-butyl ether (MTBE). The collected
eluant was dried down and reconstituted in mobile phase yielding clean, concentrated samples with reproducible recoveries for accurate quantitation. The MMA was
chromatographically separated from the co-eluting interference SA using a Restek
Organic Acids column via high-pressure liquid chromatography and detected with
electrospray mass spectrometry. The MMA was extracted from spiked serum samples at concentration levels of 1 µg/ml. Averaged sample recoveries were 90% for
MMA with percent relative standard deviations less than 10%.
321
Matrix Effects and Optimization of Sample Preservation for Trace
Level Analysis of Hexavalent Chromium in Drinking Water
According to USEPA Method 218.7
Jay Gandhi, MetrohmUSA, 4738 Ten Sleep Ln., Friendswood, TX
77546, Matthew Johnson
Have you seen the movie “Erin Brockovich”? Did you hear more recent news regarding Hexavalent chromium in drinking water? Chromium is a one of the essential
micro nutrient required by living cells to convert fat into energy. Chromium required
by living cells is supposed to be in its +3 oxidation state; however, chromium in its
+6 oxidation state causes health risks for living cells especially in humans. This
poster presentation highlights various experiments for sample preservation studies.
322
What is this Stinky Smell? Air Monitoring of Thio Compounds
Matthew Johnson, United States Environmental Protection Agency,
10625 Fallstone Dr., Houston, TX 77079, Jay Gandhi
Living around a petrochemical industry area could be annoying for “stinky” smell,
especially during night hours. Even though industry is in compliance for air monitoring, reality of “stinky” smell is still there. This presentation highlights analysis of “thio”
compounds in air using ion chromatography.
323
Unattended Quantitative Determination of VOCs in Cigarette
Carton and Packaging Samples Using a Robotic Sampler for
Automatic Standard Addition and Subsequent Headspace Analysis
Massimo Santoro, Thermo Fisher Scientific, Strada Rivoltana, Rodano,
20090 Italy, Stefano Pelagatti, Robert Biniakewitz, Eric Phillips, Matthew
Lambing
The headspace analysis by means of a dedicated auto sampler is a standard technique for the determination of volatile organic compounds (VOC) possibly present
in the food packaging materials. The packaging sample is generally cut in square
pieces and placed in headspace vials for the incubation at determined temperature
before the headspace sampling. The main problem for this kind of analysis is the
quantitation of volatile compounds present, as these samples are normally layered
solids. The external calibration is not reliable as it does not consider the matrix
effect, which is significant for these samples. The standard addition calibration is
instead a reasonable quantitation procedure for these difficult matrixes, as it uses
the real sample for the calibration procedure. Until now, preparation of the samples
for the standard addition calibration was performed manually by the operator off-line
before the headspace analysis of the samples. This was a time consuming and error
prone procedure. The possibility of performing the sample preparation by means of
the same robotic sampler used for the headspace analysis allows the quantitation
sequence to run automatically in an unattended way. In this poster the use of a
robotic auto sampler for the standard addition calibration and the related gas chromatography mass spectrometry headspace analysis of volatile organic compounds
in cigarette carton and packaging are presented.
326
Extraction of Testosterone and Other Endogenous Steroid
Hormones from Plasma Using Supported Liquid Extraction (SLE)
prior to UPLC-MS/MS Analysis
Adam Senior, Biotage, 10430 Harris Oaks Blvd., Charlotte, NC 28269,
Lee Williams, Victor Vandell
Steroid hormones are important physiological compounds controlling: inflammation,
immune functions, salt and water balance, and development of sexual characteristics. Here we demonstrate a rapid and reliable 96 well supported liquid extraction
assay for the extraction of endogenous steroids from human biological fluids. Extraction conditions were evaluated using 100 µL matrix pre-treated (1:1, v/v dilution)
with various buffers to provide effective pH control (from pH 3 to 10.5); 200 µL was
applied to each well of the plate. Extraction was investigated using 1 mL volumes
of various water immiscible extraction solvents and solvent combinations: ethyl acetate, butyl acetate, hexane, methyl tert-butyl ether, diethyl ether, hexane, DCM
and chlorobutane. This study investigated the extraction of the following steroids
from human plasma and serum: aldosterone, 11 deoxycortisol, 21 deoxycortisol,
androstendione, testosterone, DHEA, 17α hydroxyprogesterone, androsterone
and progesterone. The lipophilicity of these molecules has been shown to result
in non-specific binding to plastic surfaces. For this reason it was decided to evaluate extraction of endogenous steroids using the ISOLUTE SLE+ extraction plate
combined with a variety of pre-treatment conditions and extraction solvents with
differing characteristics and polarities. Matrix pH (both plasma and serum) was adjusted with: 1% formic acid (aq); 0.1% formic acid (aq); water; or 0.5 M ammonium
hydroxide (aq). Both matrices showed similar behaviors, although slight differences
were observed. Overall, the data suggests that a neutral loading pH and mid-polarity
extraction solvent combination result in recoveries in excess of 80% for endogenous
steroids with relative standard deviations below 10% for both matrices.
50
2012 EAS Abstracts
November 2012
327
Redefining Solid-Phase Extraction Performance in Bioanalytical
and Clinical Workflows
Ken Meadows, Thermo Fisher Scientific, Tudor Rd., Runcorn, WA7 1TA
United Kingdom, Tim Liddicoat, Robert Wiedmer
Solid-phase extraction (SPE) plays a vital role in sample preparation for many fields
of analytical chemistry including, bioanalytical, clinical, forensic, environmental and
food safety. SPE usually involves cartridges or well plates packed with a loose powder of silica or polymeric material positioned between two frits. These packed beds
are potentially prone to settling and voiding in production or transport, leading to
phase channeling as well as irreproducibility in packing, all resulting in poor recovery and reproducibility in analytical results. The work presented in this poster
describes and demonstrates the advantages of a novel SPE cartridge and well
plate device (Thermo Scientific SOLA) that eliminates the issues associated with
classical SPE beds. These SPE beds have significant advantages for the analyst
when processing compounds in complex matrices particularly in high-throughput
bioanalytical and clinical liquid chromatography mass spectrometry assays in which
reduced failure rate, higher-analysis speed and lower sample/solvent requirements
are critical in gaining greater confidence in analytical results and reducing cost.
Data presented also compares this new material to classical loose packed material
and exemplifies: improved reproducibility; improved recovery even at low elution
volumes; lower solvent consumption; higher extract cleanliness from high-quality
media and reduced extractables.
330
Analysis of Volatile Organic Compounds from Gas Sampling
Canister
Using
Solid-Phase
Microextraction
and
Gas
Chromatography
Jianying Guan, University of Florida, Department of Chemistry,
Gainesville, FL 32611, Greg Slack, Alan Rossner
Finding affordable and effective ways to identify and quantify the amount of volatile
organic compounds (VOCs) in an indoor environment is important to evaluating the
sustainability of a building design. Indoor air samples often include minute concentrations of VOCs, many with concentrations in the range of parts per billion. These
low VOC concentrations make detection challenging and costly for common methods like gas chromatography (GC). This project focuses on finding an affordable
and effective way to identify and quantify the amount of VOCs in indoor air. The
analytes of interest are collected with a 6L gas sampling canister and transferred to
GC via solid-phase microextraction (SPME). The SPME fibers used to transfer the
samples were 65 μm polydimethylsiloxane/ divinylbenzene (PDMS/DVB), which is
used to detect aromatics in liquids and organic materials. Toluene was used as the
sample analyte with the GC being held at an isothermic temperature of 100 °C for
four minutes. The accuracy and precision of this method was tested by developing
a calibration curve by plotting the known concentration of the analyte by the area
under the GC curve. The preliminary results show that this could potentially be a low
cost and effective method to analyze VOCs in indoor air, but further studies need to
be done to understand the variability associated with different VOCs.
328
331
Automated Disposable Pipette Extraction for the Detection of
Mycotoxins in Food Using LC-MS/MS
Oscar G. Cabrices, Gerstel, 701 Digital Dr., Suite J, Linthicum, MD
21090, Fredrick D. Foster, Edward A. Pfannkoch, John R. Stuff, William
E. Brewer
One of the major food safety challenges is the abundance of mycotoxins, including but not limited to Aspergillus flavus and Aspergillus parasiticus molds, which
are among the fungi that produce toxic secondary metabolites known as aflatoxins.
Several mycotoxins are known to be human carcinogens or to cause other human
disease. Mycotoxin levels in food and animal feed are regulated in most countries
and there is great interest in a fast, sensitive, and selective analysis method. Determining mycotoxin concentrations at trace levels in the presence of large amounts
of sample matrix is a challenging task. The accuracy and precision of the analytical results depends largely on the extraction and cleanup methods used to isolate
the mycotoxins from the complex food and animal feed matrices. In this work, the
automated extraction of mycotoxin residues from contaminated corn samples using
disposable pipette extraction (DPX) is described. DPX is a solid-phase extraction
(SPE) technique that is based on loosely contained sorbent inside a pipette tip fitted
with a screen. This device provides faster extraction because only minimal conditioning steps are needed. The automated DPX extraction and analysis method for a
panel of 11 mycotoxins (aflatoxins, fusarium toxins and fuminosins) provided extraction efficiencies greater than 70% for all mycotoxins screened in corn samples with
relative standard deviations less than 15%. In addition; good linearity was achieved
(R2 values of 0.98 or greater) with limits of quantitation lower than confirmation
cutoff concentrations for most analytes. The ability to automate the dispersive SPE
clean-up of corn sample extracts and to couple the extraction directly to liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical methods, results in
improved laboratory productivity by streamlining the complete analytical process.
Analysis of Difficult Matrices with a New QuEChERS Product
Emily Barrey, Supelco/Sigma-Aldrich, 595 North Harrison Rd.,
Bellefonte, PA 16823, Katherine K. Stenerson, Michael Ye, Jennifer
Claus, Daniel Vitkuske
The concept of dispersive solid-phase extraction (dSPE) cleanup (otherwise known
as QuEChERS method) for the analysis of contaminants in food samples is an example of “just enough” cleanup before the end-analysis is performed. The method
was first introduced to pesticide analysis in produce, but in recent years has been
expanded to other applications and matrices. Here we introduce the use of a new
QuEChERS (quick, easy, cheap, effective, rugged, and safe) sorbent, Z-Sep+, and
compare it to the most common phases used for cleanup. We have found that ZSep+ has better capacity for retention of lipids and colors then the commonly used
C18 sorbent. Therefore, we looked at two specific applications of the new sorbent
towards samples with higher-fat contents. The first is analysis of pesticides in highfat foods such as avocado and olives, and the second is the analysis of veterinary
drugs as contaminants in animal tissue samples. Both applications are described in
detail with regards to the analytical protocol and the obtained results.
332
Comprehensive Analysis of Acid, Neutral and Basic Drugs of
Abuse by Disposable Pipette Extraction (DPX) and Liquid
Chromatography Tandem Mass Spectrometry (LC-MS/MS)
William E. Brewer, University of South Carolina, Department of
Chemistry, 631 Sumter St., Columbia, SC 29208, Yujing Wen, Stephen
L. Morgan
We developed a five minute LC-MS/MS method for simultaneous analysis of 50
drugs and their metabolites in urine by using a fast DPX procedure. Among the
three DPX resins tested, DPX-RP-S resin provided the fastest clean-up procedure
without conditioning and elution steps and with sufficient recovery for all 50 drugs.
The direct comparison of DPX with a dilute and shoot method shows that DPX can
concentrate samples and increase their sensitivity by about five times for all drugs.
LC parameters were optimized to achieve best separation among isobars, such
as morphine-hydromorphone and codeine-hydrocodone. Optimal multiple reaction
monitoring transitions were selected to both increase signal intensity and decrease
background interferences. Some interference, especially isobaric compounds, are
hard to remove by LC or sample clean-up procedure, but can be removed by careful
MRM transition optimization as was observed for amphetamine, 11-nor-9-carboxyTHC, and pentazocine. For multiple chlorine containing compounds, such as lorezapam and α-hydroxy-triazolam, it is necessary to choose appropriate internal
standard ion transitions to maintain linear calibration plots due to their isotope effects. The LC/MS/MS-DPX method is specific with no interferences for all tested
drugs. The accuracy was 97 to 107% for all 50 drugs, and percentage of relative
standard deviations were less than 20%, even at the low concentration range. The
correlation coefficients were greater than 0.990 for all drugs, and the method was
cross-validated by comparing the patient results with other reference labs and commercial companies.
329
SiliaPrepTM Family of Polymeric Phases Designed to Enhance
Solid-Phase Extraction Analytical Performances
David Dubé, SiliCycle, 2500 Blvd du Parc-Technologique, Quebec,
PQ G1P 4S6 Canada, Vincent Bédard, Geneviève Gingras, François
Béland, Denis Boudriau, Cale Barnes
The use of polymeric sorbents with hydrophilic and lipophilic balance or with ion
exchange functions in solid-phase extraction (SPE) has been growing in many fields
of the analytical domain since the addition of new environmental and Food and
Drug Administration legislations. Moreover, the treatment of complex matrices such
as blood, urine, soils and food are requiring robust and polyvalent sorbents. These
changes present a constant challenge to obtain cleaner extracts and higher recovery with reduced ion suppression. SiliCycle has developed a complete family of advanced hydrophilic and lipophilic polymer sorbents for specific SPE of acidic, basic
and neutral analytes from biological fluids, food matrices and aqueous samples. The
unique hydrophilic and lipophilic backbone coupled with uniform surface coverage
of ion-exchange functions allows efficient mass transfer and rapid exchange of the
analytes into the polymeric matrix. This poster presents different applications of the
SiliaPrepXTM polymeric phases for the solid-phase extraction of carbendazim in
orange juice, quantification of quinolone residues in salmon and determination of
pesticides from drinking water.
51
2012 EAS Abstracts
November 2012
333
semi-quantitative and quantitative inductively coupled plasma mass spectrometry
(ICP-MS) methods for evaluating trace metals during the early process development of APIs. By establishing limit specifications for residual metals during routine
chemical intermediate synthesis and controlling through in-process monitoring by
ICP-MS, chemists are able to ensure that metal content specification is met in the
final product, API. Routine well established and standardized sample preparation
methods are critical for the analysis of trace metals. Current wet digestion methods using open-vessel or closed-vessel digestion systems have their own distinct
advantages and depend on a number of factors such as sample chemistry, sample
size, quantitative versus qualitative analysis, the trace metals to be analyzed and
the nature of sample matrix. Examples of digestion techniques currently used within
our lab for in-process ICP-MS metals testing are shown. Additionally, the rational for
choosing the type of digestion as well as considerations concerning key parameters
for improved recovery and lab safety are discussed.
Characterization of Novel Porous Carbon-Based Materials for use
in Sample Preparation
Dwight Stoll, Gustavus Adolphus College, 800 West College Ave., St.
Peter, MN 56082, David C. Harmes, Jon Thompson, Doug Fryer, Conor
Smith
We have recently developed a series of novel carbon-based materials for use as
solid-phase extraction media. These materials, which are based on porous inorganic substrates, exhibit superior performance compared to existing pure carbon supports. Specifically, these materials are highly mechanically robust, while maintaining
other desirable properties including high surface area, high carbon load, and good
mesopore size distribution. This improved mechanical robustness results in higher
and more consistent flow through characteristics under pressure, and makes the
material easier to handle due to the absence of fines. In this work we focus on the
use of these new materials in food analysis applications involving the QuECheRS
(quick, easy, cheap, effective, rugged, and safe) method of sample preparation.
The performance of the carbon-based extraction medium is assessed using pesticide recovery data determined by gas chromatography mass spectrometry (MS)
and liquid chromatography tandem MS, characteristics of the extracted sample, and
ease of use. We find that the new materials described here exhibit equivalent or
better recoveries compared to pure carbon materials for all of the pesticides tested
in this study.
337
A Critical Assessment of Closed Microwave Digestion Vessels with
Controlled Pressure Release Capabilities
Reynhardt Klopper, Anton Paar USA, 10215 Timber Ridge Dr., Ashland,
VA 23005, Markus Michaelis
Microwave digestion vessels featuring pressure release capabilities are popular in
atomic spectroscopy laboratories. This presentation critically assesses both technologically and analytically important parameters of these digestion vessels. The
assessed parameters include challenges of construction materials’ properties, pressure and temperature relations, and removal of reaction gases versus potential loss
of analytes. It concludes with practical recommendations and supporting analytical
data.
334
Automated Sample Preparation Using a Digital Syringe with
Embedded SPE Capability
Rob Freeman, SGE, 2007 Kramer Ln., Austin, TX 78758, Dan R. DiFeo, Jr.
Popular sample preparation techniques such as solid-phase extraction (SPE) can
be time consuming and expensive, whether implemented on automated platforms or
performed manually. Furthermore, method development to improve the efficiency of
the sample preparation process is often difficult and time prohibitive, with long evaporation steps throughout the process. A key objective for all laboratories is to implement efficient sample preparation where there is a real need to improve productivity and minimize waste. While the introduction of automated and semi-automated
platforms into the laboratory for sample preparation enables improved efficiencies,
they generally come at a significant cost. We have combined the advantages of
automation and SPE using a digital syringe with an embedded SPE cartridge which
not only improves efficiencies, but virtually eliminates solvent use and waste. The
miniaturized format is ideal for small valuable samples such as biological extracellular fluids. Here we demonstrate the advantages in combining the automation of a
handheld, digital syringe with miniaturized SPE sorbent embedded in the needle of
the syringe. Method development is rapid and inexpensive, enhancing laboratory
workflow, while increasing the accuracy and reproducibility of the SPE process.
338
Increasing Productivity by Outsourcing the Preparation of
Formulations
Anthony R. Kemperman, Honeywell, Burdick and Jackson, 1953 S.
Harvey St., Muskegon, MI 49442, Jeremy N. Diringer
Formulations are prepared by various functions within a company. Departments performing this task include research and development (R&D), method development,
quality control and the pilot plant. Companies are continuously looking for ways
to apply lean manufacturing principles to the other supporting functions within the
organization. Depending upon the application, the process of preparing formulations in-house is often considered a non-value added activity. The task of making
buffers and chemical solutions reduces productivity and creates hidden costs within
a department. This poster examines the process of preparing formulations in the lab
and identifies the non-value added activities that are part of that process. These are
the activities that decrease overall productivity and create hidden costs. Other factors that must also be considered when evaluating the preparation of formulations
include worker safety, lot-to-lot formulation consistency, inventory management and
reliability. A solution to this dilemma is to utilize commercially prepared formulations.
Honeywell LabReady Blends by Burdick & Jackson is an example of an offering
that is specifically suited to eliminate these wastes while addressing these other
factors. When organizations choose to buy versus make their formulations, they are
able to deploy their resources toward more value added activities, thus enhancing
productivity. Honeywell LabReady Blends can be used from the R&D lab through
quality control and into the manufacturing area. The poster demonstrates how an
organization that adopts Honeywell LabReady Blends can eliminate the non-value
added steps required in formulation preparation, freeing resources for more value
added activities.
335
Evaluation of a New Technique in Semi-Automated, Miniaturized
Solid-Phase Extraction
Dan R. DiFeo, Jr., SGE, 2007 Kramer Ln., Austin, TX 78758, Rob
Freeman
Solid-phase extraction (SPE) is a very popular method of sample preparation. Conventional SPE is performed by passing a liquid sample across a sorbent bed to
retain analytes of interest. Once bound to the adsorbent material, the sample can be
further concentrated or washed to eliminate interferences. Although, conventional
SPE has several advantages over liquid-liquid extractions, namely time and solvent
savings, SPE methods can still be tedious and time consuming when performed
manually. Also, sample preparation as a whole remains a limiting factor in sample
throughput in the modern laboratory. Micro extraction by packed sorbent (MEPS) is
a new development in the field of SPE. The MEPS technology consists of a modified removable needle syringe and a needle assembly (Barrel In Needle) that contains the SPE sorbent material. MEPS performs the same function as SPE, namely
the purification or speciation of samples, but with some significant improvements.
MEPS works with much smaller samples sizes than conventional SPE. Due to the
smaller sample sizes, MEPS significantly reduces the volume of solvents needed,
from milliliters to microliters, and ultimately the amount of waste product that is generated. Also, the solvent eluant which contains the sample extract is often compatible with common gas chromatography or liquid chromatography injection volumes
and therefore can be introduced directly into the instrument for sample analysis,
eliminating the need for time consuming sample concentration step.
339
Statistical Assessment of Dissolution Profiles: Effect of Varied
Dilution and Mixing Efficiency
Aditya A. Marfatia, Electroblab, Goregaon (East), Maharashtra, Mumbai,
400063 India, Kavita H. Singh, Tejashri Gursallkar, Amrita N. Bajaj
Investigations are needed to understand the effect of dilution and mixing efficiency; and assess the underlying statistical parameters while carrying out dissolution
studies on high dose drugs. Dilution of dissolution aliquots is a critical step in UV
spectrophotometric analysis and it requires stringent absorbance range of 0.2-0.8 to
ensure linear concentration range and coefficient of correlation close to 0.999 (Beer
Lambert’s law). To fit into this narrow range of analysis and expedite routine analysis
of dissolution aliquots, it’s a common practice to dilute and analyze the aliquots. Effective mixing of aliquots with the diluent/buffer is extremely vital in order to achieve
accurate dissolution results. In the present investigation, two independent sets of
studies were carried out for % drug release analysis of Naproxen sodium tablets
550 mg. Results from both the methods, i.e., manual dilution and mixing versus
automated dilution and mixing of dissolution aliquots were obtained and statistically evaluated for any variations in data point values. The similarity and difference
factors were found to be 92.94 (f2) and 1.10 (f1) respectively. The two tailed paired
student t-test was applied to compare both the methods of dissolution aliquot mixing. Non-significant difference was found with the student t statistics with p = 0.2349.
The statistical analysis of the data ensured a statistical closeness of the results
336
Sample Digestion Optimization for ICP-MS Analysis During Early
Stage Pharmaceutical Development
Keith B. McKellop, Boehringer Ingelheim, 39 Briar Ridge Rd., Danbury,
CT 06810
The controlling and the monitoring of residual metal impurities in early stage pharmaceutical development are important. The common practice is to set specifications for the trace metals in the final active pharmaceutical ingredients (API). This
approach can be costly, time-consuming, and may require re-working large batches
of an otherwise acceptable batch of API. An alternative approach is to develop quick
52
2012 EAS Abstracts
November 2012
obtained and empowered use of an automated dissolution system capable of diluting and effectively mixing dissolution aliquots for UV spectrophotometric analysis.
ment properly adjusted and prepared, sample preparation steps appropriate for the
material to be measured are undertaken. Proper sample filling, either manually or
by automatic sample filling unit, is important to ensure repeatability. Taking into account the physical properties of the sample and the possible presence of dissolved
gasses prevents errors that could invalidate the measurement. The final step, cleaning, makes sure that the density meter is prepared to accurately measure the next
sample to come. Following these simple steps helps to provide greater accuracy
and repeatability in density measurements, a longer and better instrument life span,
and greater ease of operation for a digital density meter.
340
Importance of Visual Observations in Dissolution Testing
Aditya A. Marfatia, Electroblab, Goregaon (East), Maharashtra, Mumbai,
400063 India, Kavita H. Singh, Amrita N. Bajaj
The dissolution test is a vital tool for formulation optimization, method development,
formulation changes, manufacturing changes, process modifications to comply with
regulatory requirements and compliance, in-vitro in-vivo correlation (IVIVC) and
scale up and post approval changes (SUPAC). One commonly overlooked parameter that can provide a wealth of information about the dosage unit under study is
a visual observation of the façade of dosage forms during dissolution process. We
have used a dissolution tester (EDT-08Lx, ELECTROLAB) coupled with iDisso-06
(ELECTROLAB) for the visual evaluation of osmotically controlled bi-layered extended release tablets. In addition to uninterrupted video recording of the dissolution test, the preprogrammed snapshot facility allowed comparison of the impact of
process and manufacturing changes on the dissolution profile. Visual observation
was complimentary to chromatographic data in evaluation of out-of-specification
results. Hence, it is imperative to visually observe the behavior of the dosage form
throughout the dissolution testing run which is not a common practice with researchers using conventional dissolution apparatus. Thus, visual imaging techniques have
potential applications in assessment of dissolution behavior of novel drug delivery
systems. Moreover, research and development, quality control and quality analysis
departments could periodically record the dosage forms undergoing dissolution for
record keeping purposes as a supplement to the analytical results.
344
Interactive Charts: A Powerful New Tool for Understanding
Chromatography Data
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, James A. Schibler, Ryo Komatsuzaki, Jay Lorch
Most laboratories are under pressure to increase productivity, which can be defined
in terms of results delivered per unit time. Advances in methodology, instrumentation, and automation have significantly reduced analysis time, often resulting in
much greater volumes of data generated every day. This has created a compelling need for fast, efficient data management tools that can help analysts keep up
with the data deluge. A new interactive charting capability provided in a modern
chromatography data system facilitates rapid visual screening of results, enabling
analysts to quickly gain an overview of trends and patterns, and to spot anomalies
or other specific items of interest. Single-click switching to different chart types or
source data, combined with instantaneous redraw, allows large volumes of data to
be scanned very quickly. Dynamic links between the charted values and their source
data provide an interactive experience in which analysts can instantly access details
behind the data points, choose different data sets to plot, and gain understanding
of the correlations between different aspects of their data. This solution provides
advantages over traditional tools for reviewing data, such as tabulated results or
externally-generated charts. Various applications of interactive charting for data
management and chromatographic troubleshooting are shown.
341
Analytical Instrument Qualification and Common GDP Issues
Vladimir Veselov, Johnson & Johnson, 1000 US Route 202, Raritan, NJ
08869, Helen Roytman, Lori Alquier
Most of the analytical instrument qualification activities (installations, operation and
performance testing) are executed by instrument qualification vendors; however, the
quality of qualification documentation provided by vendors may not meet corporate
requirements for good documentation practices (GDP) and compliance with Food
and Drug Administration regulations. Instrument qualification service providers that
are not the original manufacturers of the laboratory instrumentation may simplify
important procedures. As a result, the instrument qualification outcome might not be
adequate for the intended use. This presentation provides an example of qualification process and requirements, traceability analysis, and summarizes the most frequent problems with qualification. Practical case studies are presented to illustrate
best practices to release analytical instrumentation into production for GxP testing.
345
Evaluation of a ReactArray Workstation for Automation of Solution
Forced Degradation in an Active Pharmaceutical Ingredient
Shirley A. Rodriguez, Boehringer Ingelheim, 900 Ridgebury Rd., PO Box
368, Ridgefield, CT 06877, Jessica Bonilla, James Mullis, Scott Pennino
The ReactArray Workstation is a fully integrated synthesis high-pressure liquid
chromatography (HPLC)-data handling system which can be utilized to perform pH
and oxidative forced degradation stress studies in active pharmaceutical ingredients
(APIs). This poster presentation describes the system used and the experimental
design to eliminate much of the manual labor associated with sample preparation,
quenching, dilutions, and vialing of samples for injection onto the HPLC system.
This system offers flexibility with independently controlled reaction vessels. The profiles for assay and degradation impurities for a typical API are presented.
342
Methodologies for Improving Laboratory Safety
Tracey Jacksier, Air Liquide, 200 GBC Dr., Newark, DE 19702
The use of analytical instruments in the lab often necessitates the use of cylinder
gases. Analysts often worry about lack of sufficient training when using a new analytical instrument; however, training on the use of compressed and liquefied gases
is equally important. Compressed and liquefied gases, such as nitrogen stored in
dewars, can pose significant safety hazards if used or stored improperly. Inert gases
can displace air and cause an oxygen deficient atmosphere if there is a significant
leak (or venting from a dewar) and the cylinders are not stored in a well-ventilated
area. Oxygen deficiency monitors are critical for use in these areas to clearly identify potential safety hazards. Some analysts may improperly use the gas regulator
instead of the cylinder valve to shut the supply of gas to an analyzer. This may cause
premature failure of expensive equipment allowing leaks, and air and moisture entrainment. Air Liquide is striving to help minimize risks to our customers. I-SEE Gas
Handling Safety audits have been developed to survey how the gases used in your
facility are stored, used and distributed to your point-of-use with the objective of
identifying potential health and safety hazards. This presentation emphasizes the
use of SMARTOPTM, oxygen deficiency monitors and i-See Gas audits to improve
and enhance safety in Laboratories.
346
Using Micro-Flow Wafer Technology to Simplify the Determination
of Sub-ppm Levels of Oxygenated Compounds in Petroleum
Streams by Gas Chromatography
Mamdouh Farag, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484,
Andrew Tipler, John Irion
Gasoline contaminated with unapproved oxygenated compounds can negatively
affect the environment. Feed stocks of butenes, propylene, and naphtha contaminated with oxygenated compounds decrease catalyst activity in downstream
polymerization and olefinic cracking processes. The ability to detect precise subppm amounts of methyl tert-butyl ether, methanol, acetaldehyde and other C1-C5
oxygenated hydrocarbons in gasoline or in other petrochemicals is a very complex
problem. A simple separation with a single column cannot be used because of peak
co-elution. To address these limitations, more robust analytical methods must be
employed. A gas chromatograph equipped with a capillary split/splitless injector,
FID, a precolumn, a SwaferTM microfluidic switching device, and a high selectivity
column was used to enable these oxygenates to be monitored to sub-ppm levels.
Results demonstrated include the standard methods such as American Society for
Testing and Materials (ASTM) D7423-09, and UOP960-06, and the advantages of
the SwaferTM micro-flow technology over traditional valving or Dean Switches are
presented and discussed.
343
Good Density Measurement
Bill Kurtz, Anton Paar USA, 10215 Timber Ridge Dr., Ashland, VA 23005,
Dennis Meyers, Nicholas Groseclose
Today’s digital density meters are highly complex instruments designed to provide highly accurate and reliable measurements. However, many factors can work
against these instruments and influence the accuracy of their measurement. Performing good density measurements with a digital density meter requires care and
attention to detail in order to insure accuracy and reproducibility every time. Anton
Paar has specialized in digital density meters for over forty years and, with that
experience, has determined five basic areas of concentration in order to insure the
best possible density results. The first step is a confirmation of instrument stability
and accuracy by means of a water check. Water check failure is often a result of
insufficient cleaning. Should a water check fail repeatedly, especially after cleaning,
adjustment with appropriate reference media becomes necessary. With the instru-
347
Optimizing GC Parameters for Faster Separations with
Conventional Instrumentation
Anila Khan, Thermo Fisher Scientific, Tudro Rd., Runcorn, WA7 1TA
United Kingdom, Stephen Aspey, Robert Wiedemer
Cost is a significant factor when analyzing any sample. In gas chromatography
(GC), a typical analysis time can be more than 60 minutes, which means that cost
per sample is high and productivity is low. Analysis time can be reduced by careful
selection of the stationary phase, column geometry, temperature program and gas
flow. We demonstrate that by maintaining the phase ratio of the column, but by re-
53
2012 EAS Abstracts
November 2012
ducing its diameter that faster mass transfer and better efficiency can be achieved,
allowing for the use of shorter columns and producing faster separations.
fold. Conversely, choosing the wrong liner geometry can significantly decrease the
reproducibility and quality of a given analysis. In this poster, through a series of controlled injection parameters, we evaluate the differences between various GC inlet
liner designs for a group of analytes across a wide boiling point range.
348
Thermal Gravimetric Analysis/Mass Spectrometry Simulation and
Direct Polymer Analysis Using the GERSTEL Automated Pyrolyzer
John R. Stuff, Gerstel, 701 Digital Dr., Linthicum, MD 21090, Jacqueline
Whitecavage, Michael McAdams, Eike Kleine-Benne
This study shows the use of the GERSTEL MPS 2/TDU/CIS with pyrolysis module
for generating both simulated thermal gravimetric-mass spectrometry data (TGAMS) and for direct analysis of polymer samples. For simulated TGA-MS analysis, a
short piece of uncoated capillary is attached from the gas chromatography (GC) inlet to the mass spectrometer. A relatively slow temperature ramp is run similar to that
used for actual TGA analysis. The total ion chromatogram can be graphed against
temperature to produce simulated TGA-MS data. The mass spectral data is used
to identify degradation products at various temperatures. Direct thermal analysis
involves flash pyrolysis of the sample. The injection port is connected to the mass
spectrometer with a short piece of fused silica, The GC injection port and oven are
kept hot so that little or no chromatography is performed on the pyrolysate. This
configuration is similar to analysis by direct insertion probe. The spectra across the
“chromatogram” can be averaged and searched against a library for polymer identification. A set of standard conditions can be used to build a user specific library.
Several types of polymers are examined in this study. Simulated TGA-MS data are
compared with actual TGA data.
352
Simplifying Connections in the GC
Rob Freeman, SGE, 2007 Kramer Ln., Austin, TX 78758, Dan R. DiFeo, Jr.
Analytical chemists are increasingly turning to the use of multi-column capillary gas
chromatography (GC) systems to address the increasing demand for speed, selectivity, and/or sensitivity of analysis. Electronic pressure control has reached a
level that has permitted a revival of multi-column approaches. However the success
or otherwise of a multi-column GC approach now often depends upon something
seemingly mundane: column connections. Without proper care (or sufficient skill)
increasing the number of connections, unions and connecting conduits in the GC
system can increase the likelihood of leaks, un-swept volumes, and/or active sites.
Those using multi-column systems warmly welcome technologies that alleviate
these problems. Here we discuss our approach using micro-fabrication processes to
develop a number of robust solutions ideally suited to the harsh environment of the
GC oven including: stainless steel micro channel devices; leak free finger tight metal
ferrules; metal surface deactivation. We briefly discuss these technologies by providing case studies from our experiences developing multi-column GC approaches.
353
Use of an Innovative Reverse Flow Split/Splitless Injector for the
Simplification of ASTM D3606 Method
Massimo Santoro, Thermo Fisher Scientific, Strada Rivoltana, Rodano,
20090 Italy, Suresh Seethapathy, Andrea Caruso, Eric Phillips, Matthew
Lambing
A number of complex mixture analyses are today resolved using gas chromatography (GC) multi-column switching solutions. Even if apparently the analytical flow
is simple, these solutions require a rather complex hardware, from a construction
stand point, and dedicated software solutions to guide the chemists in setting up
method parameters. In case of system maintenance, for instance a simple trimming
of a column, the complexity of hardware solution sometimes makes it difficult and
time consuming for the chemist to set-up the system again so that often service
assistance is required. Simplification of analytical workflow for complex mixture
analysis is one of the challenging tasks leading the development of new instrumentation. Typical complex analysis involving two analytical packed columns and flow
switching systems can be replaced by this reverse flow split/splitless solution with a
large savings in terms of capital investment, system set-up and user training. Capability of this solution has been tested on the American Society for Testing and Materials (ASTM) method D3606. Effect of main parameters on system performance
is discussed in terms of cutting time and recovery of target compounds while still
preserving the necessary repeatability and reproducibility performance. This injector capability is part of a new gas chromatograph platform, incorporating compact
injectors and detectors, electronics and plumbing which make some standard multicolumn switching solutions looking obsolete. The unique level of modularity in GC
instrumentation does not require any longer dedicated packed column analyzers to
those laboratories with a handful of samples per day.
349
How Does Adding Water Change the Taste of Whisky?
David J. Scott, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484,
Andrew Tipler
The two senses of taste and smell are heavily involved in the enjoyment of potable
spirits and it is common to “nose” many beverages as part of the consumer experience. The nose of a beverage can describe much of the flavor and quality of the
drink but is in many ways a subjective test that requires experience to develop. Part
of the challenge is that blends of compounds produce different aromas due in part
to different odor thresholds and while it is possible to recognize certain compounds
others are not easily described. In this paper we describe a gas chromatography
olfactory accessory used in parallel with mass spectrometry to correlate the human experience with compound identification. There are several difficulties to be
overcome with respect to the sample analysis. Injection of neat whisky into the injector will inject a significant amount of water to the detriment of the column and
instrumentation. Headspace sampling would then be an excellent option but may
only inject a small volume that may not provide the same consumer experience, so
in this case, we use a headspace trap instrument that samples a larger volume of
the headspace. Active splitting of the analytes post column enables optimum control
of the carrier flow for effective separation and optimum detector performance. In
this example we apply the instrumentation to investigate the change in consumer
experience caused by adding water to whisky and attempt to settle the argument of
whether you should or not add water to whisky.
350
Advances in Ionic Liquid Capillary Columns
Leonard M. Sidisky, Supelco/Sigma-Aldrich, 595 North Harrison Rd.,
Bellefonte, PA 16823, Greg A. Baney, James L. Desorcie, Daniel
Schollenberger, Katherine K. Stenerson
Ionic liquids have received considerable interest as new green solvent systems in
the areas of organic reactions and separation technologies along with a number of
other areas. These materials are a class of non-molecular solvents that consist of
organic and inorganic cations and anions and have been found to possess negligible volatility, non-flammability, high thermal stability, and low melting points. There
are numerous combinations of cations and anions possible, so tailoring the material
to a specific application or function is a potential benefit of these materials.
354
A New Modular Approach to GC Instrument Design and Operation
Massimo Santoro, Thermo Fisher Scientific, Strada Rivoltana, Rodano,
20090 Italy, Dwain Cardona, Eric Phillips, Matthew Lambing
Gas chromatographic (GC) equipment requires specific hardware configuration with
appropriate inlets or detectors selected based on the application in use. Preventive maintenance and changing a system configuration to follow a new analytical
need or application is a difficult and time consuming operation. If different injector
and detector configurations are required, often a new system requirement results. A
new approach to the GC instrumentation design that allows the user an easy component change for preventive maintenance or configuration upgrade is presented.
As in modern modular high-pressure liquid chromatography, in this fundamentally
new GC design philosophy the individual instrument components are independent
modules that are combined in one instrument to deliver the desired analytical layout.
Inlets and detectors are in fact modular devices incorporating all flow, pressure,
temperature, and signal control. The injector and detector modules are then housed
on a GC oven where they connect to power supply, gas feeds and provide the
analytical column terminals. Details on the technical solutions offered by this easeof-use approach are described in this presentation. Particular emphasis is provided
to the design aspects of the injector and detector modules for ease of configuration
changes and preventive maintenance work. Data showing the integrity of the analytical performances also for most critical GC and GC-mass spectrometry applications are presented and discussed.
351
Inlet Liner Geometry and the Impact on Gas Chromatography Mass
Spectrometry Sample Analysis
Rob Freeman, SGE, 2007 Kramer Ln., Austin, TX 78758, Dan R. DiFeo, Jr.
The function of the gas chromatography (GC) injection port or inlet is to vaporize a
liquid sample and introduce a portion of that sample onto the GC capillary column
so that an effective separation can take place. Today there are a multitude of GC
inlet liner geometries and packing options available on the market. Coupled with
the various injection modes that are available, choosing the optimal inlet liner for
a given application is increasingly difficult. Choosing the correct liner design and
packing can significantly impact analytical performance. The use of glass wool in
inlet liners is well documented. Glass wool on the positive side helps volatilization,
as long as it is properly positioned inside the liner. On the negative side, glass wool
even if it is fully deactivated can cause breakdown of very active chemicals. Liner
choice also affects molecular weight discrimination. The best liner allows all compounds regardless of boiling point to load onto the column equally and in a sharp
band. In some cases optimization of the inlet system can improve sensitivities three
54
2012 EAS Abstracts
November 2012
355
Background for this includes the transformation from initial lab involvement with
traditional batch high-pressure liquid chromatography (HPLC) and subsequent SFC
technology transformation to that of a full-fledged, global purification support efforts
for Lilly Discovery Chemistry. Recent efforts include the current application of SFC
as well as the continued application of other “green” HPLC approaches like closed
loop peak shave recycling and steady-state recycling (SSR) to our chiral purification
workflow. In addition, mass guided SFC is utilized in a high-throughput manner in
part of our achiral purification workflow. SFC technology has allowed us to provide
mg-kg quantities of purified compounds for chemical synthesis support and biological study in a pharmaceutical discovery environment. We have done extensive
comparison of these techniques for various projects to determine the optimal chromatographic approach. Factors include throughput, productivity, suitability, solvent
consumption, and potential for scale up.
Water Determination in API Using GC TCD Detector
Heather (Huifang) Wang, Merck, 126 East Lincoln Ave., Rahway, NJ
07065, Yuri Bereznitski, Roy Helmy
Moisture determination in active pharmaceutical ingredients (APIs) and other
chemicals is commonly done by Karl Fisher (KF) titration using volumetric or coulometric titration methods. However, volumetric KF titration requires relatively large
amount of sample, and coulometric titration results might be affected by side reactions between sample components and titration reagents. In this regard gas chromatographic (GC) methods offer an advantage of direct interference-free analysis
of water and other volatile components in the sample matrix. In the current study the
authors evaluated suitability of gas chromatographic methods with thermal conductivity detection for water determination in pharmaceutically relevant solid and liquid
samples. The study was performed using both traditional and novel ionic liquid gas
chromatography phases of different polarity. Chromatographic data were critically
evaluated to the select method with best sensitivity and peak shape. Special considerations were given to sample preparation and diluent choice to improve method
quantitation. Limit of detection studies indicated that 10 ng of water can be readily detected on-column, which corresponds to 0.1%w limit of quantitation in solid
sample when 10 mg/ml sample prep is used. This method also shows good linearity.
The gas chromatographic approach of simultaneous determination of water and
residual solvents in pharmaceutical samples seems to be especially attractive for
early discovery areas where sample quantities are limited.
360
Improved Resolution Two-Dimensional Acquisition for CASE and
Manual Structure Elucidation
Steve Cheatham, DuPont Crop Protection, Stine-Haskell Research
Center, Newark, DE 19714, Michael Kline
Successful use of computer assisted structure elucidation (CASE) is very dependent on accurately peak picking spectra. In complex systems this becomes very
difficult because of spectral overlap. There are a large variety of methods published
to improve resolution in the F1 domain of a two dimensional (2-D). We have investigated the practical aspects of optimizing 2-D acquisition for resolution using a
combination of selective acquisition and non-uniform sampling. While this can be
optimized for individual samples the goal is to achieve a method that works for the
majority of samples and provides high-resolution data in a time frame consistent
with standard 2-D acquisition.
356
Analytical and Preparative SFC in Support of Pharmaceutical
Discovery and Development
Christopher Welch, Merck, 126 East Lincoln Ave., Rahway NJ 07065
Supercritical fluid chromatography (SFC) continues to develop as an essential tool
for supporting pharmaceutical discovery and development. In this presentation
we describe, using actual case studies, the evolution of the use of analytical and
preparative SFC to support discovery and development pharmaceutical synthesis,
highlighting recent trends, existing unmet needs, and future prospects for this technology.
361
NMR Spectroscopy of Small Molecules in Anisotropic Media: A
Powerful Tool for the Configurational, Conformational and
Constitutional Analysis of Small Organic Molecules
Roberto A. Gil, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh,
PA 15213
Residual dipolar couplings (RDCs) provide extremely powerful structural information in the analysis of small molecules, particularly when traditional nuclear magnetic resonance (NMR) experiments, such as 3J coupling constant analysis and nuclear
overhauser enhancement fail to provide a unique configuration and/or conformation.
The information obtained by the later methods is of local character and is restricted
to structural information in the local environment of a molecule. However, RDCs
provide information of non-local character and it is possible to determine the relative
configuration of stereocenters no matter how far they are located one from each
other. These NMR parameters help to lift the local information limitations provided
by traditional methods. Since RDCs are not directly observed in conventional liquidstate NMR experiment (isotropic conditions), the sample needs to be exposed to an
anisotropic medium to reveal their values. Anisotropy can be induced in the NMR
sample either by using stretched or compressed cross-linked polymers (gels) or
liquid crystal solutions of homo-polypeptides such as PBLG or PELG in CDCl3. The
presentation covers basic concepts on the origin of RDCs, as well as the features
and limitation of their use for stereochemical analysis of small molecules in organic
solvents. Sample preparation, NMR experiments for the acquisition of RDCs in
aligned media, as well as computationally based RDCs data analysis procedures to
determine relative configuration and conformation of small molecules are presented. Examples of the applications of RDCs to the configurational and conformational
analysis of natural and synthetic small molecules are discussed.
357
Separation of Ten Ionic Sulfated Estrogens by Packed Column
Supercritical Fluid Chromatography (SFC)
Manisha A. Patel, Pfizer, 224 Eastern Point Rd., Groton, CT 06340,
Mark Hardink, Frank Riley, Loren Wrisley, Mehdi Ashraf-Khorassani,
Christopher J. Hudalla, Jacob N. Fairchild, Jason F. Hill
This presentation discusses the separations of ten sulfated sodium salts of various estrogens using a variety of stationary phase packed columns were obtained
using supercritical fluid chromatography coupled with evaporative light scattering
detector and UV detection. In all separations, 10 mM ammonium acetate and H2O
were used as co-additives. Different modifier programs, temperatures, and water
percentages were employed to optimize the separations. It was determined that a
properly conditioned 2-ethylpyridine column provided the best separation compared
to new conditioned bare silica, diol, or cyano columns. The employment of both salt
and water as additives to the predominant CO2 mobile phase suggested a mixed
mode of separation involving both ion pairing of each anionic sulfated estrogen with
ammonium cation and hydrophilic interaction facilitated by partitioning of analyte
between the aqueous solvated stationary phase and the aqueous component of
the mobile phase. Optimization of the separation was also evaluated using sub2-micron particle size columns and advanced analytical SFC platforms.
358
Applying SFC in the Achiral World: Stationary Phases for
Supercritical Fluid Chromatography
William Farrell, Pfizer, 10777 Science Center Dr., San Diego, CA 92121
The use of supercritical fluid chromatography (SFC) is increasing for achiral applications within the pharmaceutical industry due to its orthogonal selectivity relative to
reversed-phase high-pressure liquid chromatography, its broad range of applicability for small molecules and its low organic solvent consumption. Analyte selectivity
can be further enhanced by modifying the functionality of the stationary phase to
obtain rapid separations of structurally similar compounds. Since both retention time
and peak elution order can be affected by changing the stationary phase, a number
of novel SFC phases have been developed to address specific separation challenges, several of which are now commercially available. In order to examine the
effects each phase has on selectivity, this presentation highlights the preparation of
these novel phases and present a comparison of small molecule selectivity to that
of similarly prepared 2-ethylpyridine and (1, 2)-propanediol phases.
362
Determination of Relative and Absolute Stereochemistry in the
Pharmaceutical Industry by NMR Methods
R. Thomas Williamson, Merck, 126 East Lincoln Ave., Rahway, NJ
07735, Gary E. Martin, Alexei V. Buevich, Christopher J. Welch
Stereochemical determination remains as one of the most daunting tasks in the
structure elucidation of pharmacologically relevant molecules. Recently, a number
of new or improved nuclear magnetic resonance (NMR)-based methods have been
developed or advanced for exploring both the relative and absolute stereochemistry
of structurally complex molecules. Often, these tools are used in a complementary fashion to provide a complete molecular description of the entity of interest. A
number of NMR methods have been developed recently to facilitate the extraction
of long-range nJCH heteronuclear coupling constants. These tools are essential
for the routine determination of relative stereochemistry through application of the
J-based configuration analysis method. Of these new methods, the heteronuclear
single quantum multiple bond correlation based-experiments appear to have made
the most significant impact. A review of the development and current state of these
pulse sequences are presented along with several pertinent examples. We also
discuss the utility of 3JCC coupling constants for the determination of relative stereochemistry. This 13C-13C homonuclear coupling information was previously unattainable due to sensitivity constraints. However, with the advent of high sensitivity
cryogenically cooled NMR probes and optimized pulse sequences, these data can
359
SFC and its Application to Preparative Separation of Pharmaceutical
Compounds
Eric Seest, Eli Lilly, Lilly Research Laboratories, Indianapolis, IN 46285,
Thomas Castle, Thomas Perun, Matthew Belvo, Douglas Schmidt, Paul
McDermott
The purpose of this presentation is to discuss the evolution of supercritical fluid
chromatography (SFC) preparative chromatography efforts within our laboratories.
55
2012 EAS Abstracts
November 2012
366
now be acquired on mg quantities of material. The extremely powerful nature of this
information when combined with density functional-theory (DFT) calculations is discussed. Finally, progress towards a new class of chiral derivatizing agents (CDAs)
is presented. These new CDAs provide the advantage of being stable enough to
handle in the open-air but reactive enough to derivatize most primary amines and
secondary alcohols directly in an NMR tube with straightforward interpretation of
results. A discussion of their development and application is presented.
Meeting Good Manufacturing Practice Requirements with Handheld
Spectroscopic Instrumentation
Mark Kemper, Thermo Fisher Scientific, Dieselstrasse 4, Karlsruhe
76227 Germany
Increasingly, regulatory authorities are placing more and more emphasis on qualityby-design. This concept begins with raw materials and works its way through the
processing stage before reaching the final product. The previous position of most
manufacturers was to wait until the final product was made to make sure quality
manufacturing had been accomplished. The analytical tools to make efficient and
effective raw material analysis possible are now available for point-of-use testing.
Miniaturized handheld instrumentation with extraordinary performance has begun
to make raw material testing much more effective. Not only has this streamlined
compliance, it also gives manufacturers more confidence in their overall operations.
This miniaturization has also reached the processing area and allows manufacturers the ability to use the same process monitoring in the development area as will be
used in scale-up and production. These tools are making pharmaceutical operations
much more flexible than was the case five years ago. All aspects from development
to production have realized the benefits.
363
Inverted 1JCC 1,n-ADEQUATE and 1JCC-Edited HSQC-1,nADEQUATE: A New Paradigm for Establishing Molecular Structure
Mikhail Reibarkh, Bruker BioSpin, Structure Elucidation Group, Rahway,
NJ 07065, R. Thomas Williamson, Gary Martin, Peter G Dormer, Merck,
Wolfgang Bermel
Undesired 1JCC correlations unavoidably “leak” into 1,n-ADEQUATE spectra because of the oscillatory nature of the amplitude transfer functions of the 1JCC and
nJCC coupling constants. Asymmetrical modification of the amplitude transfer delays provides a means of selectively inverting 1JCC correlation responses in 1,n-ADEQUATE spectra. The modified pulse sequence, in addition to editing the spectrum,
also provides a cleaner noise floor than the original 1,n-ADEQUATE experiment.
Unsymmetrical (UIC) or generalized indirect covariance (GIC) processing of a nonedited gradient heteronuclear single-quantum coherence (gHSQC) spectrum with
an inverted 1JCC 1,n-ADEQUATE spectrum affords a 1JCC -Edited HSQC-1,n-ADEQUATE spectrum. Direct (1JCC) correlations between pairs of protonated carbons
are diagonally symmetric and have negative phase; 1JCC correlations between
protonated and adjacent non-protonated carbons are diagonally asymmetric and
negative; long-range (typically via 3JCC) correlations between pairs of protonated
carbons are diagonally symmetric and positive; long-range correlations between
protonated and non-protonated carbons are diagonally asymmetric and positive.
Strychnine is used as a model compound to illustrate the results obtained. The resulting 1JCC -Edited HSQC-1,n-ADEQUATE spectrum provides unequivocal access
to 1JCC correlation data equivalent to 2JCH correlations in a gradient hetero-nuclear
multiple bond coherence (gHMBC) and predominantly 3JCC long-range correlations
equivalent to 4JCH gHMBC correlations. When used to complement a readily acquired gHMBC spectrum, the synergy of these data provides a powerful new paradigm for establishing molecular structures.
367
Withdrawn by the author.
368
On-Column N-Nitrosylation of Secondary Amines Observed in
High pH HPLC Impurity Separations
David P. Myers, Eli Lilly, 1400 W. Raymond St., Bldg 110, Indianapolis,
IN 46221, Zhongming Liang, Evan M. Hetrick, Chad E. Hadden, Steven
Bandy, Craig A. Kemp, Steven Baertschi, Thomas M. Harris
Data is presented that demonstrates that compounds containing secondary amines
can exhibit an artifactual on-column reaction during high-pressure liquid chromatography (HPLC) analysis when utilizing ammonium hydroxide, acetonitrile and columns with hybrid silica particles capable of operation in higher pH regions. These
common HPLC conditions were utilized in a column screening strategy for impurities method development; and therefore, the investigation and alleviation of this oncolumn reaction was important. This work provides strong evidence that the column
hardware is facilitating the reaction by a metal-catalyzed oxidation reaction. The
reaction appears to be aided by the presence of acetonitrile as the reaction is eliminated by the replacement of acetonitrile with methanol in the mobile phase. This
presentation discusses the impurity formation, its dependence on HPLC conditions
and studies to ascertain the mechanism of the artifactual impurity formation.
364
UV Resonance Raman Studies of Explosive Materials: Spectra
Detection Limits and Photochemistry
Sandy Asher, University of Pittsburgh, 219 Parkman Avenue, Rm. 701,
Pittsburgh, PA 15260, Luling Wang, Manash K. Ghosh
Standoff Raman detection of explosives and other threat chemical and biological
species requires maximizing the spectral signal-to-noise ratios (S/N). This requires
maximizing the intensity of the analyte signals while minimizing interference and
background noise. This optimization can be accomplished by deep UV resonance
Raman excitation that simultaneously increases the Raman cross sections, increases the spectral selectivity and avoids relaxed fluorescence interference. Unfortunately, the intensity is attenuated by self-absorption. We discuss the dependence of
the observed intensities and spectral S/N in standoff detection of resonance Raman
spectra of energetic molecules on these phenomena and the trade-offs required to
maximize spectral S/N.
369
Use of High Resolution LC-MS in Elucidation of Mysterious Peaks
caused by Trace Amounts of Impurities in Mobile Phase, HPLC
Vials, and Product Samples
Bin Chen, Merck, 181 Passaic Ave., Summit, NJ 07091, Xin Wang, Xin
(Jack) Yu, Min Li, Angela M. Harmon
Quick identification of unknown peaks that exceed certain levels (so-called identification thresholds as defined by the International Conference on Harmonization
guidelines) during high-pressure liquid chromatography (HPLC) analysis of pharmaceutical products is of critical importance from both the compliance and safety
perspectives. High-resolution liquid chromatography mass spectrometry (LC-MS)
is becoming an essential and powerful tool in expediting identification of unknown
peaks, especially the “mysterious” unknown peaks. Most unknown peaks are either
related to the active pharmaceutical ingredients, or excipient ingredients, or typical
leachables and extractables (e.g., plasticizers) from the packaging material. The
“mysterious” peaks are unknown peaks caused by impurities that fall out of the
aforementioned categories. These kinds of unknown peaks are often extremely difficult to be identified without high-resolution LC-MS analysis. Three case studies are
presented to demonstrate the critical roles of high-resolution LC-MS during elucidation of mysterious peaks caused by trace amounts of impurities in mobile phase,
HPLC vials, and product samples.
365
Impact of Design of Experiments on Pharmaceutical Calibration
Models Based on Near-Infrared Spectroscopy
Benoit Igne, Duquesne University, 410C Mellon Hall, Pittsburgh, PA
15282, Robert W. Bondi Jr., Carl A. Anderson, James K. Drennen III
The objective of this work was to determine how the predictive performance of partial least squares (PLS) models is affected by the experimental design. Design of
experiments are widely used to produce a set of calibration samples for generating
empirical models based on vibrational spectroscopy (e.g., near-infrared). There are
numerous experimental designs that can be utilized, but the choice often depends
on the available resources and the intended use of the model. However, the predictive performance of the model may be influenced by the designed experiment
and number of replicates at each design point. The following experimental designs
were evaluated for a model pharmaceutical composite system: 5-level full factorial,
3-level full factorial, central composite, and D-Optimal. The factors for all designs
were acetaminophen content and ratio of microcrystalline cellulose (MCC) to lactose monohydrate. Other constituents included croscarmelose-Na and magnesium
stearate (content remained constant). Five 13-mm compacts were produced at each
level (5 levels for a total of 150 compacts). PLS-based models were generated according to each design using near-infrared spectroscopy. Models were generated
using data from individual design of experiments. The predictive performance of
each model was evaluated based on an independent validation set for both spectroscopic techniques. The effect of each designed experiment was evaluated by determining the statistical significance of the difference in bias and standard deviation for
its model prediction performance.
370
Quality Investigation of PF-04991532 Reference Standard:
Challenges in the UPLC Method Development for Polar Impurities
Yun Huang, Pfizer, Eastern Point Rd., Groton, CT 06340, Elise Clement,
Tasneem Patwa, Angel Diaz, Wendy Wang, Karen M. Alsante
High-pressure liquid chromatography (HPLC) has been the predominant analytical
tool for pharmaceutical analysis in many aspects. Recent years, to fully utilize the
advantages of increased speed, resolution, sensitivity and efficiency over HPLC,
ultra-PLC has gained widespread acceptance in pharmaceutical industry and has
started to enter the quality control laboratories as a standard instrumentation. Polar
impurities analysis is a well-known challenge for chromatographic method development since the polar impurities tend to elute in the front region of the chromatogram,
causing the difficulty in the separation and peak integration and the risk of missing
key impurities or degradation products in the drug substances and drug products.
This presentation discusses a quality investigation on PF-04991532 reference
standard for a polar impurity that was eluted in the solvent front and missed to be
56
2012 EAS Abstracts
November 2012
reported. A UPLC method development to separate the polar impurities presented
in the drug substance and drug products as well as the correction and prevention
actions associated with the investigation are highlighted.
are demonstrated on a low dose tablet formulation where solid form conversion during manufacturing and storage was anticipated and investigated. In this case, the
multimodal chemical imaging approach quickly identified fluorescence imaging as
a sensitive solid form indicating method which allowed for the support of systematic
process development activities with chemical imaging methodology. Additional examples of the application of multimodal imaging in pharmaceutical product development may be discussed.
371
Artifactual Degradation of an Active Pharmaceutical Ingredient:
Impact of Glass Surface Activation
Michael Watkins, Eli Lilly, Lilly Corporate Center, Indianapolis, IN 46285,
Xia Dong, Carolyn Stobba-Wiley
During the development of the impurity and potency method for an active pharmaceutical ingredient (API) a solution stability study was performed in the analytical
sample solvent. This experiment demonstrated solution stability for up to five days.
However, during method validation the API was found to degrade rapidly in solution,
generating a degradation product above 0.1% over the duration of the validation
study. The discrepancy between the stability observed in the original solution stability study and the instability observed in the validation experiments was found to
be rooted in the type of glassware in which the sample was stored. This presentation discusses the mechanism of degradation, examine the physical and material
properties of the glass contact surfaces and identify the reason for the observed
API solution stability (or lack thereof) when stored in the various glass containers.
375
Transmission Electron Microscopy (TEM) of Nanoparticles in
Polymers
Barbara Wood, DuPont Central Research & Development, Experimental
Station, Route 141, Wilmington, DE 19880
Polymer nanocomposites hold the promise for lightweight materials with a very desirable balance of properties, but the properties of greatest interest – mechanical,
electrical, barrier, and others – depend on the dispersion of inorganic nanoparticles.
TEM of thin sections has emerged as a preferred technique for assessing nanoparticle dispersion. The detailed, un-averaged microstructure revealed by TEM allows
comparison of nanoparticle morphology near sample surfaces versus in the bulk.
Sampling and imaging of typical nanoparticles and their dispersions in epoxies and
thermoplastic polymers are discussed together with examples of dispersion problems in commercially available nanocomposites.
372
Exploring Iridescence in Hummingbird Feathers and Its
Relationship to Effect Pigments
Jerome Jourdan, BASF Corporation, 1609 Biddle Ave., Wyandotte, MI
48192
Hummingbirds are considered jewels of the avian world for their ability to fly with
blinding speed, and for their bright, iridescent colors. Scientists have studied iridescence and coloration in bird feathers since the early 1900’s. But until now, they
have relied on spectroscopy, and light and electron microscopies to understand the
mechanisms that produce such colorations. This presentation focuses on the use
of atomic force microscopy for characterizing the microstructures in the barbules of
Ruby-throated Hummingbirds to explain the differences in observed color between
the bright red gorget feathers and the green back feathers. We also attempt to answer the question of why the gorget feathers of some male hummingbirds appear
bright orange in late summer prior to molt and how these color differences relate to
effect pigments produced in the laboratory today.
376
Dichroism Spectroscopy of Microscopic Particles
Dale Purcell, John Jay College of Criminal Justice, The City University
of New York, 445 W 59th St., New York, NY 10019
Microscope photometry is recognized internationally by various technical and scientific working groups as a valuable instrumental analysis technique to microspectroscopically compare color through a quantitative and objective method. Dichroism
is the selective absorption of plane polarized light between two orthogonal directions by an anisotropic material. Ordinarily the absorption of light in a dyed fiber or
polymeric thin film is dependent upon a sample’s thickness and concentration, in
contrast, the ratio of absorbancies along the two principle directions is a function
of the orientation only and is independent of sample thickness and concentration.
In this presentation we discuss how dichroic ratio analysis may increase the classification and discrimination power of color analysis of microscopic particles using
microscope photometry.
373
Minor and Trace Element Qualitative Analysis in VP-ESEM - Part 1
Robert A. Carlton, GlaxoSmithKline, 709 Swedeland Rd., King of
Prussia, PA 19406
Primary electron beam scatter in the variable pressure: environmental scanning
electron microscopy (VP-ESEM) degrades the spatial resolution of chemical analysis using energy dispersive X-ray spectrometry. The resulting X-ray spectrum can
include X-rays from regions outside the feature of interest. One qualitative analysis
strategy is dependent upon the observation that X-ray count peaks due to scattered
electrons increase with increasing chamber pressure whereas X-ray peaks due to
the primary electron beam decrease. This rule is reasonably well-established for
major elements, but there has been little reported extending it to minor and trace
element qualitative analysis. An initial study of the accuracy of qualitative analysis
of minor and trace elements using the X-ray counts versus chamber pressure rule
is reported herein. In a previous study using NIST SRM 2066 glass and an ESEM
equipped with a gas path reducer, the slope of X-ray counts versus chamber pressure behaved as expected for elements with compositions as low as ~9%. In this
current study, NIST SRM 1873 K963 glass and an Hitachi low vacuum SEM with
no gas path reducer were used along with a Bruker Xflash 30 mm2 EDS detector. A
sample of the glass was mounted and polished. The mounting medium contained C,
O, Si, Ca, Al. The results of this study clearly indicate that the aforementioned rule
for qualitative analysis does not hold for trace elements and cannot be completely
depended upon for minor element analysis. Strategies for minor and trace element
analysis in VP-ESEM are proposed.
377
A Short History of Forensic Microscopy
Skip Palenik, Microtrace, 790 Fletcher Dr., Suite 106, Elgin, IL 60123
The microscope and microchemistry have been utilized since at least the time of
Raspail (who coined the term chemical microscopy) when he appeared in court
cases against the famed father of toxicology in Paris in a number of high profile
cases in the 1830s. Using benchmark cases from the past, the historical roots of
forensic science are traced through the pioneers of forensic science who were all
experts in the use of the microscope. They are introduced to the audience though
their cases, all of which illustrate the scientific method at its best and the pivotal role
of the microscope as forensic science developed and matured. The cases serve as
inspiring examples of the power of reason when it is fed by facts developed through
the use of the microscope.
378
Microscopical Examination of the Explosive Triacetone Triperoxide
Peter Diaczuk, John Jay College of Criminal Justice, The City University
of New York, 445 W 59th St., New York, NY 10019
The use of improvised explosives for criminal, domestic and international terrorism is an ever-increasing problem. Methods for homemade manufacture of these
non-military and non-industrial explosives often require little-to-no background in
chemistry. The clandestine manufacture of triacetone triperoxide (TATP) is no exception. This rather exotic explosive is currently receiving considerable attention
because of its ease of synthesis and procurement of starting materials. The research presented here was spawned by earlier work detailing the optical properties
of two identified TATP polymorphs.[1-2] This study examines the crystal morphology
of TATP synthesized using different acid catalysts. The potential linkage between
crystal morphology and the specific acid catalyst used during synthesis could be
used as an investigative lead when attempting to determine provenance of a TATP
sample submitted as evidence.
374
Multimodal Chemical Imaging in Pharmaceutical Development –
Application of Fluorescence Microscopy
Mark Strohmeier, GlaxoSmithKline, 709 Swedeland Rd., King of
Prussia, PA 19406
The distribution of components and their solid form in solid drug dosage forms are
often critical for the performance of the drug product and require control during manufacturing, storage and its intended use. This presentation outlines a multimodal imaging approach that utilizes spatially correlated Raman mapping and fluorescence
imaging for drug product analysis. While Raman mapping is a well-established
techniques for chemical imaging of solid dosage forms, the fluorescence properties
of active ingredients and excipients have not been utilized for chemical imaging
in drug product analysis routinely. The combination of these two techniques takes
advantage of the strengths of both techniques namely chemical specificity of Raman
and sensitivity of fluorescence imaging and can overcome the limitations of both
methods namely slow data acquisition and chemical specificity that often restrict
their effective utilization in development efforts. The advantages of this approach
[1] Hietpas et al. 2005.
[2] Speir et al. 2006.
379
The Use of Mid-IR Microspectroscopy for Forensic Glass Analysis
Brooke W. Kammrath, University of New Haven, 300 Boston Post
Rd., West Haven, CT 06516, John A. Reffner, Nicholas D.K. Petraco,
Thomas A. Kubic
Silicate glass is a common and valuable class of transfer evidence discovered in
criminal and civil litigation. Glass is an excellent source of physical evidence because it is brittle and fractures into fragments that are easily transferred from the
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2012 EAS Abstracts
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383
scene to victim and perpetrator. The challenge to the forensic scientist is to associate these fragments with their source. The analysis of the molecular structure of
glass is a novel forensic method that is not currently employed by forensic scientists.
Attenuated total reflection (ATR) mid-infrared (mid-IR) microprobe analysis could
be easily incorporated into the analytical scheme of glass because it requires only
minimal additional sample preparation to that which is already done for refractive
index analysis. Mid-IR microprobe analysis can also be used for samples too small
for elemental analysis as it is capable of analyzing samples as small as 10-μm in
length which is smaller than the ablated hole made in laser ablation-inductively coupled plasma-mass spectrometry. With the use of chemometric methods, specifically
principal component analysis-canonical variate analysis or partial least squares
analysis, ATR mid-IR spectroscopy provides both end-product classification and
glass discrimination. ATR mid-IR spectral analysis offers information about the molecular structure of soda-lime silicate glass to support other traditional analysis and
strengthen the association of this evidence.
Insights into Amyloid Formation Mechanisms from Studying Fibrils
by Solid-State NMR
Patrick C. A. van der Wel, University of Pittsburgh, 3501 Fifth Ave, BST3
Room 2044, Pittsburgh, PA 15116
Various disorders are characterized by the formation of amyloid fibril plaques: e.g.,
Alzheimer’s and Huntington’s Disease (HD), but also prion diseases. In each case
one or more proteins undergo aggregation, which seems to correlate with disease
onset. Intriguingly, independent of the protein, the final amyloid fibrils seem to share
many structural features. Much of our knowledge of amyloid fibril structure has been
obtained from solid-state nuclear magnetic resonance (SSNMR), but it is sometimes
uncertain to what degree fibril structures can help us understand the toxic species, which may not be the fibril itself, but rather a transient oligomeric precursor.
In certain amyloid-related diseases, the amyloidogenic protein has a well-defined
native structure, which somehow gets subverted or destabilized during aggregation.
In a number of such proteins, an intriguing correlation between an amyloidogenicity and the ability to form domain-swapped multimers has been noted. This could
indicate a role for native-like structure in the mature fibril or (importantly) in the toxic
oligomers, or both. I discuss our SSNMR studies of one of these fibrils, which turn
out to be non-native in structure. In other experiments we have probed HD-related
aggregates, revealing some surprising structural features of the final fibrils. Here,
the aggregating protein fragment is disordered prior to aggregation. I discuss how
the characterizations of these two quite different amyloid systems knowledge of the
fibril structure provide insight into both the mechanism of assembly and the (possibly toxic) oligomeric precursors.
380
Structure Determination of Uniaxially Aligned Biological
Macromolecules by Solid-State NMR
Alexander A. Nevzorov, North Carolina State University, Department of
Chemistry, 2620 Yarbrough Dr., Raleigh, NC 27695, Robert Knox
Oriented-sample nuclear magnetic resonance (OS NMR) has emerged as a powerful technique for structure determination of membrane proteins in their native lipid
environment. In OS NMR, the structural information is directly contained in the
angular-dependent NMR frequencies. Several issues of critical importance for OS
NMR are addressed. The first one is line narrowing due to uniaxial ordering and rotational diffusion. We have derived closed-form expressions for the NMR line widths
of uniaxially diffusing proteins and performed line shape simulations using the more
general Stochastic Liouville Equation. It was found that the line widths contain as
much structural information as the OS NMR frequencies, and can be used as a
complementary structural restraint. Second, we have developed a method for the
spectroscopic assignment of OS NMR spectra using magnetization transfer under
the mismatched Hartmann-Hahn conditions. This allows one to transfer magnetization between the dilute spins that are over 6 Angstroms apart. The method is
exemplified by solid-state NMR spectra of a single crystal and assignment of OS
NMR spectra of Pf1 coat protein in the phage form and reconstituted in magnetically aligned bicelles. Finally, an algorithm that extracts three-dimensional structure
entirely from the dipolar couplings is presented. Effect of experimental uncertainty
on the quality of the calculated structures is assessed.
384
Light-Induced Phase Transition and Deposition of Polymers
Chaoying Ni, University of Delaware, 201 DuPont Hall, Newark, DE
19716
Photon-material interactions have been exploited in energy conversion, signal
transmission, photonic devices, and material processing and reconstruction. As an
application example of material modifications, light-induced simultaneous surface
activation of carbon nanotubes (CNTs), vaporization of candidate polymers and uniform re-deposition onto CNTs in CNT-forest or CNT-yarn are realized in the absence
of solvents and high temperature. The energetic photons with a narrow wavelength
distribution from a vacuum ultraviolet (VUV) lamp are found to be capable of activating the CNT surface by breaking the C-C bonds to graft the polymer molecules
de-bonded from the surface of a candidate polymer. Poly(methyl methacrylate)
(PMMA), polystyrene (PS), and poly(3-hexylthiophene) (P3HT) are successfully
deposited onto the surface of the CNTs in a N2 environment for 30 minutes and
the resulting polymer coating homogeneously covers the CNT circumference. Xray photoelectron spectroscopy (XPS) data reveal that the atomic bonding occurs
between the CNT and the coating material. Molecular dynamic simulation elucidates
a wrapping process of the P3HT on CNT.
381
Solid-State NMR Analysis of HIV Protein Assemblies
Marvin Bayro, National Institutes of Health, 9000 Rockville Pike,
Bethesda, MD 20892, Robert Tycko
We describe the application of solid-state nuclear magnetic resonance (NMR) spectroscopy to the structural and dynamical characterization of protein assemblies
formed by the matrix and capsid domains of type 1 human immunodeficiency virus (HIV-1) Gag polyprotein. Matrix domain (MA) proteins self-associate and bind
phospholipid bilayers to anchor Gag assembles, while capsid domain (CA) proteins
self-assemble into the closed lattice of the viral capsid. The main limitation to the
analysis of MA complexes and CA assemblies in a physiological constructs is their
large size and insoluble nature. However, solid-state NMR spectroscopy is a highresolution technique that circumvents these limitations. We present our initial results
in characterizing the molecular structures of MA and CA assemblies via solid-state
NMR, with particular emphasis on elucidating supra-molecular interactions.
385
Terahertz Scanning Reflectometry: A New Frontier for Non-Invasive
In-Depth Investigation
Anis Rahman, Applied Research & Photonics, 470 Friendship Rd., Suite
10, Harrisburg, PA 17111, Aunik K. Rahman, Bozena B. Michniak-Kohn
Terahertz scanning reflectometry offers an opportunity to investigate both the surface and the sub-surface of biological tissues (e.g., skin) in a non-invasive fashion.
The non-ionizing nature of T-ray eliminates radiation damage of sensitive tissues
while be able to probe disease conditions in the deeper layers leading to an effective real-time diagnostic tool. For example, measurement of permeation kinetics and
concentration gradient in a non-invasive mode is important in several areas such
as in the study penetration behavior of an active ingredient through human skin
or other tissue. Two critical factors are: the concentration gradient of permeating
ingredient across the depth of skin; and the kinetics (or rate) of such permeation.
These factors are essential for quantitative analysis, for example, via Fick’s laws
of diffusion. In this study, a terahertz scanning reflectometer (TeraScan®) has been
used for direct measurement of both concentration gradient across the thickness of
stratum corneum and permeation kinetics of an active ingredients. Two analytes,
hydrocortisone and caffeine were investigated. Some details are discussed in terms
of experimental data.
382
Structural Studies of Multidrug Resistance Membrane Proteins by
Oriented and Magic-Angle-Spinning Solid-State NMR Spectroscopy
in Lipid Bilayers
Nathan J. Traaseth, New York University, 100 Washington Sq. E, New
York, New York 10003, Anindita Gayen, James R. Banigan
Multidrug resistance (MDR) is a pervasive clinical problem that reduces the effectiveness of treatment against bacterial infections, viral infections, and cancer.
Efflux of drugs across the lipid bilayer by MDR membrane protein transporters is
one way in which resistance is conferred to the host organism or cell. To derive a
structure/function relationship for this class of proteins, our study uses the small
multidrug resistance (SMR) family as a model system for deciphering the mechanism of transport with the long-term goal of decoding the evolutionary importance of
the transporter family. We used solid-state nuclear magnetic resonance (SSNMR)
spectroscopy to study the topology and structure of EmrE in liposomes (magicangle-spinning or MAS) and magnetically aligned lipid bilayers (oriented SSNMR or
O-SSNMR). The MAS experiments were used to map chemical shift perturbations
upon drug binding while the O-SSNMR experiments revealed the change in helix
orientation upon binding and transport. Together these complementary techniques
showed that drug binding perturbs both the structure of EmrE as well as the helical
orientations with respect to the lipid bilayer.
386
Activation and Passivation of a Biosensor Model Surface on a
Crystalline Silicon Substrate
Yue Liu, University of Delaware, Department of Chemistry, Newark, DE
19711, Andrew Teplyakov, Junghuei Chen
Si(111) substrate was used to build a biosensor model surface. After preparing
H-terminated Si(111) by standard wet chemistry methods, it was modified with
11-amino-1-undecene monolayer followed by a reaction with sulfo-succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (Sulfo-SMCC) cross-linker.
Then a single strand DNA oligomer with 5’ and 3’ end modified with biotin and thiol
respectively was covalently attached to the surface through the robust thiol chemistry. Then the entire system was exposed to the streptavidin-coated gold nanopar-
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2012 EAS Abstracts
November 2012
390
ticles to label the biotin DNA through the biotin-streptavidin interactions. Finally, the
remaining surface reactive sites were chemically blocked with 1-octadecanethiol
(ODT). X-ray photoelectron spectroscopy (XPS) data showed an increasing nitrogen signal after labeling the DNA with streptavidin coated gold nanoparticles. Sulfur
presence from the thiol ssDNA and ODT was detected and analyzed by using the
time-of-flight secondary ion mass spectroscopy. Fourier-transform infrared spectra
indicated a well-ordered self-assembled monolayers formation after passivation of
the unreacted surface sites with ODT. Atomic force microscopy showed approximately 1 nm decrease in the apparent height of gold nanoparticles after ODT passivation. Transmission electron microscopy (TEM) was used to observe the ODTpassivated surface with gold nanoparticles on silicon. Further experiments follow
the in-situ DNA hybridization events by using this biosensor model surface.
Obtaining Federal Funding to Support Your Research
Frederick H. Walker, Air Products and Chemicals, 7201 Hamilton Blvd.,
Allentown, PA 18195, Marcie F. Walker
This presentation provides a framework for developing a winning proposal to obtain federal assistance to support your research and development efforts. Specific
topics include: 1) an overview of the federal grant portals, such as grants.gov; 2)
identifying appropriate funding opportunity announcements (FOA); 3) working with
federal agencies and program officers; 4) engaging your organization; 5) becoming
a principal investigator; 6) assembling a proposal-writing team; and 7) the description of a process to ensure that an optimal proposal meeting all the requirements of
the FOA is submitted.
391
Strategies for Preparing Successful Proposals
Alexis Nagengast, Widener University, One University Pl., Chester, PA
19013
Funding rates are at an all-time low and it is increasingly important to submit a
high quality proposal to maximize the likelihood of funding. Practical approaches for
submitting a successful proposal are discussed with emphasis on considerations
for first time applicants. Additionally, methods for completing funded work are presented.
387
Mechanistic Understanding of Protein-Silicone Oil Interactions
Archana Jaiswal, Biolin Scientific, 514 Progress Drive, Suite G,
Linthicum Heights, MD 21090
Prefilled glass syringes are commonly used as delivery devices for therapeutic
agents. Silicon oil, a common lubricating material, often lines surfaces of these syringes. Unfortunately, this oil can adversely affect formulations by causing protein
loss. Interfacial adsorption of proteins is the key step and maybe responsible for the
phenomenon of soluble-protein loss when contacting silicone oil and the irreversible
adsorption of protein may be associated with protein denaturation and aggregation. Quartz crystal microbalance with dissipation and interfacial tension measurements were used to study the interactions between abatacept and two surfactants,
polysorbate 80 and poloxamer 188. Both polysorbate 80 and poloxamer 188 were
observed to reduce the interfacial tension between silicone oil and abatacept in buffer. Polysorbat 80 was more effective than Poloxamer 188 in preventing abatacept
adsorption. Moreover, it was noted that some of the adsorbed abatacept molecules
were not desorbed readily upon buffer rinse. No homogeneous aggregation was observed at room temperature and a slight increase of aggregation was only observed
for samples measured at 40 °C which can be prevented using Polysorbate 80.
392
Insights into Grants and Funding at the National Science
Foundation
Charles D. Pibel, National Science Foundation, 4201 Wilson Blvd., Suite
1055, Arlington, VA 22230
This talk will provide background information on opportunities for funding for research in analytical chemistry and allied fields at the National Science Foundation.
Different funding mechanisms are discussed, as well as emerging priority research
areas in the Foundation, where analytical chemists can actively participate.
393
NIH 101: Turning a Good Idea into a Funded Research Project
Charles G. Edmonds, National Institutes of Health, 45 Center Dr.,
Bethesda, MD 20892, Douglas M. Sheeley
While the federal government is a major source of research support, the agencies
and processes involved may seem arcane. Just as there is more to a good novel
than plot, a successful grant application needs more than the germ of a good idea.
The application and review process at the National Institutes of Health requires understanding of many details, including the policies governing submission of applications and the review process, technical aspects of online systems used for submission and subsequent communications, and perhaps most importantly, the staff who
are available to help you and when to contact them. This presentation discusses the
primary components of the process, including the identification of appropriate agencies and programs, writing an effective application, responding to reviewers’ criticisms, and taking full advantage of program administrators. The speakers explore
these issues from the perspectives of the applicant, reviewer, and administrator,
with some emphasis on the young investigator. References to additional resource
388
Enhanced XPS Depth Profiling of Biological and Polymeric
Materials Using an Argon Cluster Ion Beam Source
Brian R. Strohmeier, Thermo Fisher Scientific, 5225 Verona Rd.,
Madison, WI 53711, Richard G. White, Tim S. Nunney, Paul Mack
Detailed knowledge of surface chemistry is a crucial factor for the successful production and optimization of innumerable advanced materials, organic coatings, and
multi-layer thin film devices. X-ray photoelectron spectroscopy (XPS), with its high
surface sensitivity and chemical state specificity, is the ideal analytical technique for
evaluating the qualitative and quantitative surface composition of materials. XPS
depth profiling, where XPS is combined with argon ion sputtering, allows the identification of chemical variations from the topmost surface of solid materials to many
micrometers deep into the bulk, which facilitates characterization of complex layered structures. However, in-depth analysis of many materials by XPS depth profiling is often problematic when using traditional monatomic argon ion beam sources
because of chemical damage caused by the ion beam sputtering process. A recently
developed argon cluster ion beam source was used in this study for soft XPS depth
profiling of a variety of biological and organic materials to minimize ion-induced
damage during the analyses. Comparisons were made to samples analyzed using monatomic argon ion beam depth profiling. The materials examined include: a
multi-layer amino acid thin film structure; poly(methyl methacrylate) (PMMA) coatings for contact lens; and hydrophobic fluoro polymer plasma coatings for textiles.
XPS depth profiling results obtained with the argon cluster ion beam source were
consistent with minimal ion beam damage and preservation of important chemical
state information throughout the profiles.
394
Nuclear Magnetic Resonance: Rich in History, Rich in Application
Jeffrey Reimer, University of California-Berkeley, Department of
Chemical and Biomolecular Engineering, 201 Gilman Hall, Berkeley, CA
94720
Nuclear spin, as probed by magnetic resonance, have now been associated with
Nobel Prizes in Physics, Chemistry, and Medicine. Magnetic resonance spectroscopy and magnetic resonance imaging (MRI) have impacted virtually every field
of science and engineering, and enjoy a plethora of applications, ranging from
medicine and biological sciences, physical sciences and engineering, to industrial
sensing and process control. I illustrate the rich history of magnetic resonance by
juxtaposing great works of antiquity with current applications of magnetic resonance
in material and chemical sciences.
389
Dynamic Reorientation of Stearic Acid on a Gold Surface Induced
by Ambient Water Molecules Studied by Infrared Spectroscopy
Takeshi Hasegawa, Kyoto University, Laboratory of Solution and
Interface Chemistry, Gokasho, Uji 611-0011 Japan, Takafumi Shimoaka
Gold surface is conveniently used as a substrate of Langmuir-Blodgett (LB) film for
measuring infrared reflection-absorption (RA) spectra with a high sensitivity and
the useful surface-selection rule. In addition, a gold surface is chemically inert and
stable, which is another good character as an LB film substrate. Nevertheless, at the
same time, a gold surface is known to have a relatively hydrophobic characteristic,
on which some mono-layers cannot be fabricated. In the present study, we focus
on a poorly transferred LB film (or molecular adsorbates) of stearic acid on gold.
When the poor LB film was analyzed in ambient and dried conditions by using the
polarized-modulated infrared reflection-absorption spectrometry and the conventional RA techniques respectively, the spectra exhibited dramatic changes with a
low rate particularly during the drying process. The spectra changes suggested that
the orientation and the molecular conformation were both greatly changed irreversibly by the adsorbed ambient water molecules.
395
Solid-State NMR Structural Characterization of Designer SelfAssembling Peptides
Anant Paravastu, Florida State University, College of Engineering, 2525
Pottsdamer St., Tallahassee, FL 32310
We report solid-state nuclear magnetic resonance (NMR) structural measurements
on the peptides RADA16-I and MAX8, which were rationally designed to self-assemble into nanofiber matrices. These matrices have technological applications
in regenerative medicine: they can support 3-dimensional cell cultures, stem cell
encapsulation, and neuron repair. The amino acid sequences are characterized by
alternating hydrophobic and hydrophilic residues, intended to promote β-strand formation and self-assembly through backbone hydrogen bonding.[1, 2] MAX8 has the
additional feature of salt-triggered self-assembly, ostensibly triggered by dielectric
screening to overcome side chain repulsion and promote hairpin structure necessary for self-assembly. Through NMR spectroscopy of natural abundance samples
as well as samples with strategic placement of 13C isotopic labels, we have analyzed
spectral features and 13C-13C dipolar couplings. These data were used to constrain
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2012 EAS Abstracts
November 2012
all-atom computer models of peptide nanofibers. Despite the simplicity of amino
acid sequence motifs, nanofiber molecular structures have significant unpredicted
features. Molecular models will enable further studies of nanofiber adaptive dynamic
reassembly and triggered self-assembly.
Farmworkers wore SERS sensors into the field as dosimeters, urine samples were
collected and vapor sweeps of the farmworker barracks were performed. Some preliminary results are presented herein.
400
Rapid Detection of Food Pathogens by Surface-Enhanced Raman
Spectroscopy
Atanu Sengupta, Real-Time Analyzers, 362 Industrial Park Rd., Unit
8, Middletown, CT 06457, Chetan Shende, Hermes Huang, Stuart
Farquharson, Frank Inscore
Food borne diseases pose major health and public safety concerns. According to a
new statistic released by the Center for Disease Control and Prevention (CDC), as
many as 9.4 million episodes of foodborne diseases were reported in 2011, which
resulted in 55,961 hospitalizations, and 1,351 deaths. The challenge to prevent
foodborne illnesses lies in the fact that even a single bacterial cell in contaminated
food products can rapidly multiply and exceed the infectious dose limits within a few
days. Traditional cell culture based methods can detect and identify a single bacterial cell but unfortunately the detection process also takes a similar time period (1-4
days). Hence to ensure food products are safe for people, there is a critical need
for a pathogen analyzer that can rapidly extract, detect, and identify (within 1-2
hours) a broad range of food pathogens at exceptionally low concentrations, and
with a specificity that differentiates from indigenous microflora, so that false alarms
are eliminated. To address this need, we have been developing a sampling system that isolates such pathogens from food, and selectively binds these pathogens
to a sensor substrate, which produces surface-enhanced Raman spectra (SERS).
In this presentation we show preliminary SERS measurements of four important
food pathogens - E. coli, Listeria, Salmonella, and Campylobacter at less than 1000
colony forming units per gram of food.
[1] Ellis-Behnke, R. G., Liang, Y. X., You, S. W., Tay, D. K. C., Zhang, S. G., So, K.
F., and Schneider, G. E. (2006), Proc. Natl. Acad. Sci. U.S.A. 103, 5054-5059.
[2] Haines-Butterick, L., Rajagopal, K., Branco, M., Salick, D., Rughani, R., Pilarz,
M., Lamm, M. S., Pochan, D. J., and Schneider, J. P. (2007), Proc. Natl. Acad.
Sci. U. S. A. 104, 7791-7796.
396
Long-Lived Signals and Metal MRI
Alexej Jerschow, New York University, Department of Chemistry, 100
Washington Square East, New York, NY 10003
We have recently discovered that long-lived signals can be excited in certain solid
systems, including bone tissue samples. The appearance of such signals in dipolar
coupled networks is at odds with commonly accepted thought about such processes. The origin of these signals is currently unknown, but they present a great opportunity for nuclear magnetic resonance (NMR) and magnetic resonance imaging
(MRI) interrogations of rigid systems, including rigid tissues in-vivo. In the second
part of this presentation, we focus on recent work on MRI of Lithium-ion batteries, in
which the location of microstructure buildup upon charging is visualized.
397
Nuclear Magnetic Resonance of Heavy Metals: Experiment and
Calculation
Cecil Dybowski, University of Delaware, Department of Chemistry,
Newark, DE 19716, Shi Bai, Fahri Alkan, Jaclyn Catalano, Nicholas
Zubulyadis, Yao Yao, Anna Murphy, Robert Taylor, Silvia Centeno
The investigation of nuclei such as Pb-207 and Hg-199 opens a wide range of nuclear magnetic resonance (NMR) studies, which leads to examination of a wider
range of materials. We report application of nuclear magnetic resonance to a variety
of lead- and mercury-containing materials, from semiconductors to artists’ paint formulations. The connection of experimental results to chemical processes requires
models which predict the NMR parameters for a given chemical structure or condition. The calculation of chemical shielding for multi-electron systems, using models
of such as lead- and mercury-containing materials is intensive, but the calculations
can lead to qualitative correlation of NMR parameters with chemical state. We show
examples from our recent work.
401
Understanding the Role of Physical and Chemical Adsorption on
the Raman Enhancement from Nano-Metallic Surfaces
Jason Guicheteau, United States Army Edgewood Chem Bio Center,
Attn: RDCB-DRD-L, 5183 Blackhawk Rd., APG, MD 21010, Steven
Christesen, Jerry Cabalo, Augustus W. Fountain III, Craig Knox, Erik
Emmons, Ashish Tripathi
We present the results of a three year collaboration between the US Army Edgewood Chemical Biological Center, and the US Army Research Laboratory-Aldelphi
Laboratory Center on the evaluation of various nano-metallic surfaces developed
for the Defense Advanced Research Programs Agency (DARPA) surface-enhanced
Raman spectra (SERS) Science and Technology Fundamentals Program. The primary role of the two Army labs was to develop the analytical and spectroscopic
figures of merit to compare the sensitivity and reproducibility of the various SERS
substrates submitted by the program participants. We present the design of a procedure for evaluating SERS active surfaces. We also introduce the use of receiver
operating characteristic curves to define an effective enhancement factor for substrate comparison.
398
Enhancement Factors for SERS Substrates
Stuart Farquharson, Real-Time Analyzers, 362 Industrial Park Rd (#8),
Meriden, CT 06457, Hermes Huang, Chetan Shende
Surface-enhanced Raman spectroscopy (SERS) provides exceptional specificity
and sensitivity and promises to be a powerful analytical chemistry tool. Specificity is
provided by Raman scattering producing rich molecular information, while sensitivity
at the part-per-billion concentration level is provided by the plasmon field surrounding silver or gold nanoparticles. SERS has been successfully demonstrated for biotechnological, environmental, homeland security, military, medical, and pharmaceutical applications. However, the exceptional sensitivity provided by SERS is mostly
due to “hot spots,” plasmons at structures such as particle gaps or junctions that can
provide as much as 10-orders of magnitude enhancement. Unfortunately, hot spots
are not evenly distributed across substrates, and hence signal intensities cannot be
quantitatively reproduced. This presents difficulties in defining sensitivity when one
is concerned about detecting low analyte concentrations. In an effort to better define
sensitivity; several methods have been developed to determine enhancement factors (EF), so that the performance of various substrates can be compared. In this
presentation, we will compare these EF methods applied to several commercial
products, using two standard analytes, benzenethiol and bispyridylethylene.
402
Impact of Secondary Metal Oxides on the Oxidative Reactivity of
Manganese Dioxide
Huichun (Judy) Zhang, Temple University, 1942 N 12 St., Philadelphia,
PA 19122, Saru Taujale
Manganese oxides (MnO) typically exist as mixtures with other metal oxides in soilwater environments; however, information is only available on their redox activity as
single oxides. To bridge this gap, we examined five binary oxide mixtures containing
MnO2 and a secondary metal oxide (Al2O3, FeOOH, Fe2O3, SiO2 or TiO2). The goal
was to understand how these secondary oxides affect the oxidative reactivity of
MnO2. Using triclosan as the probe compound, pseudo-first-order kinetic results
showed that the secondary metal oxides typically lowered the reactivity of MnO2 by
surface complexation/precipitation and/or heteroaggregation. For Al2O3 and SiO2,
comparable inhibitory effects were observed for hydroxylated Al3+ and Al2O3 and for
SiO2 and silicate species, most likely through complexation/precipitation of soluble
Al/Si species with MnO2 surface which blocks the active reactive sites. For goethite
and hematite, soluble Fe(III) concentrations in the oxide suspensions were less than
0.6 micromolar (at pH=5), but significant inhibition of MnO2 reactivity was not observed until [Fe(III)] had reached 1 micromolar. Heteroaggregation is thus the most
likely interaction mechanism between FeIII oxides and MnO2. TiO2 inhibited MnO2
reactivity only when a limited amount of triclosan was present. Due to the strong
adsorption and slow desorption of triclosan by TiO2, precursor-complex formation
between triclosan and MnO2 was much slower and became the new rate-limiting
step (as opposed to electron transfer in all other cases). Our ongoing work explores
surface complexation modeling to identify the dominant surface complexes formed
and also the extent of heteroaggregation based on the DLVO theory.
399
Surface-Enhanced Raman Spectroscopy as a Pesticide Monitor
Kevin M. Spencer, EIC Labs, 111 Downey St., Norwood, MA 02062,
Anthony R. Rowey, James M. Sylvia, Susan L. Clauson
Persistent pesticide presence is a fact of life for the majority of farm workers. Longterm health care predictors are dependent on accurate pesticide identification and
quantification, including the percentage of pesticide ingested and the extent of exposure. As the farmworker performs chores, how much pesticide becomes airborne?
Of the airborne pesticide, how much enters the farmworker through oral, dermal or
other routes? How much of the pesticide residuals are transported to the farmworkers residence and are other family members exposed? An inexpensive sensor is
required to provide significant data on the farmworker populace as a whole that
can: 1) act as a dosimeter to quantify pesticide presence; 2) detect pesticides/metabolites in urine to determine ingestion levels; and 3) provide instantaneous data
on pesticides present within dwellings. EIC Laboratories, in conjunction with Wake
Forest University and the North Carolina Farm Workers Bureau, have been testing
surface-enhanced Raman spectroscopy (SERS) as a monitor for all three concerns.
60
2012 EAS Abstracts
November 2012
403
Reactivity of scCO2 with Iron Bearing Minerals
Kristin D. Lammers, Temple University, Department of Chemistry,
Philadelphia, PA 19122, Leanne VanBuren, Daniel R. Strongin
Mineral carbonation reactions of iron oxides and fayalite (Fe2SiO4) involving “wet”
CO2 and aqueous sodium sulfide was investigated by in-situ attenuated total reflection Fourier transform infrared spectroscopy. Mineral trapping carbon dioxide
as a carbonate mineral is a permanent and secure method of CO2 sequestration.
Morphological changes were observed with transmission electron microscopy. The
reaction between scCO2 with dissolved water and either ferrous silicate and a ferric oxyhydroxide was followed at 35 and 50 ˚C and 80 bar. The results show water
concentration is a critical parameter in mineral carbonation and siderite (FeCO3) formation. The lack of carbonation during exposure to “dry” scCO2 showed the importance of the presence of water in the scCO2 on reactivity. The addition of aqueous
sulfide in contact with “wet” scCO2 and iron oxyhydroxides was also investigated in
the present study. It was experimentally determined that siderite (FeCO3) formation
was dependent on sulfide concentration in the wet scCO2. The observations of the
formation of FeCO3 based on in-situ and ex-situ evidence illustrates the relatively
short timescale for both iron reduction and mineral transformation during exposure
to scCO2 in the geological subsurface.
lary to suppress electroosmotic flow (EOF) allows sample injection from each end
of the capillary. Therefore, a sample can be subjected to appropriate amounts of
pressure and time of injection from opposite ends of the capillary to allow equal
concentrations of each active ingredient to be introduced into the capillary. Once
voltage is applied, the active ingredients will migrate from their respective injection
site at opposite ends of the capillary toward the detector. Representative electropherograms from a separation of Advil® Cold & Sinus Liqui-Gels®, where each gel
contains almost seven times larger amount of ibuprofen, a non-steroidal anti-inflammatory drug, compared to pseudoephedrine are shown. Due to the large difference
in active ingredient amount between the analytes of interest, quantitative analysis
under conventional CZE conditions would be more difficult than under the proposed
DOI-CZE method.
407
Flexibility and Scalability of Core-Shell Based Particles for HPLC
and UHPLC Separations
Jason A. Anspach, Phenomenex, 411 Madrid Ave., Torrance, CA 90501,
Jeff Layne, Lawrence Loo, Carl Sanchez, Tivadar Farkas
The last ten years of high-pressure liquid chromatography (HPLC) development
has seen a massive increase in HPLC separation performance. At first these improvements were powered by small fully porous particles in conjunction with new
high-pressure instrumentation. This technology came to be known as ultra (UHPLC). In 2006 there was a reemergence of particles based upon core-shell technology. These particles consist of a non-porous silica core wrapped in a porous
silica layer. This latest generation of core-shell materials is able to provide UHPLC
performance, with significantly lower resistance to flow (lower operational pressure).
The lower pressure provides a greater flexibility in terms of column architecture
as well as system compatibility essentially decoupling performance from pressure
requirements. Core-shell technology also benefits from the decoupling of the shell
porosity from the particle size, allowing for application specific particle engineering.
In this talk we show several different types of core- shell particles that are specifically designed for ultra high-performance applications, lower pressure applications,
and medium to large molecule (bio) applications. We demonstrate that the synthetic
flexibility of the core-shell materials allows for optimization of the particle performance attributes for each specific application while maintaining backpressures that
are acceptable for the instrumentation typically used in each industry.
404
Precipitation of Layered Fe(II)-Al(III)-Hydroxides during Reaction of
Aqueous Fe(II) with Al-Oxides and Clays
Evert J. Elzinga, Rutgers University, 101 Warren St., Newark, NJ 07102
The reactivity of aqueous Fe(II) with aluminum oxide and montmorillonite clay in
anoxic solutions was investigated with batch experiments and Fe K edge X-ray absorption spectroscopy (XAS) measurements for characterization of Fe(II) sorption
trends and products. The XAS data indicate the formation of Fe(II)-Al(III)-layered
double hydroxide (LDH) phases during Fe(II) sorption with the mineral substrates
under conditions common to reduced geochemical environments. These secondary Fe(II) phases are composed of brucite-like Fe(II)-hydroxide sheets with partial
substitution of Al(III) for Fe(II), charge balanced by anions coordinated along the
basal planes. The Fe(II)-Al(III)-LDH precipitates form fast (on time scales < 24 h)
and are therefore expected to be an important sink of Fe(II) in suboxic and anoxic
geochemical environments, where reductive dissolution of Fe(III)-oxides leads to
the buildup of high concentrations of dissolved Fe(II) in solution. In addition, owing
to small particle size, layered structure, and high Fe(II) content, these phases are
likely to be highly reactive towards redox-active contaminants such as Cr(VI) and
U(VI), and may control retention of divalent metals such as Ni(II) and Zn(II) through
adsorption and co-precipitation reactions. The research presented here characterizes formation of these phases in relation to observed macroscopic sorption trends
of Fe(II) as a function of Fe(II) concentration, pH and reaction time.
408
Expanding the Versatility of SFC Through New Enhancements to
MS Directed, Open Bed Fraction Collection Systems
Steven Zulli, Waters, 159 Lukens Dr., New Castle, DE 19720, Jonathan
L. Jones, Cuping Luo, Timothy P. Martin, Ziquang Wang, Harbaksh
Sidhu
Several new design and software features are demonstrated, creating versatile
preparative, semi-preparative and analytical scale supercritical fluid chromatography (SFC) systems, utilizing mass directed, open bed fraction collection. These
enhancements for the Waters Prep 15, 30, and 100 SFC-mass spectrometry (MS)
Directed systems are the first with a method selectable capability to perform injections in either modifier (co-solvent) or combined stream, enabling the choice of the
advantages of either injection mode, allowing method development and purification
of a wider range of compounds. The stacked injection module, which can be configured for modifier or combined stream injections, allows high-throughput purifications
with repetitive injections and collections in a single run, with fractions repeatedly
collected into a single vessel per compound. The new Stacked Injector Graphical
Editor is an easy mouse click driven interface which simplifies optimization of injection and collection parameters, such as injection interval and run time, supporting
all FractionLynx timed events, such as fixed time window or peak threshold determined collections, and different collection thresholds for each compound. The
stacked injector can operate as automated, or can be controlled in real-time for the
optimization of injection parameters during a run. These systems utilize the MassLynx/FractionLynx software, featuring open-bed automated sample handling and
fraction collection, supporting Mass, UV, and evaporative light scattering detector
directed purifications, and OpenLynx walk-up operation. These system’s analytical
scale capabilities and the AutoPurify software enable automation of pre-purification
sample screening and focused gradient selection, and purity determinations of collected fractions. Presented are examples and limitations of these new capabilities,
and typical performance results.
Coupled Redox Transformation of Arsenite and Chromate on
Ferrihydrite
Daniel R. Strongin, Temple University, Department of Chemistry,
Philadelphia, PA 19096, Narayan Bhandari, Elizabeth Cerkez, Richard
J. Reeder
The simultaneous redox transformations of arsenite (As(III)) and Chromate (Cr(VI))
in the presence of the iron oxyhydroxide, ferrihydrite, have been investigated. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray
absorption near edge structure (XANES), and solution phase analysis have been
used to characterize the surface bound and aqueous phase species. Both ATRFTIR and XANES show that the exposure of ferrihydrite to As(III) and Cr(VI) for
up to 12 hours in the dark leads to significant changes in the oxidation state of the
adsorbed or aqueous phase As and Cr species. The majority of the surface bound
As(III) is oxidized to arsenate (As(V)) and also a substantial amount of Cr(VI) being
reduced to chromite (Cr(III)). Exposure of the As(III)/Cr(VI)/ferrihydrite to simulated
solar radiation results in faster kinetics and therefore more complete redox transformations compared to the dark experiments. At a solution pH of 5, the conversion of
As(III) to As(V) and Cr(VI) to Cr(III) on ferrihydrite results in minimal amount of Fe(II)
being released into the aqueous phase, which contrasts to previous studies, where
just As(III) and Ferrihydrite present, results in the noteworthy release of Fe(II). The
lack of Fe(II) release into the solution shows that Cr(VI) reduction is not occurring in
the solution phase and hence suggests that the reaction is occurring on the surface.
In addition, the majority of the As(V) and Cr(III) products remain bound to the ferrihydrite surface.
405
409
Two-Dimensional Chromatography and the Life Cycle of Complex
Polymeric Materials
Stephan Moyses, SABIC, 1 Noryl Ave., Selkirk, NY 12158
There are typically several stages in the life cycle of a polymeric material during
which the molecular weight or the composition distributions can be modified. The
first stage is the synthesis which can occur in several sequential steps. The polymer
can then be compounded and this may also include several steps, for example, one
for blending with another polymer and one for the addition of inorganic fillers. The
next stage is processing (e.g., via injection molding) which can impact significantly
406
Simultaneous Determination of Oppositely Charged Active
Ingredients in Advil® Cold & Sinus Liqui-Gels® Utilizing DualOpposite Injection Capillary Zone Electrophoresis
Donna M. Blackney Beckett, Drexel University, Department of Chemistry,
3141 Chestnut St., Philadelphia, PA 19104, Joe P. Foley
The ability of dual-opposite injection capillary zone electrophoresis (DOI-CZE) to simultaneously separate oppositely charged active ingredients in an over-the-counter
pharmaceutical drug is demonstrated. The use of a poly(vinylalcohol)-coated capil-
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2012 EAS Abstracts
November 2012
the molecular weight or composition distributions. Finally, the resulting part might
be exposed to harsh chemicals or heat as part of the manufacturing process or
during its active duty as a component depending on the application. In this paper, I
illustrate how two-dimensional chromatography (2-D) with gradient polymer elution
chromatography in the first dimension and size exclusion chromatography (SEC) in
the second dimension can provide invaluable insight into a material’s composition
and how it can improve our understanding of the changes it undergoes during the
different stages of its life cycle. I discuss the example of a star styrene-butadiene
block copolymer (SB)n where the 2-D data evidences several populations that are
clues left by the sequential anionic polymerization process. The thermo-oxidative
degradation of this (SB)n are also investigated. I also discuss poly(phenylene ether)
to illustrate how compounding and processing can alter the original polymer.
designing a near 100% current efficient electrodialysis unit and applying a fixed potential, the approach results in a current signal that directly measures the transient
species. In this presentation we discuss the utility of the charge detector for analysis
of both fully and weakly dissociated ionic species. Further, we compare our results
from the charge detector with a suppressed conductivity setup. Example analysis of
real life samples such as drinking water, waste water, fruit juices and wine are also
presented here.
[1] Anal. Chem., 2010, 82 (3), pp. 951–958.
413
Development and Evaluation of an SEC Method for the
Determination of Monomer Conversion in Polymerization Systems
Containing Non-Volatile Reactants and Additives
Walter B. Shepherd, Organomation Associates, 266 River Rd., West,
Berlin, MA 01503
Monomer conversion in polymerization reactions has typically been achieved by
weighing of the subject polymer following isolation either by evaporation of the reaction solvent, unreacted monomer and other volatile components of the polymerization system or by precipitation of the polymer in a non-solvent. In either case the
determination of the weight of polymer formed is dependent on the “isolatibility” of
the polymer which in turn is dependent on the volatility of the monomer and other
system components and/or limited solubility of the polymer. In systems where the
monomer and/or system components are not sufficiently volatile or have solubility
characteristics equal to that of the polymer the determination of the monomer conversion is difficult and time consuming. A new method has been developed for the
determination of monomer conversion based on the direct analysis of the monomer
and other system components isolated by size-exclusion chromatography (SEC).
Volatile system components are either removed by selective evaporation at atmospheric pressure or together with the non-volatile components are used as internal
standards. The method is applicable to the analysis of polymerization systems containing high boiling/non-volatile monomers, solvents and system additives and the
Mw, Mn, Mw/Mn and monomer conversion are determined simultaneously.
Multiple Headspace GC Analysis Meeting the Needs of Advanced
Drug Delivery Technologies
Elizabeth C. Banks, Bristol-Myers-Squibb, Reeds Lane, Wirral, CH46
1QW United Kingdom
Accurate quantification for equilibrium headspace gas chromatography (GCHS)
depends on a several factors including sample volume, the partition coefficient of
the compound of interest between the gas and liquid phases in the sampling vial,
response factors of the detector to the analytes of interest and instrumental constants. With advancing drug delivery technologies, e.g., the application of spray
dried dispersions (SDD’S), which utilize polymer properties in drug product formulations, sample matrix is becoming increasingly important as a factor in accurate,
low level detection of volatiles. Traditional pharmaceutical analysis has relied upon
volatile sample preparation utilizing liquid injection and GCHS for increased sensitivity or viscous/solid samples. As GCHS does not introduce the sample matrix to
the column, which may result in interference and column contamination, it provides
the opportunity for cleaner analyses with greater sensitivity and accuracy which is
ideally suited to the complex characteristic of SDD’s . Furthermore, the technique
of multiple headspace extraction (MHE), commonly used in the analysis of solid
samples such as vinyl chloride monomers and PVC resins, presents an additional
more robust and accurate way to measure volatiles in SDD’s. This presentation
discusses the advantages and disadvantages of liquid and GCHS techniques for the
analysis of complex matrices, such as SDD’s and shows evaluations of the application of MHE as an alternative. The presentation contains example determinations
of multiple volatiles in several current and active SDD systems by both GCHS and
MHE techniques. Conclusions regarding method robustness, accuracy and manipulation of the data generated are presented.
411
414
410
A High-Performance Anion-Exchange Chromatography with
Pulsed Amperometric Detection Assay for Carbohydrates in Urine
as a Measure of Intestinal Permeability
Michael Hvizd, Thermo Fisher Scientific, 3000 Lakeside Dr.,
Bannockburn, IL 60015, Peter Bodsky, Deanna Hurum, Jeffrey S. Rohrer
A wide range of diseases and pharmaceutical therapies can cause damage to the
small intestinal mucosa leading to changes in intestinal permeability. Examples include celiac disease, rheumatoid arthritis, HIV, food allergies and intolerance, nonsteroidal anti-inflammatory drug use, and stress. These changes have additional
consequences, including poor absorption of nutrients, further complicating potential
treatments for the original disease. Measuring the absorption of metabolically inert carbohydrates, such as D-mannitol and lactulose, is a non-invasive method for
evaluating intestinal damage. In this work a high-performance anion-exchange with
pulsed amperometric detection (HPAE-PAD) chromatographic assay was developed to quantify carbohydrates in urine. The five metabolically inert carbohydrates
are resolved from each other and elute within 16 minutes with a total analysis time of
23 minutes. Linearity of the method was evaluated with coefficients of determination
greater than 0.995. Method precision, as relative standard deviation of peak area,
was less than 2%. Samples were evaluated after preparation by dilution. Carbohydrate recoveries, measured by spiking samples prior to sample preparation, ranged
from 91-115% across three days of sample analysis indicating method accuracy.
This assay allows the sensitive detection of multiple indicative carbohydrates without complex sample preparation, time-consuming derivatization, or matrix elimination steps.
Headspace GC Analysis of Ethylenediamine and Other Residual
Solvents in Polyethyloxazoline
Jeff Parish, Shimadzu Scientific Instruments, 7102 Riverwood Dr.,
Columbia, MD 21046
Ethylenediamine is a very toxic substance that is a widely used building block of
chemical synthesis. When toxic substances are used to produce products that are
ingested, verification that residual solvent levels are below required thresholds is
critical. Polyethyloxazoline is one such compound. It is used in the cosmetic and
pharmaceutical industries as a carrier for active ingredients and is water-soluble.
Headspace gas chromatography (GC) is typically used for residual solvent analysis,
but amines are very difficult to analyze by GC because the strong basic nature of
the molecules interact with acidic fused silica columns to cause significant peak tailing that reduces the limit of quantitation. In this analysis, a 60 meter column, which
was developed specifically for the separation of amines, is used with headspace
analysis to assay solvents used in the synthesis of polyethyloxazoline, including ethylenediamine, ethanol, and toluene. The monomer of polyethyloxazoline, 2-ethyl2-oxazoline, was also quantitated.
415
Development and Validation of GC Method for Metacresol Assay
Minli Liu, United States Pharmacopeia, 12601 Twinbrook Pkwy.,
Rockville, MD 20852, Shane Tan, Fatkhulla Tadjimukhamedov, Samir
Wahab
The current United States Pharmacopeia (USP) Metacresol monograph lacks organic impurities test and uses titration for assay. To modernize the monograph, USP
uses a gas chromatography (GC) method submitted from a pharmaceutical manufacturer as the organic impurities test. With some modifications, the GC method was
adopted to replace the titration assay. The modernized organic impurities procedure
uses G7 type of capillary column, helium as carrier gas, and flame ionization detection. In the assay, the oven temperature program was modified to shorten the run
time; and phenol was incorporated as an internal standard. These modifications ensured fast and accurate method for quantitation of metacresol. The assay procedure
was validated and found to be specific, precise, accurate, and linear.
412
Charge Detector, a New Detector for Ions for Ion Chromatography
Kannan Srinivasan, Thermo Fisher Scientific, 445 Lakeside Dr.,
Sunnyvale, CA 94087, Art Fitchett, Sheetal Bhardway, Mrinal Sengupta,
Barbara Shao, Peter Bodsky, Christopher Pohl
The role of a suppressor in ion analysis using ion chromatography is to remove the
influence of the background conductivity while allowing detection of ions in a weak
background. As practiced today with electrolytic suppressor devices the current applied plays an important role dictating the observed background and noise performance. The current efficiency of the device thus is an important parameter to consider when designing suppressor devices. In this presentation we discuss how to
control or optimize the current efficiency of suppressor devices. Further we discuss
applying this methodology to a novel charge detector device. Recently Dasgupta
et. al.[1] described a new charge detector concept. In this concept an electrodialysis
type unit is used to de-ionize a suppressed effluent stream containing analytes. By
416
Shape-Based Correlation of GC-MS Compositional Data to Fuel
Performance Properties Using Partial Least Squares Regression
Jeffry A. Cramer, United States Naval Research Laboratory, 4555
Overlook Ave. SW, Washington, DC 20375, Robert E. Morris
The links between a fuel’s chemical constituency and resultant fuel properties and
performance are being investigated for the purposes of comprehensive fit-for-pur-
62
2012 EAS Abstracts
November 2012
pose (FFP) fuel modeling. The goal of this research is to develop a link between
the entire composition of a fuel and how it would perform in any given application.
This approach addresses the limitations of current property specification-based fuel
assessment methodologies and provides a framework for determining the FFP parameters of a fuel in any engine or system, regardless of fuel type or source. In order
to address such a wide-ranging goal, a comprehensive analysis strategy must first
be developed that accurately correlates fuel compositional information to known
performance properties. In previous work, a chemical profiler was used to quantify
the constituencies of fuels using gas chromatography mass spectrometry (GC-MS)
data, and said constituencies were then correlated to specification properties using
partial least squares regression modeling. However, this method not only requires
the use of a separate GC-MS profiler prior to the actual data modeling, but also
requires an ad-hoc methodology to extricate usable chemical information from large
numbers of competing masking compounds. To streamline the procedure, a new,
profiler-free methodology has been developed that evaluates data shapes in a more
direct fashion. It is shown that this new procedure produces fuel property models
that are more capable of discriminating meaningful chemical relationships without
data unmasking.
from simple and complex matrices. In SDME, a single drop of solvent is suspended
from the syringe needle and exposed to the headspace in a sealed vial containing a
sample. The drop is withdrawn into a syringe and injected in to the instrument. Distribution constant and analytical figures of merit including precision, accuracy, limit of
detection and limit of quantification were determined using gas chromatography and
gas chromatography mass spectrometry for the instrumental analysis. Ionic liquids
offer an attractive alternative to traditional liquid solvents for static headspace single
drop microextraction.
420
New Functionalities on Monolithic Silica HPLC Columns
Egidijus Machtejevas, Merck KGaA, Frankfurter St. 250, Darmstadt,
64293 Germany, Karin Cabrera
Monolithic high-pressure liquid chromatography (HPLC) columns are made of one
piece of silica possessing macro- and mesopores. The macropores offer high permeabilities and low column backpressures whereas the mesopores create high
surface areas needed for a sufficient chromatographic separation process. The selectivity of these columns is determined by the surface modification, which is most
typically a C18 reversed phase type. Recently, we have developed a monolithic
column with aminopropyl - selectors which can be used for the separation of sugars
(and other components) using partitioning chromatography. Interestingly, even short
columns (25-100mm length) show high selectivities for sugars of similar structures.
In cases with limitations in the selectivity, we have coupled two or more columns for
adding separation efficiency which is generally possible due to the low column backpressure of these columns. Moreover, monolithic HPLC columns have proved to be
extremely useful for the analysis of “dirty” samples without prior sample preparation
as the macropore size is of around 2-µm. We show applications on the analysis of
sugars from serum samples as well from food and beverage samples.
417
Analysis of Fentanyl and Norfentanyl in Human Urine by
Multidimensional GC-MS
Paul V. Macek, Shimadzu Scientific Instruments, 7102 Riverwood Dr.,
Columbia, MD 21046, Carl E. Wolf, Alphonse Poklis
Multidimensional gas chromatography (MDGC) is a powerful emerging technique
for separation and quantification of complex mixtures. Most techniques involve use
of two conventional detectors or one conventional detector and one mass spectrometer. In some cases, the technique cannot be employed because the conventional detector is not sufficiently sensitive to allow detection or because the mixture
is too complex to be analyzed on a conventional detector. Such is the case with
the drug fentanyl and its metabolite norfentanyl in human urine samples. Modern
advances in mass spectrometry (MS) have resulted in mass spectrometers that
are significantly more sensitive than most conventional detectors. The sensitivity
of the Shimadzu QP-2010 Ultra gas chromatography (GC)-MS combined with a
robust vacuum system allows both the primary column and the “cut” column to be
interfaced to the mass spectrometer resulting in a fast, sensitive and comparatively
interference-free analysis. Implementing the method involved optimizing numerous
GC and MS parameters. These included GC injection technique, GC column stationary phase selection for both columns, which included column lengths, column
diameters, and column flows, as well as MS scan parameters. It was also necessary
to overcome technical limitations imposed by the vacuum system and the maximum
temperature of the available stationary phases, all while maintaining the separation
of relatively low concentration analytes in the presence of very high concentration
interferences, with an acceptable analytical analysis time.
421
Selectivity of Stationary Phases Utilized in HILIC Chromatography
David S. Bell, Supelco/Sigma-Aldrich, 595 North Harrison Rd.,
Bellefonte, PA 16823, Carmen T. Santasania, Wayne K. Way, Hugh
Cramer, Craig R. Aurand
Interest in chromatography using aqueous-organic mobile phases high in organic
content, aqueous normal-phase and hydrophilic interaction liquid chromatography
(HILIC), has continued to build in recent years.[1,2] In previous studies, significant
contribution of stationary phase chemistry toward the manipulation of retention and
selectivity in HILIC has been demonstrated.[3] The aim of this continuing study was
to further enhance our knowledge of mechanisms of retention that dominate in this
interesting mode of chromatography. When different stationary phase chemistries
are applied in reversed-phase chromatography, alternative retention and selectivity
are most often observed. The same can be said for the impact of different stationary
phase chemistries when applied in HILIC mode. Since retention and selectivity are
dependent on the retention mechanisms exhibited by the stationary phase chemistry, it is of great importance to better understand the retention mechanisms provided
by these polar phases. In this study, three different stationary phase chemistries,
including bare silica, pentafluorophenyl and pentalol, are investigated. The relative
dominance of partitioning, polar and ionic forces are determined through systematic
experimentation. The impact of variation of dominant retention mechanisms are related to real chromatographic separations and useful general trends are developed.
418
Quantitative Analysis of Steroids Using SPME Coupled to GC/MS/
MS
Shilpi Chopra, Seton Hall University, Center for Academic Industry
Partnership, 400 South Orange Ave., South Orange, NJ 07079,
Nicholas Snow
Triple quadrupole mass spectrometry (GC-MS/MS) is generating great interest as
a multidimensional technique because of its high selectivity due to the presence of
two quadrupoles. GC-MS/MS was used for the analysis of new type of steroids and
designer steroids which are a range of steroids containing minor chemical modification to evade detection. The main aim of this research was to reduce chemical
noise, thereby increasing signal-to-noise and lowering detection limit using MS-MS
to get good quantitation parameters for each designer steroid. Solid-phase microextraction (SPME) conditions were developed for each designer steroid. The fiber
was chosen according to the polarity of the steroids. Once the fiber chemistry was
chosen, extraction optimization was done using a full factorial design of experiment,
where critical parameters (volume, temperature, pH and extraction temperature)
were applied for the best optimization of each steroid. For each steroid the quantitation was done by calculating analytical figures of merit (precision, accuracy, linearity,
limit of detection, limit of quantitation, and reproducibility).
New LC Separation Technique Based on Hydrogen-Bonding
Interaction
Yury Zelechonok, SIELC, 65 E. Palatine Rd., Suite 221, Prospect
Heights, IL 60070, Vlad Orlovsky, Tatiana Sbignev
Traditional liquid chromatography (LC) techniques include reverse-phase, normalphase, and ion-exchange based mechanism of separation. We recently developed
a new approach and a stationary phase which allows doing separation entirely
based on hydrogen bonding properties of the molecules. This technique is extending the separation scope to many different molecules and it is based on presence
of hydrogen bonding capable groups in the analytes. Many classes of compounds
can be analyze and separate in a simple mobile-phase composition in gradient and
isocratic mode with unique selectivity. Some traditionally difficult separation methods can be easily achieved with this new technique. Low viscosity mobile-phase
allows ultra performance liquid chromatography type separation at traditional highpressure LC pressure boundaries. Many examples and key points of method development using hydrogen-bonding mechanism are discussed.
422
419
Ionic Liquid Static Headspace Single Drop Microextraction of
Aromatic Hydrocarbons from Water and Determination of
Thermodynamic Properties
Ramkumar Dhandapani, Seton Hall University, 54 Fielding Ct., South
Orange, NJ 07079, Nicholas Snow
Room temperature ionic liquids, also known as molten salts, are salts or mixture
of salts that are liquid at ambient temperature and consist entirely of ionic species.
They have emerged as possible “green” solvents. The high viscosity, low volatility and high selectivity of ionic liquids were utilized to selectively extract aromatic
hydrocarbons from water. In present research, ionic liquid-static headspace single
drop microextraction (IL-SHS SDME) was performed to extract analyte of interest
423
Selectivity: A Potent Ally in RPLC Method Development
Thomas J. Waeghe, MAC-MOD Analytical, 103 Commons Ct., Chadds
Ford, PA 19317, Robert T. Moody, Carl L. Zimmerman
Selectivity has been demonstrated to be the most powerful ally in the development
of fast, reliable reversed-phase liquid chromatography (RPLC) methods in an expeditious fashion. Moreover, a careful, thorough, and systematic evaluation of a
combination of key parameters that affect reversed-phase selectivity has also been
shown to produce rugged and robust separations that deliver accurate and precise
results, with fewer surprises during validation and everyday use. A viable method
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2012 EAS Abstracts
November 2012
development strategy should include screening and evaluation of a variety of orthogonal stationary phases, as well as other critical parameters such as gradient
steepness, mobile phase pH, organic modifier type (acetonitrile (ACN), methanol
(MeOH), ACN/MeOH blend, and so-called green solvents such as ethanol and isopropanol), and column temperature. In this talk we present results that demonstrate
the application of several unique column stationary phase chemistries (C18-phenyl,
C18-pentafluorophenyl, and others) in fast ultra high-pressure liquid chromatography method development. An experimental strategy that utilizes those stationary
phases as an integral part of a systematic method development approach is also
discussed.
compound in a shorter run time compared to the full gradient, ultimately resulting in
higher sample purification throughput.
427
Insight into the Allosteric Mechanism of Scapharca Dimeric
Hemoglobin
Francesca Massi, University of Massachusetts Medical School, 364
Plantation St., Worcester, MA 01605, Jennifer M. Laine, Brittany R.
Morgan
Allosteric regulation is an essential function of many proteins that control a variety
of different processes such as catalysis, signal transduction, and gene regulation.
Structural rearrangements have historically been considered the main means of
communication between different parts of a protein. Recent studies have highlighted
the importance of changes in protein flexibility as an effective way to mediate allosteric communication. Scapharca dimeric hemoglobin (HbI) is the simplest possible
type of allosteric system, with cooperative ligand binding between two identical subunits. In this study, we used nuclear magnetic resonance spin relaxation methods
and computer simulations to investigate how changes in HbI flexibility contribute
cooperatively and how changes in protein dynamics are coupled to and correlated
with structural changes upon ligand binding.
424
Improving Sensitivity and Throughput in LC/MS/MS while Reducing
Solvent Usage with Microflow-UHPLC
Khaled S. Mriziq, Eksigent part of AB SCIEX, 5875 Arnold Rd., Dublin,
CA 94568, Steve Hobbs, Remco van Soest, David Neyer, Tina Settineri
Recent developments in the bio-analysis of drugs and their metabolites in physiological fluids place a continuous demand on liquid chromatography mass spectrometry (LC-MS) vendors to improve instruments to meet the growing demand for
higher throughput and greater sensitivity. Microflow LC-MS with column diameters
≤1mm is a new approach for high-throughput LC-MS in bio-analytical development.
Key needs for microflow separations include low delay volumes, fast gradient generation and low dispersion electrospray ionization probes. We have developed two
new small diameter electrospray ionization electrodes (25-µm ID and 50-µm ID) to
minimize dispersion, enabling high-performance separations at 5-200 µL/min with
sub-2 second peak widths. These new electrodes are hybrid electrodes, since they
are made up of PEEKsil tubing, with a short stainless steel tip. The short stainless
steel tip provides minimal residence time to reduce any possible electrochemical
degradation of analytes. We report in this presentation the benefits of an ultra highpressure liquid chromatography (UHPLC) system that can deliver fast and accurate
gradients at microflow rates with very low gradient delays and fast re-equilibrations.
We examine the use of a new auto sampler injection system for small volume handling without sample losses, low carryover and sub-minute cycle times. This presentation describes the advantages of combining a microflow-UHPLC with mass
spectrometry using these small ID hybrid electrodes. We demonstrate equivalent
peak widths and chromatographic resolution compared to high-flow UHPLC. Several examples of using 0.5 and even 0.3 mm columns showing fast, high resolution
chromatography, improvement in sensitivity and throughput, while substantially reducing solvent usage, are discussed.
428
Dynamics of the Equine Infectious Anemia Virus (EIAV) Capsid
Protein: Structural and Functional Implications for the Conserved
尾-Hairpin
Nico Tjandra, National Institutes of Health, Bldg. 50, Room 3503, 50
South Dr., Bethesda, MD 20892, Kang Chen, Grzegorz Piszczek, Carol
Carter
A mature retrovirion encapsulates its genomic RNA within a core structure assembled from the capsid (CA) protein. CA proteins from different retroviruses are structurally similar and consist of a highly conserved N-terminal 尾-hairpin and two helical
domains. The CA proteins of HIV-1 dimerize in solution and form hexagonal and
pentagonal assemblies. Its proteolytically refolded 尾-hairpin structure is necessary
for assembly of the functional mature core structure; however, it does not form any
inter-molecular contacts in the crystal structures of the dimer or hexamer. Therefore
the 尾-hairpin must be involved indirectly to drive assembly. We performed hydrodynamic and solution nuclear magnetic resonance (NMR) measurements on CA
proteins from Equine Infectious Anemia Virus (EIAV) which, like HIV-1, is a member
of the lentivirus subgroup. The 26-kDa EIAV-CA protein contains the correct folding
of the 尾-hairpin. The NMR chemical shift data on both the wild-type EIAV-CA and
a variant lacking the 尾-hairpin demonstrated that the native 尾-hairpin specifically
induced more helical structures at conserved residues N-terminal to helix-1. NMR
relaxation dispersion data suggested that the more helical structured helix-1 was
crucial to initiate proper hexamerization and promote intermolecular interactions
at sub-millisecond time scales for hexamer-core helices within the CA N-domain.
At time scales of microseconds or faster dimerization was promoted along helix-9
within the CA C-domain and the domain linker. Thus, the rate-limiting step in the
initial capsid assembly in solution is the CA N-domain hexamerization induced by
the 尾-hairpin structure, independent of CA C-domain dimerization.
425
High-Pressure Capillary Ion Chromatography: A New Platform for
High-Throughput,
High-Resolution
Separations
of
Ionic
Compounds
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94087
There has been increasing interest in the development of capillary ion chromatography (IC) systems and methods for determination of ionic species. The practice of ion
chromatography in the capillary format offers a number of advantages. Because the
eluent consumption is very low, capillary IC systems can be operated continuously
and thus are always on and ready for analysis. Capillary IC systems offer improved
compatibility with applications where the amount of sample is limited. Capillary IC
systems also provide improved performance for determination of target analytes at
trace levels. Now with the introduction of high-pressure capillary IC, new smaller
particle size columns can improve separation efficiency and/or speed. The speed
and efficiency of these separations greatly improves the system compatibility with
a mass spectrometer. In addition, the use of capillary separation columns opens
the door for the possibility of offering new selectivity for difficult applications using
new columns packed with stationary phases which are more costly and difficult to
prepare.
429
Protein Spin Relaxation in Anisotropic Media
Joel Tolman, Johns Hopkins University, 3400 N Charles St., Baltimore,
MD 21218, Luke Arbogast
Weakly orienting alignment media are commonly employed in protein nuclear magnetic resonance (NMR) spectroscopy for the measurement of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (rCSAs). Concentrations of
alignment media are typically kept low to avoid deleterious broadening of resonances due to unfavorable changes in T1 and T2 relaxation times or from unresolved
long range RDCs (lrRDCs). By employing perdeuteration or other selective labeling
schemes, the effects of lrRDCs can be greatly diminished, but higher media concentrations are still generally avoided due to the potential for unfavorable increases in
relaxation rates. We have measured relaxation rates (15N: T1, T2, NOE, η and 1HN:
T2, η) on the small protein GB1 (70% perdueterated) in strongly aligned DMPC/
DHPC (3:1) bicelles (>15% w/v) in order to assess the effects of alignment on relaxation rates. Remarkably, for solutions of up to 20% bicelles we observed little
diminishment in the quality of the 1HN-15N heteronuclear single quantum coherence
spectra and only marginal changes in relaxation rates relative to a buffered solution.
Above 20% spectral quality decreases significantly due to substantial increases in
the 1H T2 relaxation times yet 15N relaxation times remaining largely unchanged.
Numerical simulations of expected 1HN-1HN lrRDCs using the 1HN-15N RDC derived
alignment tensor show a good correlation with the observed increases in 1H T2
rates. We discuss the results of our ongoing studies of protein spin relaxation in
strongly aligned media.
426
Rapid UPLC Screening and Method Transfer to Preparative HPLC
using CSH Column Technology and Focused Gradients
Mia Summers, Waters, 34 Maple St., Milford, MA 01757, Kenneth J.
Fountain
Improving throughput in the purification laboratory requires streamlining the steps
involved with compound screening and preparative separations. In this seminar,
a fast ultra performance liquid chromatography (UPLC) screening of sample mixtures at high and low pH using charged surface hybrid (CSH) column technology
is presented. The resulting separations are then efficiently scaled up to preparative high-PLC using focused gradients. CSH columns are employed as they have
a wide pH stability range and lack the retention time shifts that can be seen with
traditional C18 stationary phases when repeatedly switching between high and low
pH. The use of the same CSH column stationary phase on the preparative scale
maintains the separation selectivity from the UPLC screening run, alleviating the
need for method re-development across systems. Transferring the UPLC method to
preparative HPLC using focused gradients offers cleaner purification of the target
64
2012 EAS Abstracts
November 2012
430
interest is growing in its physical and electronic properties also. GO has a narrow radical signal with a linewidth of approximately 2 G at X-band that apparently
arises from unpaired electrons that are delocalized in the conjugated pi orbitals of
“graphene islands” that remain after oxidation. GO supported Photosystem II shows
an increase in the GO radical signal after illumination and we became intrigued by
the mechanism of radical formation in GO. The GO electron paramagnetic resonance (EPR) signal is diminished by treatment of graphene oxide with amines and
enhanced by the addition of mild chemical reductants. A mild oxidant can either
increase or decrease the production of radical signal, depending on the presence or
absence of redox active metal ions like Mn(II). We describe a chemical mechanism
for the production of graphene oxide radicals that is consistent with the data.
Structure, Dynamics and Function of Large Protein Signaling
Complexes
Wolfgang Peti, Brown University, 70 Ship St., Providence, RI 02903,
Rebecca Page
Approximately 70% of all eukaryotic cellular proteins are regulated by phosphorylation. While structural data for kinases and phosphatases are readily available, only
a single protein:protein complex structure, which is necessary to understand the biological function of the underlying regulation, has been described. Here we report our
efforts to close this gap by describing our novel, structural insights into the mechanisms by which the kinase interaction motif family of tyrosine phosphatases (KIMPTPs: HePTP, STEP, PTP-SL) regulate mitogen-activated protein kinases (MAPKs)
(p38α and ERK2). Specifically, we have integrated nuclear magnetic resonance
spectroscopy for the analysis of basic components with lower resolution techniques
(small angle X-ray scattering) for constraints on the dynamic, multi-protein assemblies. This has allowed us to determine the first detailed structures of MAPK:MAPK
regulatory protein complexes (p38:HePTP, ERK2:HePTP and pTpY-ERK2:HePTP).
We are now extending these studies to the remaining KIM-PTPs (STEP/PTP-SL)
and complementing these results with a comprehensive ITC study, to understand,
at a molecular level, how residues outside of common interaction sites contribute to
MAPK specificity. Finally, through structural and cell biology studies, we have also
discovered how the extracellular-signal-regulated kinases (ERKs) scaffolding protein KSR1 is targeted to the membrane in activated cells. Collectively, these studies
are enabling us to identify the key, molecular determinants that direct the MAPK
pathway specificity and fidelity in the cell.
434
The Effect of Sr2+ Substitution on Structure of Oxygen Evolving
Complex of Photosystem II Studied by 1H HYSCORE Spectroscopy
Sergey Milikisiyants, Rensselaer Polytechnic Institute, 110 8th St., Troy,
NY 12180, Ruchira Chatterjee, Christopher S. Coates, K. V. Lakshmi,
Faisal H.M. Koua, Jian-Ren Shen
In Photosystem II (PSII), the solar water oxidation reaction occurs in the oxygen
evolving complex (OEC). The OEC consists of a tetra-nuclear manganese calciumoxo (Mn4Ca-oxo) cluster surrounded by amino acid residues. During the catalytic
cycle the OEC goes thought five different oxidation states S0 - S4. The recent 1.9
resolution X-ray structure of the OEC in the S1 state[1] indicates that the Ca2+ ion
forms a distorted cubane structure with three Mn ions, while the fourth Mn ion is
“dangler.” Despite the remarkable breakthrough in the determination of the structure of the S1 state, the mechanism of water oxidation is yet to be unraveled. The
role of Ca2+ ion in the catalytic reaction of the OEC remains one of the most interesting questions that are yet to be answered. The structural and functional differences induced by metal ion substitution could provide valuable insight into the
role of Ca2+ ion for catalysis of water oxidation in the OEC. The only divalent ion
capable of restoring catalytic activity after substitution of Ca2+ ion is Sr2+ ion, however, the catalytic turnover is significantly slow upon Ca2+/Sr2+ substitution. In this
study, we apply hyperfine sublevel correlation (HYSCORE) spectroscopy to probe
the structural changes in the OEC in the S2 state induced by Sr2+ substitution. Since
hyperfine interactions are highly sensitive to any perturbation of the electronic and
geometric structure of paramagnetic centers, comparison of the HYSCORE spectra
of Sr2+/Ca2+ substituted PSII allows us to draw important conclusions with respect
to structure of the OEC in the S2 state and structural changes induced by metal ion
replacement.
431
Quantum Chemical Calculations of Nuclear Magnetic Resonance
Parameters for Heavy Nuclei: Challenges and Applications
Fahri Alkan, University of Delaware, Department of Chemistry, Newark,
DE 19716, Shi Bai, Cecil Dybowski
Computation of nuclear magnetic resonance (NMR) parameters of heavy nuclei
such as Pb-207 and Hg-199 is a challenge for the current level of quantum theory,
as it is necessary to include the relativistic nature of the electron. Accurate prediction of chemical shielding tensors is an important tool to explore the molecular
and structural properties of a system, and a significant aid in assigning complex
NMR spectra. We present predicted NMR chemical shift parameters (isotropic shifts
and chemical-shift tensor elements) of Pb-207 and Hg-199 nuclei for several solid
systems modeled as molecular clusters. These results demonstrate that one can
qualitatively predict trends in these parameters, but that quantitative agreement with
experiment is more difficult to achieve with the current level of theory. Parsing the
parameters into contributions from various molecular orbitals allows one to infer
which molecular orbitals dominate observed effects. Additionally, we discuss the
practical effects of modeling an extended solid system with finite clusters, which limit
the accuracy of the calculations.
[1] Umena et al. Nature, 2011, 473, 55.
435
Tracking Light-Induced Charge Separation in Polyoxotitanate
Clusters
Karin Young, Yale University, 225 Prospect St., New Haven, CT 06520,
Robert C. Shoeberger III, Laura J. Allen, Robert H. Crabtree, Victor S.
Batista, Gary W. Brudvig, Jiji Tang, Jason B. Benedict
Functionalized titanium dioxide nanoparticles have been widely used for solar energy technology in both dye-sensitized solar cells and for light-driven water oxidation. While some of the properties of molecular chromophores anchored to titanium
dioxide surfaces have been studied, the heterogeneity of the surface sites can
complicate the interpretation of spectroscopic study. Polyoxotitanates such as Ti17,
[Ti17O24(O(iPr))20], have been proposed as models for titanium dioxide nanoparticles because they contain similar coordination and connectivity features as the
bulk crystal and also possess defined stoichiometry as determined by single crystal X-ray crystallography. In order to characterize the similarities between Ti17 and
nanoparticulate titanium dioxide, we have used electron paramagnetic resonance
(EPR) spectroscopy to study the species formed during light-induced charge separation. EPR spectra of unfunctionalized and functionalized Ti17 are presented and
compared to EPR signals observed in nanoparticulate titanium dioxide.
432
EPR Techniques in the Study of the A1A and A1B Phylloquinone
Sites of Photosystem I
John H. Golbeck, Pennsylvania State University, S328 Frear Laboratory,
University Park, PA 16802
Photosystem I is a light-driven plastoquinone (cytochrome c6):ferredoxin (flavodoxin) oxidoreductase. It functions in series with Photosystem II to split water for
the purpose of reducing NADP+ to NADPH. Photosystem I contains phylloquinones
(2-methyl-3-phytyl-1,4-naphthoquinone) in the A1A and A1B sites that accept electrons from the primary accetors, A0A and A0B, respectively, and passes them to FX,
an interpolypeptide [4Fe-4S] cluster. They function as single electron acceptors,
shuttling between the naphthoquinone ground state and the naphthosemiquinone
anion radical at midpoint potentials of ca. -650 mV (A1A) and ca. -800 mV (A1B). In
this talk, I describe the use of phylloquinone pathway delation mutants that allow the
incorporation of a wide variety of substituted naphthoquinones into the A1A and A1B
sites and the subsequent kinetic and structural characterization of the modified Photosystem I complexes by CW, ESE, hyperfine sublevel correlation (HYSCORE), and
transient electron paramagnetic resonance (EPR) methods. The relevant issues under study are: 1) how the protein differentially modulates the midpoint potentials of
the two phylloquinones; 2) how the protein allows electron transfer to occur between
the ground and semiquinone states but not between the semiquinone and hydroquinone states; and 3) how the protein is able to accommodate substitute naphthoquinones in the A1A and A1B site with widely varying ring substitutions and tail lengths.
436
Clinical EPR: Challenges and Progress
Harold Swartz, Geisel Medical School at Dartmouth, HB7785, Hanover,
NH 03755
The development and use of in-vivo techniques for experimental applications of
electron paramagnetic resonance (EPR) in animals has been very successful and
now has led to attractive clinical applications. This presentation provides an overview of the challenges, opportunities, and results as in-vivo EPR is extended into
use in human subjects. The most widespread clinical use is oximetry, where EPR
can make repeated and accurate measurements of pO2 in tissues, which provides
clinicians with information that bears directly on diagnosis and therapy, especially
for oncology, peripheral vascular disease, and wound healing. The other area of
importance in human subjects is the ability of in-vivo EPR to measure clinically significant exposures to ionizing radiation ‘after-the-fact’, due to accidents, terrorism,
or nuclear war. The unique capabilities of in-vivo EPR to detect and characterize
free radicals could be applied to measure free radical intermediates from drugs and
oxidative processes, including measurements of nitric oxide. These unique capa-
433
Radical Formation in Graphene Oxide
Donald Hirsh, The College of New Jersey, Department of Chemistry,
PO Box 7718, Ewing, NJ 08628, Michele Vittadello, Kamil Woronowicz,
Manish Chhowalla
Graphene oxide (GO) is formed by the chemical oxidation of graphite. It is watersoluble and a potential intermediate in the synthesis of graphene, a two-dimensional
and nearly transparent form of pure carbon with high electron mobility and many
potential uses in electronic devices. GO is itself an insulator or semiconductor and
65
2012 EAS Abstracts
November 2012
bilities, combined with the sensitivity of EPR spectra to the immediate environment
(e.g., pH, molecular motion, charge), have resulted in productive applications in
animals that may be adapted for use in humans. The challenges for achieving full
implementation in clinical research and practice include adapting the spectrometer
for safe and comfortable measurements in human subjects, achieving sufficient
sensitivity for measurements at the sites of the pathophysiological processes, and
establishing a consensus on the clinical value of the measurements, e.g., for establishing the effectiveness of therapies involving oxidative processes or monitoring
patients’ progress or responsiveness to such treatments.
deposited limited by the amount of surface hydroxyl groups. X-ray photoelectron
spectroscopy studies show that Cu(0) and Cu(I) species are present on the surface
following deposition. Scanning electron microscopy and energy-dispersive X-ray
spectroscopy results confirm the formation of nanoparticles. This study provides a
novel highly controlled approach to deposition of metallic nanoparticles on powder
samples.
440
Ultrafast Vibrational Spectroscopy and Dynamics of Mineral/
Aqueous Interfaces
Eric Borguet, Temple University, Department of Chemistry, Philadelphia,
PA 19122
The properties and behavior of water at interfaces are important. However, it is not
clear what effects the presence of the surface, the charge that can develop on the
surface, the solution ionic strength, and the interfacial electric field have on how
interfacial water molecules communicate with each other, e.g., how thermal (vibrational) energy flows. To address these issues we have investigated the ultrafast
vibrational dynamics of the O-H stretch using infrared pump-vibrational sum frequency generation (vSFG) probe at the water/silica interface. Contrary to previous
reports, the vibrational lifetime of the O-H stretch at the silica/water interface is approximately 600 fs when the surface is neutral, a factor 2-3 slower than bulk water.
[1]
Charging the interface appears to lead to a dramatic acceleration of vibrational
relaxation. Experiments on the effect of ionic strength, at pH=6, suggest that the
primary reason for accelerated dynamics at charged surfaces is the sampling of
water that has bulk-like solvation within the Debye length.[2]
437
Time-Resolved Laser Spectroscopy of Molecular Titania Clusters
Piotr Piotrowiak, Rutgers University, 73 Warren St., Newark, NJ 07102,
Jianhua Bao, Zhihao Yu, Lars Gundlac
Polyoxotitanate clusters such as the Ti17 allow one to investigate quantum size
effects in truly monodsiperse nanometer size systems. Condensed phase photo
physics of such small clusters which bridge the gap between molecules and polydisperse nanoparticles, is virtually unexplored. The behavior of the excited state
and the excess electron in the confined space of Ti17 was studied using femtosecond pump-probe transient absorption, pulse radiolysis, fluorescence spectroscopy, Raman microspectroscopy and density functional-theory calculations. Due to
pronounced quantum size effects the electronic spectra of both the excited state,
Ti17*, and the excess electron, Ti17• , are blue-shifted in comparison with bulk TiO2.
The 0.7 eV difference in the position of the maxima Ti17* and Ti17• indicates the
presence of strong Coulomb interaction between the excess electron and the hole
in the ~1nm diameter cluster. The vibronic features of the emission spectrum and
ground state Raman spectra suggest that the ligand modes are strongly coupled to
the overall electron density of the cluster. This finding further supports the chargeseparated nature of the exciton and the involvement of the interface in its stabilization. The implications of the large ground state ligand-to-metal charge shift at the
five-coordinate centers evident in the Raman data extend beyond the excited state
dynamics of Ti17. These sites behave as reactive “hot-spots” on the surface of the
cluster and have a major influence on the redox and potential catalytic activity the
cluster. Our results show that studies on polyoxotitanate clusters help to understand
the complex behavior of dye-sensitized metal oxide solar cells, especially when it
comes to the role of electron transfer and charge trapping at the ligand-substrate
interface.
[1] Effect of Surface Charge on the Vibrational Dynamics of Interfacial Water, Ali
Eftekhari-Bafrooei and Eric Borguet, Journal of the American Chemical Society,
131 (34), 12034–12035 (2009).
[2] The Effect of Electric Fields on the Ultrafast Vibrational Relaxation of Water at a
Charged Solid-Liquid Interface as Probed by Vibrational Sum Frequency Generation, A. Eftekhari-Bafrooei and E. Borguet, Journal of Physical Chemistry Letters, 2, 1353-1358 (2011).
441
Tailoring Surface Plasmon Resonance Wavelength and Penetration
Depth
Laurel L. Kegel, University of Delaware, Department of Chemistry, 105
Brown Lab, Newark, DE 19716
Surface plasmon resonance (SPR) sensors are widely used for the detection of
analyte binding to recognition elements on the sensor surface or refractive index
change near the surface. Additionally, SPs cause localized field enhancement,
which may be exploited by surface enhanced Raman spectroscopy (SERS) or ‘gap
mode’ enhancement of SPR within the space between plasmonic structures. The
properties of plasmonic nanostructures may be tailored to improve SPR sensing capabilities and Raman scattering enhancement. Specifically, SPR penetration depth
and wavelength may be tuned to maximize sensitivity and facilitate coupling of plasmonic materials. This paper presents the characterization of plasmonic materials
with tunable penetration depth and extension into the near-infrared (NIR) region
with a novel variable angle NIR-SPR accessory. Nanostructured materials, including nanohole arrays, are of interest for SPR sensors as a more sensitive platform
than conventional planar gold. SPs supported by nanostructures exhibit shorter
penetration depth and thus increased sensitivity to surface binding. A more accurate
method for determining penetration depth is shown and applied to nanohole arrays.
Generally, penetration depth is calculated from shift in SPR wavelength for given adsorbate thickness. We demonstrate the importance of including an additional factor
for the SPR-shift due to adsorbate bonding. This effect is amplified for thin layers of
adsorbate and materials with small penetration depth such as nanostructures. The
presented techniques enable accurate selection of penetration depth and vis-NIR
wavelength for optimal sensitivity of SPR and SERS.
438
XPS and REELS Surface Characterization and Imaging of Graphene
and Other Carbon-Based Materials
Brian R. Strohmeier, Thermo Fisher Scientific, 5225 Verona Rd.,
Madison, WI 53711, Andrew E. Wright, Tim S. Nunney, Richard G. White
The application potential of graphene is being extensively explored by the materials
science community. In all stages of application development, there is a requirement for material characterization. Most graphene materials need to be analyzed
for compositional homogeneity across the sample surface and through the sample
thickness. X-ray photoelectron spectroscopy (XPS) is a valuable technique for the
surface characterization of graphene and other carbon-based materials. XPS can
be used to characterize the surface chemistry of graphene and modified graphene
materials, detect surface impurities, and evaluate graphene-substrate interactions.
XPS can also be used to generate chemical-state surface images of graphene and
to determine total thicknesses for layer counting. Reflection electron energy loss
spectroscopy (REELS) is a complementary technique to XPS that can be performed
in XPS instruments that utilize a variable energy electron flood gun for sample
charge neutralization. The electron flood gun source can be used to produce high
quality REELS spectra. An important advantage of REELS is the ability to detect
and quantify surface hydrogen, which is undetectable by XPS. Hydrogen surface
maps can also be produced by REELS to supplement XPS surface images of other
elements and/or chemical states. In addition to the detection of hydrogen, REELS
can also be used to examine the level of carbon unsaturation at the uppermost
surface of organic materials to complement information obtained from high resolution C 1s XPS spectra. This presentation describes several examples of XPS and
REELS applications for the surface characterization of graphene and other organic
materials.
442
A Gentamicin Sulfate Assay Using HPLC-Charged Aerosol
Detection with an Ion-Pairing Reagent Gradient
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94087, Deanna Hurum, Jeffrey S. Rohrer, Jinyuan Wang, Michael Hvizd
Aminoglycoside antibiotics are proven human and veterinary antibiotics that have
broad-spectrum activity, particularly against gram negative bacteria. Many of these
antibiotics are manufactured by microbial culture (fermentation) processes. These
processes can produce a mixture of antibiotic compounds. One example antibiotic
manufactured by fermentation is gentamicin, which is produced by Micromonospora echinospora (Micromonospora purpurea). Gentamicin sulfate is a mixture of
four major compounds: gentamicins C1, C1a, C2, and C2a. In addition, gentamicin
C2b is commonly present as a minor component. These gentamicins, as well as
fermentation impurities and degradation products, are structurally similar and do
not possess chromophores, making chromatographic separation and subsequent
detection challenging. Alternate detection techniques are available for compounds
that are not chromophoric. In this work gentamicin sulfate samples are analyzed by
reversed-phase high-pressure liquid chromatography (HPLC) with charged aerosol
439
Formation of Copper Nanoparticles on a Surface of OH-terminated
ZnO Powder Material
Hsuan Kung, University of Delaware, Chemistry Department, 114LDL,
Newark, DE 19716, Andrew Teplyakov
The Cu/ZnO is a promising catalyst for several chemical processes, including
methanol synthesis. The common methods to grow Cu particles supported on ZnO
are based on wet chemistry, which is sometimes difficult to control. In this work,
water was used under high vacuum conditions to modify the surface of ZnO powder
with hydroxyl groups. The hydrogen of hydroxyl group was then used as a limiting
reagent in a reaction with copper hexafluoroacetylacetonate vinyl trimethyl silane,
Cu(hfac)(VTMS), a common cooper deposition precursor. At room temperature, this
process results in the formation of copper nanoparticles with the amount of copper
66
2012 EAS Abstracts
November 2012
446
detection. This detection technique does not rely on the presence of a chromophore,
does not require sample derivatization for detection, is compatible with volatile ionpairing reagents and readily detects non-volatile analytes such as gentamicin. A
method has been developed to separate the five gentamicin sulfate congeners and
sisomicin, a common impurity. The method was used to quantify gentamicin sulfate
in ointments, solutions, and creams with a total analysis time of 15 minutes using a
gradient of ion-pairing reagents.
Trace Analysis of Potential Genotoxic Impurities by LC-MS with
Atmospheric Pressure Photoionization
Ryan Cohen, Merck, 280 River Rd., Apt 49A, Piscataway, NJ 08854
The control and analysis of potential genotoxic impurities (PGIs) often poses a challenge during development of new pharmaceutical substances. Some of the issues
that frequently arise are the need for trace level quantitation (often 1 to 10 ppm),
poor recoveries, and matrix effects. In this study, mutagenic aryl nitro impurities
were analyzed. These compounds are commonly used as synthetic building blocks
for aryl amines. Due to weak electrospray ionization (ESI), atmospheric pressure
photoionization (APPI) liquid chromatography mass spectrometry (LC-MS) was employed. An additional advantage of this ionization mode over ESI was less susceptibility towards source contamination and matrix effects. Analysis of ten different aryl
nitro impurities resulted in linear calibration curves with an average detection limit
of 0.3 ppm (40 pg on column). Spiked recoveries into three model drug substances
were acceptable, ranging from 89-109%.
443
High-Resolution Separations of Complex Pharmaceuticals Using
UHPLC
Michael W. Dong, Genentech, One DNA Way, S. San Francisco, CA
94080, Derrick Yazzie, Nik P. Chetwyn
In the last four decades, the resolution of high-pressure liquid chromatography
(HPLC) separation for complex samples has been bounded by a system pressure
limit of 6000 psi - effectively limiting separation performance to column efficiency
(N) of approximately 20,000 plates or a peak capacity (n) of approximately 200. The
advent of ultra-HPLC and its successful commercialization coupled with the use of
sub-2-µm-particle or sub-3-µm fused core particle columns has set a new HPLC
performance benchmark. It is now possible to perform high-resolution separations
of very complex mixtures approaching the efficiency of capillary gas chromatography (n = 400 - 1,000). This paper focuses on the benefits and potential issues
of UHPLC in pharmaceutical analysis, highlighting instrumental designs tradeoffs
and operating nuance. Two areas of high-resolution separations in pharmaceutical
analysis are discussed. First is the analysis of complex natural products, requiring
the summation of all active ingredients to arrive at potency values for the products.
Second is the analysis of complex molecules with multiple chiral centers requiring
the separation of all diastereomers from the active pharmaceutical ingredients and
other impurities for process control and quality control release testing.
447
Analytical Methods to Qualify and Quantify PEG and PEGylated
Biopharmaceuticals
Christopher A. Crafts, Thermo Fisher Scientific, 22 Alpha Rd.,
Chelmsford, MA 01824, Marc Plante, Ian N. Acworth, Michael G. Hvizd
The use of polyethylene glycol (PEG) as a safe and low cost additive for the pharmaceutical and cosmetic industry is common practice. Over the last decade the
field of biopharmaceuticals has begun to use the process of covalently bonding
PEG to their active peptides or proteins (PEGylation) to improve bioavailability and
reduce immunogenicity. This process of PEGylation requires that the starting PEG
reagent meet several criteria including monodispersity for pharmaceutical batch reproducibility. While the characterization of the active protein is often best done by a
combination of high-pressure liquid chromatography (HPLC) with ultra violet (UV)
and mass spectrometry (MS) detection of PEG and PEG reagents is challenging as
they do not contain a sufficiently active chromophore for UV characterization. Such
limitations are readily overcome by charged aerosol detection (CAD). In this work
methods were developed to: 1) characterize a number of PEG compounds including PEG 400, PEG 3000 and PEG 8000 by reverse-phase ultra-HPLC\CAD, and 2)
simultaneously measure the presence of free PEG in the final PEGylated product
by an online 2D/LC system of size exclusion chromatography and reverse-phase
C8 with CAD. This work focused on the PEGylation of two different proteins with
an amine-reactive unbranched PEGylation reagent. The reaction monitoring was
accomplished by preparing all samples on the autosampler tray with user defined
methods. The results showed that free PEG could be easily quantified in the presence of the PEGylated protein down to low nanogram levels. As the industry keeps
evolving and searching for optimal PEGylation reagents both in size and branching
characteristics the use of CAD can help ensure quality products.
444
Counter-Ion and Residual-Ion Determination Using LC/MS Method
Weiqing H. Fu, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, William P. Fish, Anne Kelly, Kathleen A. Kelly, Scott A. Miller
The quantitative analysis of counter ions and residual ions are an important aspect
of pharmaceutical development and quality control. Within early development, the
ability to quickly confirm proper stoichiometry and identity, along with guiding the
selection of an appropriate salt form is of importance. Additionally, fast counter-ion
determination is a useful investigative tool to examine oral solid dose unit formulations, independent of the active pharmaceutical ingredient (API). Methods used to
determine counter-ions or residual-ions include titration, IC (conductivity), CE (indirect UV), AA/ICP (cations only), and reverse-phase high-pressure liquid chromatography (UV, CAD). Recently, two generic liquid chromatography mass spectrometry
(LC-MS) methods for cations and anions have been developed to analyze a variety
of counter-ions or residual-ions. The anion method can quantitatively determine
chloride, bromide, iodide, acetate, nitrite, nitrate, succinate, asparate, phosphate,
trifluoroacetate, monochloroacetate and the cation method can determine sodium,
potassium, calcium, and magnesium regardless of whether the sample is an API
or a formulated product. In this presentation, case studies utilizing these LC-MS
methods are discussed showing its applicability across drug development with an
emphasis on comparison to traditional analytical techniques.
448
Seven Common “Faux Pas” in Modern HPLC
Michael W. Dong, Genentech, Genentech, One DNA Way, S. San
Francisco, CA 94080
There are many common practices in high-pressure liquid chromatography (HPLC)
which may not make sense with improved technologies in modern HPLC. These
may be legacy or traditional ideas, myths, “half-truths” and ineffective procedures
used for years. In this paper, we highlight seven common “faux pas” or highly inefficient practices and present rationales on why they should not be followed without
questions. We support each idea with data, case studies, and exceptions to the rule.
These are: 1) use 5-µm columns, 2) use 4.6 mm i.d. columns at 1 mL/min, 3) filtering
mobile phases, 4) use buffered mobile phases, 5) prepare fresh standard solutions,
6) Shake sample vials, 7) use stainless steel ferrules for column connections.
445
Analysis of HOPO, a Low Level Genotoxic Impurity using Chemical
Derivatisation and LC and GC Mass Spectrometry
Emma S. Quirk, Bristol-Myers Squibb, Reeds Lane, Wirral, CH46 1QW
United Kingdom
Hydroxybenzotriazole (HOBt) is used as a coupling reagent in several active pharmaceutical ingredient (API) synthetic processes at Bristol-Myers Squibb (BMS). In
2008, the UN looked likely to reclassify HOBt as a Class 1 reagent, subsequently
limiting the use and availability of the material for manufacture and transport. To
mitigate against this potential outcome, the use of both alternative chemistries and
coupling catalysts were investigated to determine the most appropriate solution,
and a decision was made to directly substitute HOBt with 2-hydroxypyridine N-oxide
(HOPO). New analytical methods were developed to assay the HOPO material prior
to use in a manufacture, and to quantify HOPO in the presence of API. These methods presented analytical challenges due to the highly polar nature of HOPO and
its tendency to tautomerize in solution. As a result, new column chemistries and
analyte derivitization were employed within these methods. A genotoxic impurity
risk assessment had originally indicated there was no need to control HOPO levels
within API as it had presented negative in in-silico assessment. However, in August
2011, an Ames test was conducted on HOPO at BMS following concern from a
supplier that similar compounds were mutagenic and rendered a positive result.
Subsequently a limit of 1.2 ppm for residual HOPO in API was imposed on all material prepared from the coupling catalyst. This presentation discusses the analytical
method development, validation and transfer strategy required to determine residual
levels of HOPO in API commercial batches and demonstrates how the challenges of
sensitivity were overcome using liquid chromatography tandem mass spectrometry
(LC-MS/MS) and gas chromatography MS methods with derivitization approaches.
449
LC/MS/MS Quantitative Analysis of Vitamin D: Hidden Analytical
Errors
Eduard Rogatsky, Albert Einstein College of Medicine, Golding Bldg.,
Room G02, GCRC, 1300 Morris Park Ave., Bronx, NY 10461
Vitamin D deficiency is a widespread clinical problem and has been associated with many adverse health outcomes. Analysis of Vitamin D2 (ergocalciferol) and D3 (cholecalciferol) and their major metabolites 25(OH)D2 and 25(OH)D3
has become a high priority topic in clinical analysis. Since water loss is a favored
process, in-source self-dehydration of vitamin D can decrease S/N and such an
undesirable process was tightly controlled. In-source water loss was found to be
insignificant (~8%) within the assay linear range using a methanol/water mobile
phase under our experimental conditions. In contrast, if acetonitrile/water was used,
severe in-source water loss was observed and ions m/z 395.2 and m/z 383.2 resulting from analyte in-source dehydration became predominant. When acetonitrile
was used as mobile phase, and if only the transitions m/z 395.2→377.2 for 25(OH)
D2 and m/z 383.2→377.2 for 25(OH)D3 were used for quantitation, the assay standard curve was still acceptably linear; however, the area ratio between analyte and
its in-source water loss product was non-linear at different analyte concentrations.
The concentration-dependent water loss varied by 20% resulting in a significant
quantitation error in this provisionally valid assay. For an acetonitrile-based mobile
67
2012 EAS Abstracts
November 2012
453
phase, both transitions (for analyte and for its in-source dehydrated product) should
be summed to maintain true full range assay linearity.
IR Microscopy using External-Cavity Quantum-Cascade Lasers
Robert Shine, Jr., Daylight Solutions, 15378 Avenue of Science, San
Diego, CA 92128, Miles Weida, Dave Arnone, Tim Day
Infrared (IR) microspectroscopy has shown itself to be an important tool by providing
spectroscopic and chemical analysis in areas as diverse as investigating surface
defects in manufacturing to tissue diagnostics at the molecular level. To date, the
main tool for performing IR microspectroscopy has been the Fourier transform infrared (FTIR) microscope, but these have been limited by the relatively poor optical
quality of the incandescent light source. Recent work has used synchrotron sources
to significantly increase the performance of IR microscopy, but these sources are
not commercially viable. The development of broadly tunable, external cavity quantum cascade lasers (EC-QCLs) has created an ideal light source for IR microscopy.
Initial results were obtained by coupling an EC-QCL to a commercially available
FTIR microscope. Spectral performance and coherence effects were determined
by performing scans for different sample materials. Advantages of the EC-QCL
based spectra included higher spatial resolution (10-μm vs. 25-μm), higher spectral
resolution (1 cm-1 compared to 4 cm-1), and less averaging time (5 seconds vs. 1
minute). The laser based IR microscope was further improved by coupling to a micro-bolometer FPA detector. Images of approximately 2 mm by 2 mm were obtained
for both reference samples and reticles to determine the performance, and results
compared well to synchrotron-based IR microscopes. EC-QCL based imaging microscopes stand ready to rival the performance of state-of-the art IR microscopes
for a fraction of the cost and complexity.
450
Quantitation of Metabolites without the use of Standards:
Optimizing UHPLC- High Resolution MS Parameters for Accurate
Signal Response
Samantha M. Mahmoud, Kean University, 233 Nagle St., Union, NJ
07083, Sumithra Katragadda, Dil Ramanathan
In drug metabolism studies, liquid chromatography mass spectrometry (LC-MS)
is the mainstream analytical technique to quantify metabolites which are formed
through phase I and phase II reactions. Quantification of an analyte requires a standard curve to be plotted based on the LC-MS or LC-MS/MS responses of reference standards for the analyte that need to be quantified. Usually a standard curve
must be generated for each and every analyte to be quantified. As the parent and
the metabolite are structurally different, the standard curve of the parent cannot be
used for the metabolite. During early stages of drug discovery there are no internal
or reference standards available for the metabolite. This study investigates the impact of mobile phase composition on LC-MS response, the organic mobile phases
composition was methanol, acteonitrile, water and 50/50 methanol/acetonitrile. Ultra
high-pressure liquid chromatography (UHPLC) flow rates were also investigated,
200-500 μL/min, with 200uL/min showing best sensitivity as well as the addition of
a reverse composition of mobile phases to achieve uniform MS responses for drugs
and their metabolites. Preliminary results so an enhancement of signal response
with the adjustment of these parameters.
454
A New Truly Easy-to-Use Dedicated Infrared Microscope
Thomas J. Tague, Jr., Bruker Optics, 19 Fortune Dr., Billerica, MA
01821, Sergey Shilov, Fred Morris, Matthias Boese
A new stand-alone infrared microscope (LumosTM) has been developed for the rapid
analysis of small samples. The new microscope was developed with the intent of
providing state-of-the-art microanalysis capabilities with a truly easy-to-use user interface. The visual image quality of the LumosTM is excellent so the important first
step in the analysis, visualization, is easily accomplished. The LumosTM utilizes a
unique objective design, where the numerical is low for sample viewing and high for
the infrared data collection. This makes it very easy to locate and view the sample
without sacrificing infrared performance. The novel Wizard user interface controls
all aspects of the microscope and guides the user through the analysis process. The
sample stage, sample focus assembly, condenser, aperture, polarizers, and attenuated total reflectance (ATR) mode are controlled in the software providing true “point
and shoot operation.” ATR microanalysis is accomplished by simply clicking on the
area of interest in the software to center it and selecting ATR. Area reflection, transmission, and ATR images are collected by simply drawing the desired analysis and
starting the desired acquisition. The image processing software interface provides
research quality analysis tools with an intuitive interface. The LumosTM also has
a unique ability to readily analyze samples with traditional sampling accessories.
A port is provided to attach accessory modules from Bruker’s Alpha FTIR Series.
Standard ATR, transmission, reflection, and even gas cell analysis can be readily
conducted with the LumosTM. Lastly, the LumosTM comes with a comprehensive validation package to support any range of validation requirements.
451
Accurate Mass GC/MS for the Identification of Unknowns in
Biodiesel
Douglas M. Stevens, Waters, 34 Maple St., Milford, MA 01757, James
D. Stuart, Anthony A. Provatas
Adaptation of gas chromatography flame ionization detection (GC-FID) methods to
high resolution GC mass spectrometry (MS) has demonstrated the ability to identify
unknowns present in biodiesels that fail such quality assurance methods as American Society for Testing and Materials (ASTM) D6584 “Test Method for Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography.” By directly implementing the existing GC method, information can more
readily be obtained for use in directing process refinements necessitated by scale
up, changes required by previously uncharacterized feedstock, and troubleshooting of process and analytical methods. In particular the identification of unknown
compounds that do not fall into the categories of well characterized analytes such
as mono-, di-, and tri-glycerides which are already known to cause problems such
as injector fouling and engine deposits, can be identified allowing more timely and
effective remedial action. Previously work in this area was performed using electron ionization (EI) and chemical ionization on GC time-of-flight (TOF). Unknowns
were identified using library searching and accurate mass measurement. That work
helped identify limitations of the approach by demonstrating that some compounds
known to cause problems with biodiesel do not have spectra in commercial EI libraries. Since quality TOF has demonstrated utility in structural elucidation of unknowns
by liquid chromatography mass spectrometry (LC-MS), it was decided to evaluate
atmospheric pressure chemical ionization GC (APGC) on QTOF to test the feasibility of using it for identification of unknowns in biofuels. This presentation compares
and contrasts the information provided by EI GC-TOF versus APGC GC-QTOF and
discuss the suitability of each based on its fitness for purpose for process engineers
and biofuel researchers.
455
Deconvolution of Botanical FT-NIR Spectra via Analysis of TLC
Plates
Emil W. Ciurczak, Doramaxx Consulting, 77 Park Rd., Golden Bridge,
NY 10526, Cynthia L. Kradjel, Sunghee Lee, Jaclyn Robustelli, Allyson
Moffat, Kim Eastman
Botanicals are complex mixtures of natural organic compounds. Dietary supplement manufacturers use Fourier transform near-infrared (FT-NIR) to test identity
and purity of incoming botanical ingredients. Chemometric algorithms and hierarchical methods of library building improve the specificity of FT-NIR identity tests. The
FT-NIR spectra of botanicals represent composite spectra of the complex mixture
as samples are scanned as is with no sample preparation. We designed a study to
deconvolute the spectra of botanicals by spotting the botanicals on a thin layer chromatography (TLC) plate and then scanning the individual spots to obtain FT-NIR
spectra of the different components of the botanicals. Botanical standards provided
by ChromaDex were tested and then compared with samples obtained in the open
market.
452
GC-MS & PDHID Analysis of Permanent Gases and C1-C6
Hydrocarbons: Using Multiple Techniques to Achieve Quantitation
with a Single Analytical Run
Jeff Parish, Shimadzu Scientific Instruments, 7102 Riverwood Dr.,
Columbia, MD 18078
There is currently a great deal of research focused on the use of catalysis or pyrolysis to produce hydrocarbon based fuels. This research often requires the quantitation not only of the hydrocarbon sources and products, but also of permanent gas
sources and products such as H2, CO, CO2, O2, and others. A mass spectrometry
(MS) is often a requirement for this research because the reaction products need
to be identified as well as quantitated. Chromatographic separation of the reaction
products is challenging because no single column can separate both pure gases
and hydrocarbons at typical gas chromatography (GC) temperatures. In this application, both permanent gases and hydrocarbons (alkanes and alkenes) were
separated on an Alumina column at sub-ambient temperature and simultaneously
analyzed on both a pulse discharge helium ionization detector (PDHID) and a single
quadrupole electron impact (EI) mass spectrometer (MS). It was also demonstrated
that this analysis could be done exclusively on the MS with a typical GC temperature
ramp by using the discriminatory power of the MS to quantitate permanent gases
that co-elute.
456
On-Line Monitoring of Chemical Reaction Rates by Near-Infrared
Spectroscopy
Chris Heil, Thermo Fisher Scientific, 5225 Verona Rd., Madison, WI
53711, David Drapcho, Herman He, Michelle Pressler, Hilmar Geitlinger,
Martin Irrgeher
Understanding how different chemical and physical variables affect the rate of
chemical reactions is important in both the research laboratory and production
scales processes. Analytical techniques that can measure key attributes very
quickly and directly in the reaction vessel are ideal for studying chemical reaction
68
2012 EAS Abstracts
WORKSHOPS
November 2012
EAS is offering workshops on developing professional skills in two vital areas: the acquisition of
general information
available in the
text books. However
that knowledge generally
a position and progression within the chemical industry.
Full Conferee
registration
is required
in the mind of the individual or sometimes is hand written on a chart and
and space is on a first come, first serve basis; see resides
page
38
for
more
details.
filed away. Until recently, the primary method to save a spectral interpretation has
kinetics. Analysis that requires extracting samples to be brought to the laboratory lead to: delay in results on dynamically changing processes, potential sample
contamination, and analyst having to handle hazardous chemicals. Near-infrared
(NIR) spectroscopy is widely used for chemical kinetic monitoring in real-time via
direct analysis in-situ using fiber optic probes. Analysis by NIR occurs in a matter
of seconds allowing for multi-component analysis orders of magnitude faster than
laboratory analysis. This study looks at how a NIR process analyzer can be used to
study how different variables, such as time, temperature and catalysis concentration, can affect the degree and speed of reaction conversion. It is also shown how
NIR combined with process fiber optic probes is successful for challenging analysis
conditions such as high temperatures, highly basic, changing viscosity, agitation
and gaseous reagents injected into liquid reactants. Easily developed and implemented measurements based on changes in NIR absorbance spectra proved to be
effective tools for investigating how different experimental conditions can influence
the speed of a chemical reaction.
been to write notes on a chart. However, with the advent of digital spectra combined
with digital structures and along with new software tools it is now possible to create,
store and retrieve the spectrum-structure information as it is developed by in-house
experts. This paper looks at one such software tool which allows digital assignments
to be created and stored. Assignments are made electronically between spectral
peaks and attached structures or structure fragments. Assignments are retained
when the spectrum and structures are stored in a database. Searching through filing cabinets filled with charts or re-interpreting the data because it was easier than
finding the old information is now a thing of the past.
Workshop I: Career Development in the Chemical Industry
Date: Monday, November 12, 2012
Time: 9:30 am – 12:00 pm
Competencies & skills developed: Industry awareness, managing one’s career
Location: Double Tree Hotel Parlor 154
Instructors: Patricia Finnegan, Jennifer Hackett, Robert
BASF -Corporation
FTIR Spectral Searching
Assessing the Hit Quality Index
458 Ianniello,
457
Michael Boruta, ACD/Labs, 8 King St. East, Suite 107, Toronto, ON M5C
1B5 Canada
Various algorithms can be used to create a hit quality index (HQI), with the HQI
being a measure of how well the query spectrum compared against each reference
spectrum. However, HQI does not tell the full story and specifically does not tell us
much about the quality of the match between query and reference spectra. In a
ranked list of search results, the difference, or gap in the HQI between two successive hits can be used as an indicator of the quality of a match. This work looks at
progress made on using an assessment of the gap between hits to determine the
quality of a match, what represents a significant gap and when this assessment
can fail.
Workshop II: Interview This: Prepare Yourself to be Hired
IR and Raman Spectral Interpretation: What Can We Do Today
Michael Boruta, ACD/Labs, 8 King St., East, Suite 107, Toronto, ON
M5C 1B5 Canada
Interpreting infrared (IR) or Raman spectral data is a task generally done by experts
with a significant amount of experience to the ability to carry out the interpretation.
Fortunately, many experts have published textbooks on the subject to assist with
the interpretation process. In a corporate environment it is common for individuals
to have detailed knowledge about the correlation between some spectral features
and the materials they work with every day. This knowledge often exceeds the more
Date: Tuesday, November 13, 2012
Time: 9:30 am – 1:00 pm
Competencies & skills developed: Resume writing, interviewing techniques
Location: Double Tree Hotel Parlor 154
Instructors: Antonio Fernandez, Mariann Neverovitch, Bristol-Myers Squibb, Lauren Steinberg,
Widener University
Save these 2013 Dates:
th
EAS November 18-20
and
CALL FOR PAPERS
March 1- April 15, 2013
EAS seeks contributed abstracts from scientists in
multiple areas of analysis. Be a part of our Technical
Program to share your insights and innovation
Show your work and get visible!
www.eas.org
69
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