USP Method - ResearchGate

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1
ADVANCED
USPmethods
2
Also by Phenomenex
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ADVANCED
USPmethods
Published by Phenomenex
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Torrance, CA 90501
Tel: (310) 212 - 0555
Fax: (310) 328 - 7768
Copyright © 2003
4 Introduction
APPLICATION INDEX
COMPOUND
Acetaminophen
Albuterol
Alprazolam
p-Aminobenzoic acid
Amitriptyline HCl
Amoxicillin
Aspirin
Atenolol
Benzalkonium chloride
Benzoic acid
Benzophenone
Betamethasone
Butyrophenone
Caffeine
Cefuroxime axetil
Cephalexin
Cephradine
Chloramphenicol
Cortisone acetate
Desipramine
Dextroamphetamine
Dextromethorphan HBr
Diphenhydramine
Dopamine HCl
Doxepin HCl
Doxylamine succinate
Epinephrine bitartrate
Estradiol
Estrone
Ethinyl estradiol
Ethyl paraben
Fluoxetine HCl
Glyburide
PAGE NO.
10,11
14
12,13,71
65
15
16, 72
10, 73
17, 18, 19
20
10, 30, 38, 39
48
63, 64, 82
56
10
21
22, 23, 24, 74, 75
24, 75
25, 76
26, 27
50, 51
61
28
29
30
31
32
60
33
33
34
33, 34, 57
35
36, 37
COMPOUND
Guaifenesin
Hydrocodone bitartrate
Hydrocortisone
Hydrocortisone acetate
Hydrocortisone cream
1-Hydroxybenzatriazole
Ibuprofen
Imipenem (sterile)
Imipramine
Lidocaine HCl
Lorazepam
Medroxyprogesterone
Methyl paraben
Minoxidil
Naproxen
Nortriptyline
Oxacillin
Phenylephrine HCl
Phenylpropanolamine
Prednisolone
Prednisone
Procainamide HCl
Progesterone
Propoxyphene
Propranolol
Pseudoephedrine HCl
Reserpine
Salicylic acid
Tetracaine
Thiamphenicol
Triazolam
Valerophenone
PAGE NO.
38, 39
40
41, 42, 77
43, 78
44, 45
22, 23, 74
46, 47, 48, 79
49
50, 51
52, 80
53
54, 55
52, 80
54, 55
56, 57
58, 59
81
60
61
63, 64, 82
41, 42, 62, 77
65, 67
36, 37
66
67
29
68
10, 73
69
83
12, 13, 71
46, 47, 79
Introduction 5
ADVANCED
USPmethods
TABLE OF CONTENTS
APPLICATIONS SUPPLIED BY
• Application Index -----------------------------------------------
4
• About this guide ------------------------------------------------I. Method Validation --------------------------------------II. Adjusting USP Methods --------------------------------
6
7
8
• USP Methods ----------------------------------------------------
9
• Alternative Methods --------------------------------------------
70
• Notes ---------------------------------------------------------------
84
6 Introduction
ABOUT THIS GUIDE
Our goal in developing this guide was to illustrate
some of the ways which USP/NF methods may be
"modified" in order to improve system performance or
increase sample throughput. The requirements of
many of the validated HPLC methods have been left
intentionally vague in order to allow some room for
modification to fit specific circumstances. For instance,
a method may state that the flow rate should be
"around 1 mL/min". This vagueness would imply that
modifications to the flow rate are acceptable, so long
as the modified method still meets system suitability
requirements. This can be a great advantage to
analysts who, for whatever reason, are unable to meet
system suitability requirements under the specified
method. For instance, closely eluting peaks which fail
system suitability resolution requirements may be
resolved through slight modifications to mobile phase
pH, or even by going to a longer column or smaller
particle size packing for a higher efficiency separation.
In addition, simple modifications such as increasing
flow rate or reducing column length can significantly
increase sample throughput over the long run.
However, this vagueness has also led to considerable
confusion as to exactly what is considered "modified"
versus "changed". In response to this, several articles
have been published, and the USP itself has published a proposed list of acceptable system modifications in Pharmacopeial Forum vol. 25(2) and vol. 26(5).
This list, once approved, will be added to the System
Suitability section of the USP.
Although we cannot perform your validations for you,
we can act as a resource for columns and information
to better utilize USP methods. With this guide and our
Luna HPLC columns you will be able to comply with
USP requirements, as well as modernize and optimize
your methods for improved performance and increased productivity.
Introduction 7
I. METHOD VALIDATION
Chromatographic methods are routinely employed in labs
around the world for a variety of analytical and preparatory
purposes. Due to the relative ease with which most samples
can be analyzed and the robustness of many chromatographic analyses, these techniques have become the
methods of choice for combinatorial screening of potential
drug products to routine QA/QC analysis of nutritional
products.
In order to ensure the reproducibility and ruggedness of
developed methods, a process known as method validation
is carried out. Validation, as defined by the USP is "the
process of providing documented evidence that the method
does what it is intended to do" and is performed on all
instruments and methods used in an analytical method.
Thus, everything from the software and hardware system to
the method itself must be validated before the analytical
method is considered "validated". However, the focus of this
guide is specifically on the validation of HPLC methods as
defined in the USP/NF.
Method validation for HPLC methods consists of a process
designed to provide documentation that the method will
perform as intended. This process of 8 steps or "Analytical
Performance Parameters" (USP {1225}) which must be
determined in order for a method to be considered "validated". The parameters to be determined are:
1. The accuracy of a method refers to "the closeness of test
results obtained by that method to the true value" (USP
XXIII). In most cases an accuracy value of 98-102%,
determined by calculating percent recovery values of a
known standard or against a second technique, is required.
In most pharmaceutical analyses, accuracy is determined
against a standard curve generated using known amounts
of target analyte.
2. Precision represents the degree of variability in your
results using the same method. This is normally expressed
as relative standard deviation (RSD%) and must be less
than 2% for most methods. There are three different levels
of precision:
a. Method precision (repeatability) is determined by
multiple analyses of the same sample over a short
period of time.
b. Intermediate precision is the precision of the method
when run on different days by different operators using
different instruments.
c. Reproducibility is the variability when the same
method is performed by different labs.
3. Specificity is the "ability to measure accurately and
specifically the analyte in the presence of components that
may be expected to be present in the sample matrix" (USP
XXIII). In other words, the ability to separate your target
compound from interfering components. There can be no
interference from known impurities or degradation/stress
products.
4. The linearity or range of a method refers to the ability of
a method to give accurate results over a given range of
analyte concentrations. Generally, your assay must have a
correlation coefficient >0.997 for 5 points over 50-150% of
your expected target analyte concentration.
5. Limit of detection is a value given to the lowest amount
of analyte that can be detected, but not quantified, using a
given method. Often, this minimum value is specified as
three times the signal-to-noise ratio.
6. Limit of quantitation, as the name implies, represents
the lowest concentration of analyte which can be accurately
quantified using a given method. This value is usually a
minimum of 10 times the signal-to-noise ratio.
7. The ruggedness of a method is "the degree of reproducibility of the test results obtained by the analysis of the
same sample under a variety of normal test conditions,
such as different laboratories, different analysts, different
instruments, different lots of reagents, different elapsed
assay times, different assay temperatures, different days,
etc." (USP XXIII). In other words, will other labs be able to
reproduce your results? Typically, a valid method must have
an RSD% of less than 2% between labs.
8. Method robustness is a measure of the ability of a
method to withstand small changes in running conditions.
You demonstrate method robustness by varying factors
such as mobile phase pH, flow rate, temperature, etc.
8 Introduction
II. ADJUSTING USP METHODS
Why should I make modifications to USP methods?
Although the methods written in the USP compendia have
all gone through the validation process and so should, in
the best of circumstances, represent methods which will
reliably and reproducibly produce acceptable results for the
methods, there are instances where it may be worth
investigating changing or modifying existing methods. For
instance, it could be that a particular formulation for a drug
substance gives co-elution with an excipient under the USP
conditions. A method which was validated using one
particular formulation cannot take into account all of the
possible excipients which may be present in future formulations. Thus, it may be possible that a given USP method
simply will not resolve the target analyte with acceptable
specificity due to excipient compounds present in a
formulation.
Productivity issues / saving time may also be valid
considerations for USP methods. Many of these methods
were written using very old HPLC technology, and thus may
depend on the use of long columns (300 mm) with large
particle sizes (10µ). Given the numerous advances in
column technology, many of these older methods could be
more efficiently performed using modern HPLC columns
with smaller, more highly efficient (3µ) particles and shorter
column lengths. Over the long run, the time savings
resulting from using shorter columns can be substantial.
Improved results can also be an added benefit of using
more modern, state-of-the-art HPLC column technologies.
The vast advances in silica quality and bonding techniques
have resulted in columns which, for many applications, will
perform substantially better than their older predecessors.
For example, columns packed with low-purity silicas may
display extensive tailing with basic or acidic compounds,
and failure of the column to meet system suitability requirements for a given assay may delay production and product
release.
As stated previously, many USP HPLC methods are left
intentionally vague. This may be advantageous in that it
allows some flexibility in a method, allowing the analyst to
"fine-tune" and adjust it to certain situations. If you do make
adjustments which are within a reasonable range, you do
not have to entirely revalidate, although you must show an
improvement in chromatography using reference standards.
In addition, it may be advisable to show equivalence to an
existing, validated method. The problem arises in determining just what an "acceptable" modification to a method
consists of, and when a modification becomes a "change"
which would warrant re-validation of a method. For instance,
if a method calls for a 300 x 4.6mm column, do I have to revalidate if I choose to use a 250 x 4.6mm? What about a
150 x 4.6mm column?
In response to these types of questions, several adjustment
limits have been proposed and reviewed in recent publications. At this point, minor modifications to USP methods are
commonly used and accepted so long as they conform to
the method in intent. The following acceptable method
"modifications" have been proposed (Pharmocopeial Forum
25(2) and 26(5)) and are as follows :
1.
Mobile phase pH: ± 0.2 units
• pH of 7.6 can be adjusted from 7.4-7.8
2.
Concentration of salts in buffer: ± 10%
• 20mM Potassium phosphate can be 18-22mM, as
long as proper pH is maintained as above.
3.
Ratio of components in mobile phase: ± 30%
of the minor component(s), or ± 2% absolute of that
component, whichever is greater. However a change in
any component cannot exceed ± 10% absolute, nor
can the final concentration be reduced to zero.
• 60:40 Acetonitrile/Water can be adjusted to ± 12%
water (=30% of 40), but this exceeds the 10%
maximum absolute change, so can range from 30%
to 50% water in this case
4.
Wavelength of UV-Visible Detector: no deviations
permitted
5.
Column length: ± 70%
• 150 x 4.6 mm column can be varied ± 105 mm in
length
6.
Column inner diameter: ± 50%
• 150 x 4.6 mm column can be varied ± 2.3 mm
7.
Particle size: can be reduced as much as 50%
• 10µ can be switched with a 5µ particle
8.
Flow rate: ±50%
• 1 mL/min can be varied from 0.5 to 1.5 mL/min
9.
Injection volume: increased to as much as twice the
volume specified, provided no adverse effects
• Must be within stated linearity range of method!
10. Column temperature: ± 20°C
USP Methods 9
USP METHODS
10 USP Methods
- Acetaminophen, aspirin and caffeine - USP Method 2A00710
Also See ALTERNATIVE METHOD page 73
Acetaminophen
USP column specified: 100 x 4.6mm L1
Column used: 100 x 4.6mm LUNA® 5µm C18(2)
Part No.: 00D-4252-E0
Caffeine
System Suitability Requirement
USP Tailing Factor for all < 1.2
Resolution between any analyte and I.S. is > 1.4
Aspirin
USP/NF 23 page:
0021
Standard prep:
Mobile phase:
Flow rate:
Detection:
Temperature:
Injection:
Sample:
APP. ID No 3270
Benzoic acid 0.36mg/mL, all others at 0.1mg/mL
Water / Methanol / Glacial acetic acid (69:28:3)
2.0 mL/min
UV @ 275nm
45°C
10 µL
1. Acetaminophen
2. Caffeine
3. Aspirin
4. Benzoic acid
5. Salicylic acid
USP Methods 11
2A00200 - Acetaminophen capsules - USP Method
Part No.: 00G-4252-E0
Efficiency(N) > 1000 plates
USP Tailing Factor < 2.0
Column Performance:
Efficiency(N) = 5047 plates/column
USP Tailing Factor = 1.14
Resolution = NA
USP/NF 23 page:
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
APP. ID No 3259
Acetaminophen - 0.01mg/mL in mobile phase
Water / Methanol (75:25)
1.5 mL/min
UV @ 243nm
10 µL
0017
12 USP Methods
- Alprazolam - USP Method 2A03940
Column used: 250 x 4.6mm LUNA® 5µm Silica
Part N0.:00G-4042-E0
Resolution > 2.0
Column Performance:
Efficiency(N) = 8940
Resolution = 7.05
USP/NF 23 page:
0046
Standard prep: Alprazolam and Triazolam
each 0.025mg/mL in mobile phase
Mobile phase: Acetonitrile / Chloroform / Butanol / Water / Acetic acid
(850 : 80 : 50 : 20 : 0.5)
Flow rate: 2.0 mL/min
Detection: UV @ 254nm
Injection: 20 µL
Sample: 1. Triazolam
2. Alprazolam
APP. ID No 3262
USP Methods 13
- Alprazolam - USP Acceptable Modified Method
Column used: 100 x 4.6mm LUNA® 5µm Silica(2)
Part No.:00D-4274-E0
Resolution > 2.0
Column Performance:
Resolution = 5.89
USP/NF 23 page:
Standard prep: Alprazolam and Triazolam
Mobile phase: Acetonitrile / Chloroform / Butanol / Water / Acetic acid
(850 : 80 : 50 : 20 : 0.5)
Injection: 40 µL
2. Alprazolam
APP. ID No 3276
NA
14 USP Methods
- Albuterol Tablets - USP Method 2A02962
Part No.:00F-4252-E0
Efficiency(N) > 800 (was 5647)
Column Performance:
Efficiency(N) = 5647 Plates/column
Resolution = NA
USP/NF 23 page: 0039
Standard prep: Albuterol - 0.03mg/mL in mobile phase
Mobile phase: Methanol / Water with 5mM Hexane sulfonic
acid and 1% Glacial acetic acid (40 : 60)
Injection: 20 µL
APP. ID No 3260
USP Methods 15
2A09600 - Amitriptyline Hydrochloride - USP Method
Column used: 300 x 3.9mm Bondclone 10µm C18
Part No.:00H-2117-C0
USP Tailing Factor < 2.0 (was 1.56)
Efficiency(N) > 800
Column Performance:
Resolution = NA
USP/NF 23 page:
Standard prep: Amitriptyline 0.2 mg/mL in water
Mobile phase: Acetonitrile / 92 mM Monobasic
sodium phosphate pH 2.5 (42:58)
Flow rate: 2 mL/min
Detection: UV @ 254 nm
Injection: 20µL
APP. ID No 3319
0093
16 USP Methods
- Amoxicillin - USP Method 2A11300
Part No.:00G-4252-E0
Efficiency(N) > 1700
k’ between 1.1-2.8
Column Performance:
Resolution = NA
USP/NF 23 page:
0100
Standard prep: Amoxicillin - 1.2mg/mL in phosphate buffer
Mobile phase: 50mM Potassium phosphate pH 5.0 /
Acetonitrile (96 : 4)
Injection: 10 µL
APP. ID No 3258
USP Methods 17
2A17776 - Atenolol Injection - USP Method
USP column specified: 250 x 4.6mm 5µm L1
Column Performance:
Resolution = NA
USP/NF 23 page:
Standard prep: Atenolol 0.2mg/mL in citrate buffer pH 6.0
Mobile phase: Acetonitrile / 5mM 1-Octanesulfonatic acid
with 38mM Sulfuric acid (25 : 75)
Detection: UV @ 275 nm
Injection: 10 µL
APP. ID No 3284
NA
18 USP Methods
- Atenolol - USP Method 2A17774
Column Performance:
Resolution = NA
USP/NF 23 page:
NA
Standard prep: Atenolol 0.01mg/mL in mobile phase
Mobile phase: Methanol / 5mM Dibasic sodium phosphate
with 7mM 1-Heptanesulfonate and 20mM
Dibutylamine, pH 3.0 (300 : 700)
Injection: 10 µL
APP. ID No 3285
USP Methods 19
- Atenolol - USP Acceptable Modified Method
Column Performance:
Resolution = NA
USP/NF 23 page:
Standard prep: Atenolol 0.01mg/mL in mobile phase
Mobile phase: Methanol / 5mM Dibasic sodium phosphate
with 7mM 1-Heptanesulfonate and 20mM
Dibutylamine, pH 3.0 (300 : 700)
Flow rate: 0.9 mL/min (was 0.6mL/min in USP)
Injection: 10 µL
APP. ID No 3286
0320
20 USP Methods
- Benzalkonium Chloride - USP Acceptable Modified Method 7B00500
USP column specified: L10
Column used: 150 x 4.6mm LUNA® 5µm CN
Efficiency(N) > 1000 plates
Resolution > 1.5
Column Performance:
USP Tailing Factor = 1.61 and 1.27
Resolution = 2.70
Standard prep: Benzalkonium chloride - 4 mg/mL in water
Mobile phase: 100mM Sodium acetate pH 5.0 / Acetonitrile
(40 : 60)
Flow rate: 2 mL/min
Injection: 1 µL (was 20µL in USP)
Sample: 1. C12 peak
2. C14 peak
APP. ID No 9300
USP Methods 21
2C08820 - Cefuroxime Axetil - USP Acceptable Modified Method
Column used: 250 x 4.6mm Develosil TMS-UG 5µm
Part No.: CH0-4230
Resolution > 1.5
Column Performance:
Resolution = 2.69
USP/NF 23 page:
Standard prep: Cefuroxime axetil 0.24mg/mL in mobile phase
and acetanilide 0.54mg/mL in mobile phase
Mobile phase: Methanol / 0.2M Monobasic Ammonium Phosphate (380 : 620)
Injection: 10 µL
Sample: 1. Acetanilide
2&3. Cefuroxime axetil diastereomers
APP. ID No 3318
0315
22 USP Methods
- Cephalexin - USP Method 2C09200
Resolution > 5.0
Column Performance:
Resolution = 22.64
USP/NF 23 page:
0320
Standard prep: Cephalexin (0.2 mg/mL) and 1-hydroxybenzotriazole (0.1mg/mL)
in mobile phase
Mobile phase: Water / Acetonitrile / Methanol / Triethylamine (850 : 100 : 50 : 15)
with 5mM 1-Pentanesulfonic acid and adjusted to pH 3.0
Injection: 20 µL
Sample: 1. 1-Hydroxybenzotriazole
2. Cephalexin
APP. ID No 3280
USP Methods 23
- Cephalexin - USP Acceptable Modified Method
Resolution > 5.0
Column Performance:
Resolution = 17.61
USP/NF 23 page:
Standard prep: Cephalexin (0.2mg/mL) and 1-hydroxybenzotriazole(0.1 mg/mL)
in mobile phase
Mobile phase: Water / Acetonitrile / Methanol /Triethylamine (850 : 100 : 50 : 15)
with 5mM 1-Pentanesulfonic acid and adjusted to pH 3.0
Injection: 20 µL
2. Cephalexin
APP. ID No 3279
0320
24 USP Methods
- Cephradine capsules - USP Acceptable Modified Method 2C10100
USP column specified:250 x 4.6mm 10µm L1
Resolution cephalexin and cephradine must be > 2.0
Column Performance:
Resolution = 7.46
Standard prep: Mix of cephradine and cephalexin,
Mobile phase: Water / Methanol / 0.5 M Sodium acetate /
0.7 N Acetic acid (782 : 200 : 15 : 3)
Injection: 20 µL
Sample: 1. Cephradine
2. Cephalexin
APP. ID No 3264
USP Methods 25
2C11500 - Chloramphenicol - USP Acceptable Modified Method
USP Tailing Factor > 2.0
Column Performance:
Resolution = NA
USP/NF 23 page:
Standard prep: Chloramphenicol at 80 µg/mL in mobile phase
Mobile phase: Water / Methanol / Glacial acetic acid
(55 : 45 : 0.1)
Injection: 10 µL
APP. ID No 3312
0332
26 USP Methods
- Cortisone acetate - USP Method 2C27200
k’ > 2.0
Column Performance:
Resolution = NA
USP/NF 23 page:
0428
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
APP. ID No 8709
Cortisone acetate - 0.1 mg/mL
Water / Acetonitrile (55 : 45)
2 mL/min
UV @ 254nm
35 µL
USP Methods 27
- Cortisone acetate - USP Acceptable Modified Method
k’> 2.0
Column Performance:
Resolution = NA
USP/NF 23 page:
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
APP. ID No 8710
Cortisone acetate – 0.1 mg/mL
Water / Acetonitrile (55 : 45)
2.0 mL/min
UV @ 254nm
20 µL (was 35µL in USP)
0428
28 USP Methods
- Dextromethorphan HBr - USP Method 2D06700
Column Performance:
Resolution = NA
USP/NF 23 page:
0482
Standard prep: Dextromethorphan HBr-0.1 mg/mL
in mobile phase
Mobile phase: Acetonitrile / Water (70 : 30), both with
7mM Docusate sodium and 7mM
Ammonium nitrate, pH 3.4 with acetic acid
Flow rate: 1 mL/min
Injection: 20 µL
APP. ID No 8711
USP Methods 29
2D17030 - Diphenhydramine & Pseudoephedrine - USP Method
Resolution between two > 3.0
Column Performance:
Efficiency(N) = 9460 plates/column for diphenhydramine,
8867 for pseudoephedrine
USP Tailing Factor = 1.16 for diphenhydramine,
0.96 for pseudoephedrine
Resolution = 21.96
USP/NF 23 page:
Standard prep: Diphenhydramine and Pseudoephedrine
@ 25ug/mL in 0.5% acetic acid
Mobile phase: Methanol / Acetonitrile / Water w/10mM Heptane
sulfonate and 13mM Triethylamine,
pH 3.3 (10 : 26 : 64)
Flow rate: 2 mL/min
Injection: 50 µL
Sample: 1. Pseudoephedrine
2. Diphenhydramine
APP. ID No 11203
0534
30 USP Methods
- Dopamine HCl Injection - USP Acceptable Modified Method 2D0400
Resolution > 4.0
Column Performance:
Resolution = 11.94
Standard prep: Dopamine 0.16mg/mL and benzoic acid 0.5mg/mL in mobile phase
Mobile phase: Water with 1% Acetic and 5mM
Octanesulfonic acid / Acetonitrile (87 : 13)
Flow rate: 2.0 mL/min (was 1.5 by USP)
Injection: 40 µL (was 35±1 in USP)
Sample: 1. Benzoic acid
2. Dopamine
APP. ID No 8712
USP Methods 31
2D20800 - Doxepin HCl - USP Method
Part No.:00E-4249-E0
Resolution > 1.5
Column Performance:
USP Tailing Factor = 1.16 and 1.31 for isomers
Resolution = 2.24
USP/NF 23 page:
Standard prep: Doxepin 100µg/mL in mobile phase
Mobile phase: 0.2m Monobasic sodium phosphate / Methanol (61 : 39),
adjusted to pH2.5 with phosphoric acid (USP method was 70 : 30)
Flow rate: 1 mL/min
Injection: 20 µL
Temperature: 50°C
Sample: E and Z isomers of Doxepin
APP. ID No 9230
0551
32 USP Methods
- Doxylamine succinate - USP Acceptable Modified Method 2D22300
Part No.: 00D-4248-E0
Resolution > 2.5
Column Performance:
Resolution = NA
USP/NF 23 page:
0560
Standard prep: Doxylamine 0.25mg/mL in mobile phase
Mobile phase: Acetonitrile / Water (37 : 63) with 25mM
Monobasic Potassium phosphate, 10mM
Triethylamine, and 5mM Sodium lauryl sulphate
Injection: 10 µL
APP. ID No 3274
USP Methods 33
2E07900 - Estradiol - USP Acceptable Modified Method
Resolution between Estradiol and estrone > 2.0
Column Performance:
Efficiency(N) = 22223 plates/column for estradiol
USP Tailing Factor = 1.15 for estradiol
Resolution = 11.52 for 3/2, and 8.88 for 2/1
USP/NF 23 page:
Standard prep: Ethyl paraben-0.75mg/mL
Estradiol-20µg/mL
Estrone-33µg/mL
Mobile phase: Acetonitrile / Water (55 : 45)
Flow rate: 1.0 mL/min (was 1.5 by USP)
Injection: 25 µL
Sample: 1. Ethyl paraben
2. Estradiol
3. Estrone
APP. ID No 8713
0622
34 USP Methods
- Ethinyl estradiol - USP Method 2E10800
Resolution > 4.5
Column Performance:
Efficiency(N) = 10128 plates/column for ethinylestradiol
Resolution = 13.30
USP/NF 23 page:
0638
Standard prep: Ethinyl estradiol-200µg/mL
Ethyl paraben-20µg/mL
Mobile phase: Water / Acetonitrile (50 : 50)
Injection: 25 µL
Sample: 1. Ethinyl estradiol
2. Ethyl paraben
APP. ID No 3315
USP Methods 35
2F04840 - Fluoxetine HCl - USP Method
Resolution > 2.0
Column Performance:
Resolution = NA
USP/NF 23 page:
Supplement No. 8p. 4210
Standard prep: Fluoxetine HCl-110µg/mL in mobile phase
Mobile phase: 10mM Triethylamine buffer pH 6.0 w/phosphoric acid /
Tetrahydrofuran / Methanol (60 : 30 : 10)
Injection: 10 µL
APP. ID No 3273
36 USP Methods
- Glyburide - USP Method 2G2930
Resolution > 5.0
Column Performance:
USP Tailing Factor = 1.0 for glyburide
Resolution = 6.57
USP/NF 23 page:
0713
Standard prep: Glyburide (0.5mg/mL) and progesterone
(0.2mg/mL) in mobile phase
Mobile phase: Acetonitrile / 10 mM Monobasic ammonium
phosphate, pH 5.25 (55 : 45)
Injection: 10 µL
Sample: 1. Glyburide
2. Progesterone
APP. ID No 3316
USP Methods 37
- Glyburide - USP Acceptable Modified Method
Part No.:00E-4248-E0
Resolution > 5.0
Column Performance:
Efficiency(N) = 15428 plates/column for glyburide
Resolution = 8.08
Standard prep: Glyburide (0.5mg/mL) and progesterone
Mobile phase: Acetonitrile / Water with 10mM Monobasic
ammonium phosphate, pH 5.25(550 : 450)
Injection: 10 µL
Sample: 1. Glyburide
2. Progesterone
APP. ID No 3317
38 USP Methods
- Guaifenesin Tablets - USP Method 2G05800
Resolution > 3.0
Column Performance:
Efficiency(N) = 3734 plates/column for guaifenesin
Resolution = 10.83
USP/NF 23 page:
0724
Standard prep: Guaifenesin-40µg/mL
Benzoic acid-100µg/mL
Mobile phase: Water / Methanol / Acetic acid (60 : 40 : 1.5)
Injection: 20 µL
Sample: 1. Guaifenesin
2. Benzoic acid
APP. ID No 3281
USP Methods 39
- Guaifenesin Tablets - USP Acceptable Modified Method
Resolution > 3.0
Column Performance:
Efficiency(N) = 3870 plates/column for guaifenesin
USP Tailing Factor = 1.23 for guaifenesin
Resolution = 10.83
USP/NF 23 page:
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
Sample:
APP. ID No 3267
Guaifenesin-40µg/mL Benzoic acid-100µg/mL
Water / Methanol / Acetic acid (60 : 40 :1.5)
2.0 mL/min
UV @ 276nm
20 µL
1. Guaifenesin
2. Benzoic acid
0724
40 USP Methods
- Hydrocodone bitartrate - USP Acceptable Modified Method 2H06000
Column Performance:
Resolution = NA
USP/NF 23 page:
0751
Standard prep: Hydrocodone 1mg/mL in methanol
Mobile phase: Acetonitrile / Water / Diethylamine / Methanol
(440 : 2.2 : 0.55 : 45)
Injection: 5 µL(USP was 20µL)
APP. ID No 9299
USP Methods 41
2H06200 - Hydrocortisone - USP Method
Resolution > 3.0
Column Performance:
Resolution = 8.34
Standard prep: Hydrocortisone 0.1 mg/mL and prednisone 0.06 mg/mL in chloroform
Mobile phase: Butyl chloride / Water-saturated butyl chloride / THF / Methanol /
Glacial acetic acid (95 : 95 : 14 : 7 : 6)
Injection: 10 µL
Sample: 1. Hydrocortisone
2. Prednisone
APP. ID No 9295
42 USP Methods
- Hydrocortisone - USP Acceptable Modified Method 2H06200
Resolution > 3.0
Column Performance:
Resolution = 5.98
Standard prep: Hydrocortisone 0.1 mg/mL and prednisone 0.06 mg/mL in chloroform
Mobile phase: Butyl chloride / Water-saturated butyl chloride / THF / Methanol /
Glacial acetic acid (95 : 95 : 14 : 7 : 6)
Injection: 10 µL
2. Prednisone
APP. ID No 3287
USP Methods 43
2H07000 - Hydrocortisone acetate - USP Method
USP Tailing Factor > 2.0
Column Performance:
Resolution = NA
Standard prep: Hydrocortisone acetate 0.1 mg/mL
in chloroform
Mobile phase: Butyl chloride / Water-saturated
butyl chloride / Tetrahydrofuran / Methanol /
Glacial acetic acid (95:95:14:7:6)
Injection: 10 µL
APP. ID No 9298
44 USP Methods
- Hydrocortisone cream - USP Method 2H06300
None stated
Column Performance:
Efficiency(N) =13,569
USP Tailing Factor =1.02
Resolution = NA
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
APP. ID No 3271
Hydrocortisone-50µg/mL in methanol
Water / Acetronitrile (75 : 25)
2.0 mL/min
UV @ 254nm
15 µL
USP Methods 45
- Hydrocortisone cream - USP Acceptable Modified Method
None started
Column Performance:
Resolution = NA
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
APP. ID No 8714
Hydrocortisone-50µg/mL in methanol
Water / Acetronitrile (75 : 25)
1.5 mL/min (was 2mL/min USP)
UV @ 254nm
15 µL
46 USP Methods
- Ibuprofen - USP Method 2I00100
Resolution > 2.5
Column Performance:
Resolution = 8.08
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
Sample:
APP. ID No 3278
Ibuprofen 12mg/mL and valerophenone 0.35mg/mL in mobile phase
Acetonitrile / Water (600:400) with 20mM Chloroacetic acid, pH3.0
2.0 mL/min
UV @ 254nm
5 µL
1. Ibuprofen
2. Valerophenone
USP Methods 47
2I00100 - Ibuprofen - USP Acceptable Modified Method
Resolution > 2.5
Column Performance:
Resolution = 5.77
USP/NF 23 page:
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
Sample:
APP. ID No 3277
Ibuprofen 12mg/mL andvalerophenone 0.35mg/mL in mobile phase
Acetonitrile / Water (600:400) with 20mM Chloroacetic acid, pH3.0
2.0 mL/min
UV @ 254nm
5 µL
1. Ibuprofen
2. Valerophenone
0786
48 USP Methods
- Ibuprofen Oral Suspension - USP Acceptable Modified Method 2I00150
Resolution > 1.5
Peak tailing < 2.0 (was 1.03)
Column Performance:
Resolution = 7.93
USP/NF 23 page:
0551
Standard prep: Ibuprofen - 0.4 mg/mL
Benzophenone - 0.3 mg/mL in mobile phase
Mobile phase: 0.01M phosphoric acid / Acetonitrile (55 : 45)
(USP method states 63 : 37)
Injection: 5 µL
Sample: 1. Benzophenone
2. Ibuprofen
APP. ID No 3263
USP Methods 49
2I00770 - Sterile Imipenem - USP Method
Column used: 150 x 4.6mm AQUA™ 5µm C18
Efficiency(N) > 600
Column Performance:
Resolution = NA
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
APP. ID No 3272
Imipenem 0.4 mg/mL in mobile phase
7mM potassium phosphate pH 6.8
1.5 mL/min
UV @ 300nm
10 µL
50 USP Methods
- Imipramine - USP Method 2I00800
Resolution > 1.3
Column Performance:
Resolution = 2.76
Standard prep: Imipramine and desipramine 0.3mg/mL
in mobile phase
Mobile phase: 0.06M Sodium perchlorate / Acetonitrile /
Triethlamine (625 : 375 : 1), adjusted with
Perchloric acid to a pH of 2.0
Injection: 20 µL
Sample: 1. Imipramine
2. Desipramine
APP. ID No 3255
USP Methods 51
- Imipramine - USP Acceptable Modified Method
Resolution > 1.3
Column Performance:
Resolution = 2.12
USP/NF 23 page:
Standard prep: Imipramine and desipramine 0.3mg/mL
in mobile phase
Mobile phase: 0.06M Sodium perchlorate / Acetonitrile /
Triethlamine (625 : 375 :1),
adjusted with Perchloric acid to a pH of 2.0
Flow rate: 2 mL/min (was 1.5 in USP)
Injection: 20 µL
Sample: 1. Imipramine
2. Desipramine
APP. ID No 3248
0794
52 USP Methods
- Lidocaine HCl - USP Acceptable Modified Method 2L02900
Resolution Lidocaine/methyl paraben > 3.0
Column Performance:
Resolution = 22.57
USP/NF 23 page:
0887
Standard prep: Lidocaine-1.7mg/mL in mobile phase
Methyl paraben-220µg/mL in mobile phase
Mobile phase: Acetonitrile / Water with 5% Acetic acid, pH 3.4 (20:80)
Injection: 5 µL (was 20 µL in USP method)
Sample: 1. Lidocaine
2. Methyl paraben
APP. ID No 3254
USP Methods 53
2L05856 - Lorazepam Tablets - USP Method
Column Performance:
Resolution = NA
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
1.0
APP. ID No 3253
min
Lorazepam 0.1mg/mL in mobile phase
Water / Methanol / Acetic acid (55 : 45 : 2)
2.0 mL/min
UV @ 254nm
20 µL
3.0
4.0
5.0
2.0
54 USP Methods
- Minoxidil - USP Method 2M23430
Resolution > 2.0
Column Performance:
Resolution = 15.50
mL
APP. ID No 3266
Standard prep: Minoxidil 0.2 mg/mL and Medroxyprogesterone acetate 0.25 mg/
in mobile phase
Mobile phase: Methanol / Water / Acetic acid (700 : 300 : 10) with
7mM Docusate sodium and pH 3.0 with perchloric acid
Detection: UV@254nm
Injection: 10 µL
Sample: 1. Minoxidil
2. Medroxyprogesterone acetate
USP Methods 55
- Minoxidil - USP Acceptable Modified Method
Resolution > 2.0
Column Performance:
Resolution = 2.12
Standard prep: Minoxidil 0.2 mg/mL and Medroxyprogesterone acetate 0.25 mg/mL
in mobile phase
Mobile phase: Methanol / Water / Acetic acid (700 : 300 : 10) with
3.0g docusate sodium pH 3.0 with perchloric acid
Flow rate: 1.5 mL/min (was 1 mL/min in USP)
Injection: 10 µL
Sample: 1. Minoxidil
2. Medroxyprogesterone acetate
APP. ID No 3265
56 USP Methods
- Naproxen Tablets - USP Method 2N01900
Resolution > 11.0
Column Performance:
Resolution = 14.46
USP/NF 23 page:
1054
Standard prep: Naproxen 25 µg/mL and butyrophenone
0.001µL/mL
Mobile phase: Acetonitrile / Water / Glacial Acetic acid
(50 : 49 : 1)
Injection: 20 µL
Sample: 1. Naproxen
2. Butyrophenone
APP. ID No 3282
USP Methods 57
2N01950 - Naproxen Oral Suspension - USP Acceptable Modified Method
Resolution > 3.0
Column Performance:
USP Tailing Factor = 1.15 for naproxen
Resolution = 8.91
USP/NF 23 page:
1053
Standard prep: Naproxen - 50 µg/mL
Ethylparaben - 4.4 µg/mL
Mobile phase: Methanol / Water (50 : 50) with 30mM Sodium acetate pH 5.8
Injection: 20 µL
Sample: 1. Naproxen
2. Ethylparaben
APP. ID No 3283
58 USP Methods
- Nortriptyline HCl Capsules - USP Method 2N12400
Efficiency(N) > 500
Column Performance:
Resolution = NA
USP/NF 23 page:
1107
Standard prep: Nortriptyline 0.38mg/mL in methanol
Mobile phase: Acetonitrile / Methanol / 12mM Potassium
phosphate pH 6.7 (40 : 43 : 17)
Injection: 5 µL
APP. ID No 9296
USP Methods 59
- Nortriptyline HCl Capsules - USP Acceptable Modified Method
Efficiency(N) > 500
Column Performance:
Resolution = NA
USP/NF 23 page:
Standard prep: 0.38mg/mL in methanol
Mobile phase: Acetonitrile / Methanol / 12mM Potassium
phosphate pH 6.7 (40 : 43 : 17)
Injection: 5 µL
APP. ID No 9297
1107
60 USP Methods
- Phenylephrine Hydrochloride Injection - USP Method 2P13200
Resolution > 1.0
Column Performance:
USP Tailing Factor = 1.32 for epipnephrine, 0.78 for phenylephrine
Resolution = 7.32
USP/NF 23 page:
1212
Standard prep: Phenylephrine HCl and Epinephrine bitartrate,
both 0.4mg/mL in mobile phase
Mobile phase: Water / Methanol (50 : 50) with 0.1%
1-octanesulfonic acid adjust to pH3.0
with phosphoric acid
Injection: 5 µL (USP was 20µL)
Sample: 1. Epinephrine bitartrate
2. Phenylephrine HCl
APP. ID No 3246
USP Methods 61
- Phenylpropanolamine HCl-Limit of Amphetamine
- USP Acceptable Modified Method
Resolution > 5.0
Column Performance:
Resolution = 13.24
USP/NF 23 page:
1214
Standard prep: Phenylpropanolamine HCl and Dextroamphetamine sulfate, each
5µg/mL in mobile phase
Mobile phase: THF / Methanol / Water with 0.2% TMAH and 0.5% Phosphoric acid
(4 : 40 : 956)
Flow rate: 1.5 mL/min (USP was 1.0mL/min)
Injection: 10 µL (was 5 µL in USP)
Sample: 1. Phenylpropanolamine
2. Dextroamphetamine
APP. ID No 8275
62 USP Methods
- Prednisone oral solution - USP Acceptable Modified Method 2N12400
USP Tailing Factor< 2.0
Column Performance:
Resolution = NA
Standard prep: Prednisone at 40µg/mL in methanol / water
(25 : 75)
Mobile phase: 17mM Monobasic potassium phosphate /
Acetonitrile (60 : 40)
Injection: 10 µL
APP. ID No 3268
USP Methods 63
2P23800 - Prednisolone - USP Method
Resolution > 3.5
Column Performance:
Resolution = 10.30
Standard prep: Presnisolone and Betamethasone, 0.1mg/mL
in chloroform
Mobile phase: Butyl chloride / Water-saturated butyl chloride /
Tetrahydrofuran / Methanol / Glacial acetic acid
(95 : 95 : 14 : 7 : 6)
Injection: 10 µL
Sample: 1. Betamethasone
2. Presnisolone
APP. ID No 11202
64 USP Methods
- Prednisolone - USP Acceptable Modified Method
Resolution > 3.5
Column Performance:
Resolution = 6.64
Standard prep: Presnisolone and betamethasone, 0.1mg/mL
in chloroform
Mobile phase: Butyl chloride / Water-saturated butyl chloride /
Tetrahydrofuran / Methanol / Glacial acetic acid
(95 : 95 : 14 : 7 : 6)
Injection: 10 µL
2. Presnisolone
APP. ID No 3269
USP Methods 65
2P28300 - Procainamide HCl - USP Method
Resolution > 5.0
Column Performance:
Resolution = 5.99
Standard prep: Procainamide HCl - 0.05mg/mL in mobile phase
p-Aminobenzoic acid - 0.01mg/mL in mobile
phase
Mobile phase: Water / Methanol / TEA (140 : 60 : 1) adjusted to
pH 7.5 with phosphoric acid
Injection: 20 µL
Sample: 1. p-Aminobenzoic acid
2. Procainamide HCl
APP. ID No 3261
66 USP Methods
- Propoxyphene HCl Capsules - USP Acceptable Modified Method 2P30900
Part No.:00B-4251-E0
Column Performance:
Resolution = NA
USP/NF 23 page:
1319
Standard prep: Propoxyphene 6.5µg/mL in mobile phase
Mobile phase: 50mM Monobasic potassium phosphate pH 3.0
with 20mM Triethylamine / Acetonitrile
(70 : 30, was 3 : 2 in USP)
Injection: 5 µL (10µL in USP)
Sample: 1. Propoxyphene
APP. ID No 3269
USP Methods 67
2P31700 - Propranolol - USP Method
Resolution > 2.0
USP Tailing Factor for propranolol < 3.0
Column Performance:
Resolution = 15.88
USP/NF 23 page:
Standard prep: Propranolol (0.04mg/mL) and Procainamide
Mobile phase: Water / Acetonitrile / Methanol (70 : 70 : 90)
with 7mM Sodium Lauryl sulfate
and 11mM Phosphoric acid
Injection: 20 µL
Sample: 1. Procainamide
2. Propranolol
APP. ID No 3291
1327
68 USP Methods
- Reserpine - USP Method 2R01000
Column Performance:
Resolution = NA
Standard prep: Reserpine at 10µg/mL in mobile phase
Mobile phase: Acetonitrile / 1% Ammonium chloride,
pH5.6 (50 : 50)
Injection: 20 µL
APP. ID No 3292
USP Methods 69
2T05300 - Tetracaine HCl opthalmic solution - USP Method
Efficiency(N) > 500
Column Performance:
Resolution = NA
USP/NF 23 page:
Standard prep: 0.1mg/mL in water
Mobile phase: 10mM Ammonium phosphate pH3.0 / Acetonitrile
(70 : 30)
Injection: 5 µL
APP. ID No 3293
1506
70 USP Alternative Methods
ALTERNATIVE METHODS
The following Alternative Methods are based
on USP methods but have been changed to
optimize runtime, peak shape and/or provide
more rugged mobile phase conditions.
Because the methods are "changed" and not
"modified" within the proposed guidelines of
Pharmacopeial Forum vol. 25(2) and vol. 26(2),
the following methods are not USP accepted
modified methods.
These methods warrant additional validation by
the USP.
USP Alternative Methods 71
- Alprazolam - Alternative Method
Resolution > 2.0
Column Performance:
Resolution = 2.74
Standard prep: Alprazolam and Triazolam each 0.025mg/mL
in mobile phase
Mobile phase: Hexane / Methylene choride / Methanol (75 : 20 : 5)
Injection: 20 µL
2. Alprazolam
APP. ID No 3309
- Amoxicillin - Alternative Method
k’ between 1.1 -2.8
Column Performance:
Resolution = NA
Standard prep: Amoxicillin - 1.2mg/mL in phosphate buffer
Mobile phase: 20mM Ammonium acetate pH4.9 / Methanol
(95 : 5)
Injection: 5 µL
APP. ID No 3294
- Aspirin tablets - Alternative Method
Column Performance:
Resolution = 7.34
Standard prep: Aspirin 2mg/mL and Salicylic acid 1.6mg/mL
Mobile phase: 20mM Ammonium formate pH3.0 / Acetonitrile
(75 : 25)
Injection: 2 µL
Sample: 1. Aspirin
2. Salicylic acid
APP. ID No 3301
- Cephalexin - Alternative Method
Column used: 150 x 4.6mm LUNA® 5µm Phenyl-Hexyl
Resolution > 5.0
Column Performance:
Resolution = 9.61
Standard prep: Cephalexin(0.2mg/mL) and 1-Hydroxybenzotriazole
Mobile phase: Water with 0.05%formic acid / Methanol with 0.05% formic acid (70 : 30)
Injection: 5 µL
2. Cephalexin
APP. ID No 3308
- Cephradine capsules - Alternative Method
ResolutionResolution > 2.0
Column Performance:
Resolution = 5.0
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
Sample:
Mix of Cephradine and Cephalexin, each 0.1mg/mL in mobile phase
20mM Ammonium acetete pH4.9 / Methanol (70 : 30)
1.2 mL/min
UV @ 254nm
10 µL
1. Cephalexin
2. Cephradine
1
APP. ID No 3295
- Chloramphenicol - Alternative Method
Column Performance:
Resolution = NA
Standard prep: Chloramphenicol at 80µg/mL in mobile phase
Mobile phase: Water with 0.05%formic acid / Methanol with
0.05% formic acid (70 : 30)
Injection: 4 µL
APP. ID No 3312
- Hydrocortisone - Alternative Method
USP column specified:
Resolution > 3.0
Column Performance:
Resolution = 2.91
Standard prep: Hydrocortisone 0.1mg/mL in mobile phase and
Prednisone 0.06mg/mL in mobile phase
Mobile phase: Acetonitrile / Water (20 : 80)
Injection: 5 µL
2. Prednisone
APP. ID No 3303
- Hydrocortisone Acetate - Alternative Method
Column Performance:
Resolution = NA
Standard prep:
Mobile phase:
Flow rate:
Detection:
Injection:
APP. ID No 3311
Hydrocortisone acetate 0.1mg/mL in mobile phase
Methanol / Water (60 : 40)
1.0 mL/min
UV @ 254nm
10 µL
- Ibuprofen - Alternative Method
Resolution > 2.5
Column Performance:
Resolution = 7.71
Standard prep: Ibuprofen-12mg/mL in mobile phase
Valerophenone-0.35mg/mL in mobile phase
Mobile phase: 20mM Ammonium acetate pH4.0 / Acetonitrile
(60 : 40)
Injection: 5 µL
Sample: 1. Ibuprofen
2. Valerophenone
APP. ID No 3278
- Lidocaine HCl - Alternative Method
Resolution Lidocaine/methyl paraben > 3.0
Column Performance:
Efficiency(N) = 4136 plates/column for Lidocaine
Resolution = 11.16
Standard prep: Lidocaine-1.7mg/mL in mobile phase
Methyl paraben-220µg/mL in mobile phase
Mobile phase: 20mM Ammonium acetate with 30mM TFA /
Acetonitrile / Methanol (70 : 10 : 20)
Injection: 5 µL
Sample: 1. Lidocaine
2. Methyl paraben
APP. ID No 3300
- Oxacillin - Alternative Method
Column Performance:
Resolution = NA
Standard prep: Oxacillin 0.5mg/mL in mobile phase
Mobile phase: Water / Acetonitrile / Methanol,
all with 10mM Formic acid (40 : 30 : 30)
Injection: 2 µL
APP. ID No 3296
- Prednisolone - Alternative Method
Column used: 100 x 4.6mm LUNA® 3µm Cyano
Resolution > 3.5
Column Performance:
Resolution = 4.05
Standard prep: Prednisolone and Betamethasone,
0.1mg/mL in chloroform
Mobile phase: Hexane / Isopropanol (85 : 15)
Injection: 10 µL
2. Prednisolone
APP. ID No 3302
- Thiamphenicol - Alternative Method
Column Performance:
Resolution = NA
Standard prep: Thiamphenicol at 0.5mg/mL in mobile phase
Mobile phase: Water with 0.05% formic acid / Methanol with
0.05% formic acid (85 : 15)
Injection: 20 µL
APP. ID No 3297
84 Notes
Notes 85
86
87
88
ADVANCED
USPmethods
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United Kingdom
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2301_L
mail:
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USP Method - ResearchGate

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