Pharmaceutical Applications

HPLC Columns
PHARMACEUTICAL
APPLICATIONS
A P P L I C AT I O N N O T E S
Morphine, Hydromorphone and 6-MAM
1,4-Benzodiazepines in Urine
LC-MS Analysis in Plasma Samples
Conditions
LC-MSMethod
with SPE
Method
Conditions
Method
Conditions
Method
Conditions
Method
Conditions
Column:
Cogent Diamond Hydride 2.õ™, 2.2µm, 12
Method
Conditions
Catalog
No.:
70200-05P-2
Column:
Cogent
Diamond
Hydrid2
Column:
Cogent
Diamond
Hydride
Method
Conditions
Method
Conditions
Column:
Cogent
Column:
Cogent2.1
Diamond
Hydride
2.õ™,Diamond
2.2µm, 120A Hyd
Dimensions:
x 50 mm
Catalog
No.: / 0.1%
70200-05P-2
Catalog
No.:
Catalog
No.:
70200-05P-2
formic acid (v/v)
Mobile
Phase:
A:
DI H2O70200-05P-2
Catalog
No.:
70200-05P-2
Dimensions:
x 50
mmacid (v/v)
Dimensions:
2.1 x 50
Dimensions:
2.12.1
x0.1%
50
mm
B:mm
Acetonitrile/
formic
Dimensions:
2.1
xH
50
mm
Mobile
Phase:
A: DI Phase:
H
/ (min.)
0.1% A:
formic
(v/v)
2OPhase:
/ 0.1%
formic
Mobile
A: acid
DI
Gradient:
time
%B
time
(min.)
%Bac
0.1%
formic
Mobile
DI
H2O
2/O
B: Acetonitrile/
0.1%
formic
acid
(v/v)
Mobile
Phase:
A:
DI
H
O
/ 90.1%
0.1%
form
2
0
85
20form
B:time
Acetonitrile/
0.1%
formic
Gradient:
time (min.) %B B: Acetonitrile/
(min.) %B
2
6
70
10
85 fo
B:
Acetonitrile/
Gradient:
time
(min.)
%B 0.1%
0
85 time
9
20
Gradient:
(min.)
%B
timt
7
20
6
70
10
8585
Gradient:
time
(min.)
%B
0
0
85
Post Time: 7 3 min 20
06
85
70 70
PostFlow
Time:Rate:3 min 0.4 mL/min 6
7
Vol.:
1 µL
FlowInjection
Rate:
0.4 mL/min
7 6
20 20
70
Injection
Vol.:
1 µL
Spiked urine sample was
Sample:
Extraction 3
method:
Post
Time:
3 min
Post
Time:
min
7
20
Sample:
Extraction
method:
Spiked
urine
sample
was
loaded
into SPE
cartridge
I (Clean Screen
Flow
Rate:
mL/min
Flow
Rate:
0.430.4
mL/min
loaded
into SPE
cartridge
I from
(Clean
Screen
Post
Time:
min
Xcel™
purchased
UCT
Bristol, PA, USA
Injection
Vol.:
1
µL
Xcel™and
purchased
from
UCT
Bristol,
PA, USA)
Injection
Vol.:
µL
eluted1
with
0.78
mL of acetonitrile,
200
Flow
Rate:
0.4
mL/min
and eluted
withof0.78
mL
of acetonitrile,
200ofSpiked
Sample:
Extraction
method:
Spikeu
Sample:
Extraction
microL
2-propanol,
20method:
microL
ammonia
Injection
Vol.:
1
µL
microL of 2-propanol,
20
microL
of
ammonia.
loaded
into
SPE
cartridge
After the elution,
the sample
was
dried
unde
loaded
SPE
cartridge
I(
After the elution, the
sampleinto
was dried
under
N2
Sample:
Extraction
method:
Spik
gas
and dissolved
inpurchased
100
50%
Xcel™
purchased
from
U
gas and
dissolved
in 100
microL
ofmicroL
50% of
Xcel™
from
UCT
methanol/50%
DI water/0.1%
formic
acid.
Beo
loaded
into
SPE
cartrid
methanol/50%
DI water/0.1%
formic
acid.
Before
and
eluted
with
0.78
mL
and
eluted
with
0.78
mL
of
a
injection,
the spiked
10ppmsample
spikedwas
sample
filter
injection,
the 10ppm
filteredwas 20
microL
of
2-propanol,
Xcel™
purchased
from
microL
of 2-propanol,
mic
a 0.45
μm
nylon
syringe
filter20
(Micro
throughthrough
a 0.45 μm
nylon
syringe
filter
(MicroSolv
After
the
elution,
sam
and
eluted
with
0.78
m
Tech Corp).
Tech Corp).
After
the
elution,
thethe
sample
Detection:
22-propanol,
TOF mass
gas
and
dissolved
in
100
Detection:ESI – Pos
ESI –– Perkin
Posgas
– Elmer
Perkin
Elmer
AxION
TOF
m
andAxION
dissolved
in2100
mi
microL
of
2
spectrometer.
spectrometer.methanol/50% DI water/0
DI water/0.1%
After the elution,
the sa
t0: t :
0.9 min0.9 min methanol/50%
Column:
Catalog No.:
Dimensions:
Mobile Phase:
Cogent Diam
70200-05P-2
2
1
2.1 x 50 mm
A: DI H O / 0
B: Acetonitrile
3
4
Gradient:
time (min.) %
0
6
7
Post Time:
3 min
Flow Rate:
0.4 mL/min
Injection Vol.: 1 µL
Sample:
Extraction me
Detection:
ESI – POS - Agilent 6210 MSD TOF mass
spectrometer.
loaded into SP
Discussion
Peaks:
Peaks:
1. temazepam
m/z
[M+H]
Xcel™ purcha
¯ column was successfully used
Discussion
1.
temazepam301.0739
301.0739
m/z
[M+H]
The
Cogent Bidentate C18 2.o™
2. diazepam
[M+H]
2.
diazepam285.0790
285.0790
[M+H]
in the analysis of an important class of drugs in plasma samples.
3. nordiazepam 271.0633 [M+H]
0
injection,
the
10ppm
spike
3. nordiazepam 271.0633 [M+H]
eluted
¯validation
injection,
the
10ppm
spiked
Thepresented
Cogent Bidentate
2.o™
columncan
was
4. midazolam 326.0855 [M+H]
The
procedureC18
after
besuccessfully
used as a used
gas andand
dissolved
inwit
10
4. midazolam 326.0855 [M+H]
through
a 0.45
μm
nylon
in the analysis
analysisof
ofplasma
an important
class
of drugs
in plasma
Discussion
routine
samples
(or whole
blood
samplessamples.
–
through
a
0.45
μm
nylon
syr
microL DI
of wate
2-pr
Discussion
methanol/50%
after
theprocedure
extraction procedure)
for thecan
presence
of as a
Thechanging
presented
after validation
be used
Tech
Corp).
Tech Corp).
morphine,
hydromorphone,
or 6-MAM
(indicator
of heroin
routine analysis
of plasma
samples
(or whole
blooduse).
samples –
The Cogent Diamond
Hydride 2.õ™ column
was
successfully
usedelutio
After
the
injection,
the
10ppm
spu
Detection:
ESI
– Pos
– Perkin
Elme
The Cogent
Diamond Hydride
column
was
successfully
Peaks:
Detection:
ESI
– Pos
– Perkin
Elmer
A
in analysis
of
1,4-benzodiazepines
in2.õ™
urine
samples
after
SPE
after changing the extraction procedure) for the presence of Peaks:
APP A-310
APP A-305
through
a
0.45
nylo
spectrometer.
1.
temazepam
301.0739
m/z
[M+H]
in
analysis
of
1,4-benzodiazepines
in
urine
samples
after
gas
andμm
disso
extraction. Four available compounds
were well
retained
and
morphine, hydromorphone, or 6-MAM (indicator of heroin use). 1. temazepam 301.0739 m/z [M+H]
spectrometer.
For more information
For
more
information
2. diazepam
285.0790
[M+H]
extraction.
Four
available
compounds
were
well
retained
separated.
The
procedure
could
be
used
for
determination
of
this
0.9
min
Tech
Corp).
2. diazepam 285.0790 [M+H]
0:
t0: tThe
0.9
min
methanol/50%
3. nordiazepam 271.0633 [M+H]
classseparated.
of compounds
inprocedure
urine samples
andbe
other
body
could
used
forfluids.
determination of
www.MTC-USA.com or
www.MTC-USA.com
or
3. nordiazepam
271.0633 [M+H]
4. midazolam 326.0855 [M+H]
Detection:
ESI
–and
Pos
– Perkin El
4. midazolam 326.0855 [M+H]
Peaks:
other body fluids.
class
of
compounds
in
urine
samples
injection,
the 1
technical@hichrom.co.uk
technical@hichrom.co.uk
Discussion
spectrometer.
1. temazepam 301.0739 m/z [M+H]
Discussion
through
a
0.45
2. diazepam 285.0790 [M+H]
t0:
0.9 min
3. nordiazepam 271.0633 [M+H]
The Cogent Diamond Hydride
2.õ™
column
Tech
Corp).
The Cogent Diamond Hydride 2.õ™ column w
4. midazolam 326.0855 [M+H]
in analysis of 1,4-benzodiazepines in uri
Detection:
ESI – Pos
–P
in
analysis
of 1,4-benzodiazepines
in urine
Discussion
Peaks:
extraction. Four available compounds w
extraction.
Four available spectrometer
compounds were
Acetaminophen Impurities Method 1. temazepam 301.0739 m/zCough
Syrup Ingredients
[M+H] +
separated. The procedure could be used f
separated. The procedure could be used for
2. diazepam 285.0790 [M+H] +
The
Diamond
2.õ™ and
coluo
class
of compounds
inHydride
urine
tCogent
0.9samples
min
0:compounds
Robust and Easy APAP Method
Separation
of+ Antitussives, Analgesics,
Decongestants
class of
in urine samples and oth
3. nordiazepam
271.0633 [M+H]
¯
Column:
Cogent
Bidentate
2.2µm,
¯ 2.o™,
Column:
Cogent
Bidentate
C18 C18
2.o™,
2.2µm,
120A 120A
Catalog
No.: 40218-05P-2
40218-05P-2
Catalog
No.:
Dimensions: 2.12.1
50 mm
Dimensions:
x 50x mm
Mobile
Phase:
A: DI
0.1%
formic
acid (v/v)
/ 0.1%
formic
acid (v/v)
Mobile
Phase:
A: H
DI2OH/2O
B: 50%
Acetonitrile
/ 50%
Methanol
/
B: 50%
Acetonitrile
/ 50%
Methanol
/
0.1%
formic
acid
(v/v)
0.1% formic acid (v/v)
Gradient:
time
(min.)
%B
Gradient:
time (min.) %B
0 0
5 5
4
50
4
50
5
90
5
90
6
90
7 6
5 90
7
5
Post Time:
3 min
PostRate:
Time: 0.43mL/min
min
Flow
Flow Rate:
0.4 mL/min
Injection
Vol.: 1 µL
Peaks:
286.1438 m/z [M+H] +
Injection Vol.:1. Morphine
1 µL
++
2. Hydromorphone
286.1438
m/z[M+H]
[M+H]
Peaks:
1. Morphine 286.1438
m/z
3. 6-Monoacetylmorphine
(6-MAM)m/z [M+H] +
2. Hydromorphone 286.1438
+
328.1543
m/z [M+H]
3. 6-Monoacetylmorphine
(6-MAM)
Detection:
ESI328.1543
– POS - Agilent
6210 MSD
TOF mass
+
m/z [M+H]
spectrometer.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
4. midazolam 326.0855 [M+H] +
class ofCogent
compounds
in urine samples an
Phenyl Hydride™ 4µm, 100Å
Column:
Column:
CatalogNo.:
No.:
Catalog
Dimensions:
Dimensions:
MobilePhase:
Phase:
Mobile
Column: Cogent
Cogent
Bidentate
C18™,
4µm, 100A
Column:
Bidentate
C18™,
4µm, 100A
Catalog
40018-75P
Catalog
No.:No.:40018-75P
Dimensions:
75 mm
Dimensions:
4.6 x4.6
75xmm
Solvents: A: DIA:HDI
H2O/ 0.1%
Solvents:
formicformic
acid acid
2O/ 0.1%
B: Acetonitrile/
B: Acetonitrile/
0.1% 0.1%
formicformic
acid acid
Gradient:
(min.)
%B %B time (min.)
%B
Gradient: timetime
(min.)
time (min.)
%B
0 0
0
6
0
6 30
30
1 1
0
6.01 6.0110
0
10
4 4
30 30
10
1010
10
Post Time:
Post Time: 3 min
3 min
Injection Vol.: 5 microL
Injection Vol.: 5 microL
Flow Rate:
1.0 mL/min
Flow Rate: UV 280
1.0 mL/min
Detection:
(4-aminophenol, acetaminophen) and
Detection: 324 UV
(4-aminophenol, acetaminophen) and
nm 280
(4-nitrophenol)
324
nm (4-nitrophenol)
Peaks:
1. 4-aminophenol
1.072 min
Peaks:
1.
4-aminophenol
2. acetaminophen 4.6681.072
min min
2. acetaminophen
4.668 min
3. 4-nitrophenol
7.588 min
t0:
0.9 min
3. 4-nitrophenol 7.588 min
t0:
Gradient:
Gradient:
Cogent Phenyl Hydride™ 4µm, 100Å
The
Cogent Diamond Hydride 2
69020-7.5P
69020-7.5P
x 75
x 75
mmmm of 1,4-benzodiazep
in4.64.6
analysis
H /O0.1%
/ 0.1%
A: A:
DI DI
HO
TFATFA
(v/v)(v/v)
Acetonitrile/
0.1%TFA
(v/v)
extraction.
Four
available
com
B: B:
Acetonitrile/
0.1%TFA
(v/v)
time
(min.)%B%B
time
(min.)
separated.
The
procedure
coul
0
5
0
5
5
2 2
class
of5 compounds
in urine sa
11
50
Post
PostTime:
Time:
Flow
FlowRate:
Rate:
Injection
Vol.:
Injection
Vol.:
Sample:
Sample:
0.9 min
Detection:
Detection:
t0:
t0:
Discussion
Acetominophen
and two of its major impurities were analyzed
using the Cogent Bidentate C18™ column and a simple mobile
Acetominophen and two of its major impurities were analyzed
phase. The peak shapes were very high. The repeatability of
the
Cogent
Bidentate
C18™ =column
theusing
results
was
extremely
good (%RSD
0.01). and a simple mobile
phase. The peak shapes were very high. The repeatability of
the results was extremely good (%RSD = 0.01).
Peaks
APP A-249
For more information
www.MTC-USA.com or
technical@hichrom.co.uk
Discussion
procedure could be used
Method separated.
Conditions The
Method Conditions
Method Conditions
Method Conditions
Discussion
in analysis of 1,4-benzodiazepines in u
and Preservatives
extraction. Four available compounds
1. Acetaminophen
2. Pseudoephedrine
3. Guaifenesin
Discussion
Discussion
4. Benzoic Acid
5. Methyl Paraben
6. Dextromethorphan
7. Propyl paraben
APP A-174
For more information
www.MTC-USA.com or
technical@hichrom.co.uk
www.MTC-USA.com
2
2
11
50
1212
5 5
3 min
3 min
1.01.0
mL/min
mL/min
2 µL
2 µL
Stock
Solution:
1 mg/mL
solutions
of each
Stock
Solution:
1 mg/mL
solutions
of each
analyte
were
made
using
a 50/50
solvent
analyte
were
made
using
a 50/50
solvent
A/solvent
B diluent
(v/v).
A/solvent
B diluent
(v/v).
Working
Solution:
0.1 0.1
mg/mL
dilutions
werewere
Working
Solution:
mg/mL
dilutions
made
of the
stock
solutions
and and
usedused
for peak
made
of the
stock
solutions
for peak
identity confirmations. A 0.1 mg/mL mixture of
identity confirmations. A 0.1 mg/mL mixture of
all the analytes was also made from the stock
all the analytes was also made from the stock
solutions.
solutions.
UV 210 nm (0-6 min), 230 nm (6-15 min)
UV 210 nm (0-6 min), 230 nm (6-15 min)
0.9 min
0.9 min
Cold and cough formulations may contain a number of components
Cold and
formulations
may contain a number
of components
such
as cough
antitussives
(dextromethorphan),
decongestants
such as antitussives
decongestants
(pseudoephedrine,
guaifenesin),(dextromethorphan),
analgesics (acetaminophen),
and
(pseudoephedrine,
guaifenesin),
analgesics
(acetaminophen),
preservatives (methyl paraben, propyl paraben, benzoic acid). The and
preservatives
paraben,
paraben,
benzoic
acid).
method
illustrates(methyl
not only
excellentpropyl
separation
between
a variety
of The
these
compounds,
symmetric
peak between
shapes can
be of
method
illustrates but
not also
only that
excellent
separation
a variety
obtained
in each case.
in particular
is often
these compounds,
but Dextromethorphan
also that symmetric
peak shapes
can be
problematic
tailingDextromethorphan
due to the tertiary amine.
The method
obtained in terms
each ofcase.
in particular
is often
isproblematic
also very reproducible,
the five
demonstrates.
in terms ofas
tailing
duerun
to overlay
the tertiary
amine. The method
is also very reproducible, as the five run overlay demonstrates.
HPLC Columns
A P P L I C AT I O N N O T E S
Oxybenzone and Octinoxate
Assay Method for Levothyroxine
Separation of Two APIs in Chap Stick® Extract
Superior Resolution, Reproducibility & Peak Shape
Method Conditions
Method Conditions
Column:
Fig. A: MTC Phenyl Hydride™, 4µm, 100A
Column:
Fig. A: MTC Phenyl Hydride™, 4µm, 100A
Type B silica-based
Cyano,
Fig. B:Fig.
TypeB:
B silica-based
Cyano, 5µm,
100A 5µm, 100A
Catalog
A: 69020-7.5P
Fig. B: N/A
Catalog
No.: No.: Fig. A:Fig.
69020-7.5P
Fig. B: N/A
Dimensions:
4.6 xA:754.6
mmx 75
Fig.mm
B: 4.6
x 250
Dimensions: Fig. A:Fig.
Fig.
B: mm
4.6 x 250 mm
Mobile
Phase:
Fig. A:Fig.
A: DIA:water/
formic
acidformic acid
Mobile
Phase:
A: DI0.1%
water/
0.1%
B: 97% B:
Acetonitrile/
3%
DI
water/
97% Acetonitrile/ 3% DI water/
0.1% formic acid
0.1% formic acid
Fig. B: 60% DI water/ 40% acetonitrile/
Fig.
B: phosphoric
60% DI water/
0.05%
acid 40% acetonitrile/
0.05%
acid
Gradient:
Fig. A
time
(min.) phosphoric
%B
Gradient:
Fig. A 0 time (min.)
%B
20
6
0 50
20
7
6 20
50
Temperature:
Fig. A: 35 °C
Fig. B:7 ambient
20
Flow rate:
Fig. A: 1.0 mL/min Fig. B: 1.5 mL/min
Temperature:
Fig. A: 35 °C
Fig. B: ambient
Injection volume: Fig. A: 2 µL
Fig. B:100 µL
Flow rate: Mix of levothyroxine
Fig. A: 1.0and
mL/min
Fig.standards
B: 1.5 mL/min
Sample:
liothyronine
Injection volume:
Fig. 0.4
A:mg2levothyroxine
µL
Fig. B:100
Stock Solutions:
or liothyronine
dissolved µL
with 1 mLMix
10 mM
in 50:50 DI water:
methanol.
Working standards
Sample:
ofNaOH
levothyroxine
and
liothyronine
Peaks:
Peaks:
Discussion
Method Conditions
Column:
Catalog No.:
Dimensions:
Solvents:
Cogent Bidentate C18™, 4µm, 100A
40018-75P
4.6 x 75 mm
A: DI H2O/ 0.1% formic acid (v/v)
B: Acetonitrile/ 0.1% formic acid (v/v)
Gradient:
time (min.) %B
0
60
1
60
4
100
6
100
7
60
Post Time:
3 min.
Temperature: 40°C
Injection Vol.: 2 microL
Flow Rate:
1.0 mL/min
Detection:
UV 288 nm
Peaks:
1. Oxybenzone
2. Octinoxate
t0:
0.9 min
Solution (Fig. A): Aliquots of stock solutions were mixed and
Stock
0.4concentrations
mg levothyroxine
or liothyronine
dissolved
diluted with
50:50Solutions:
A:B to obtain
of 40 mg/L
and 4
mg/L for levothyroxine
and
liothyronine
respectively.
with 1 mL 10
mM
NaOH in
50:50 DI Working
water: methanol. Working
Solution (Fig.
B): Aliquots
of stock
solutions
were solutions
mixed andwere mixed and
Solution
(Fig. A):
Aliquots
of stock
diluted with the mobile phase to obtain concentrations of 10 mg/L
diluted with 50:50 A:B to obtain concentrations of 40 mg/L and 4
and 0.2 mg/L for levothyroxine and liothyronine respectively.
mg/L for levothyroxine and liothyronine respectively. Working
1. liothyronine
sodium
Solution (Fig. B): Aliquots of stock solutions were mixed and
2. levothyroxine
sodium
diluted with the
mobile phase to obtain concentrations of 10 mg/L
Discussion
and 0.2 mg/L for levothyroxine and liothyronine respectively.
1. liothyronine sodium
2. levothyroxine sodium
The USP assay method for levothyroxine requires that a resolution of
APP A-128
For more information
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not less than 5.0 must be demonstrated between levothyroxine and
related
compound liothyronine. A chromatogram obtained from
Discussion
following the USP method using a Type-B silica based L10 column is
shown
Figure
B. method
The average
resolution between
TheinUSP
assay
for levothyroxine
requiresthe
thattwo
a resolution of
compounds
five5.0
runs
is 2.8,
does not satisfy
the system
APP A-216
not lessover
than
must
be which
demonstrated
between
levothyroxine and
suitability
for
resolution
for
this
assay.
Figure
A
shows
the
five-run
related compound liothyronine. A chromatogram obtained fromFor more information
overlay obtained from a method developed with the Cogent Phenyl
following the USP method using a Type-B silica based L10 column is
HydrideTM column. The average resolution in this case was 5.3. In
www.MTC-USA.com or
shown
in Figure
B. The
averagewere
resolution
the two
addition,
the peak
shapes and
reproducibility
far superiorbetween
for the
compounds
five runs is 2.8, which does not satisfy the system
technical@hichrom.co.uk
Phenyl
HydrideTM over
method.
This method shows how two common ingredients found in
sunscreens and lip balms can be separated using the
Bidentate C18™ column. The two compounds are very
hydrophobic, so a mobile phase gradient with significant
organic content was used in order to avoid excessive retention.
Likewise, a highly organic diluent should be used to adequately
extract the compounds from the lip balm material. The figure
shows an overlay of two runs from different column lots,
demonstrating the lot-to-lot reproducibility of the Bidentate
C18™ stationary phase.
suitability for resolution for this assay. Figure A shows the five-run
overlay obtained from a method developed with the Cogent Phenyl
HydrideTM column. The average resolution in this case was 5.3. In
addition, the peak shapes and reproducibility were far superior for the
Phenyl HydrideTM method.
Atorvastatin Method Transfer
Use of Near-UHPLC 2.2µm Column
Alprazolam (Xanax®)
Robust Separation of API from USP Internal Standard
Method Conditions
Method
Conditions
Method
Conditions
¯ C18
¯ 120A
Fig. A:Fig.
Cogent
Bidentate
C18 2.o™,
2.2um,
A: Cogent
Bidentate
2.o™,
2.2um, 120A
Fig. B:
Cogent
Bidentate
C18™, 4um,
100A
Fig.
B: Cogent
Bidentate
C18™,
4um, 100A
Catalog
No.:
Fig.
A:
40218-05P-2;
Fig.
B:
40018-05P-2
Catalog No.: Fig. A: 40218-05P-2; Fig. B: 40018-05P-2
Dimensions: 2.1 x 50 mm
Dimensions: 2.1 x 50 mm
Mobile Phase: A: DI H2O / 10 mM ammonium acetate
Mobile Phase:
A: acetonitrile
DI H2O / 10
mMDI
ammonium
acetate
B: 90%
/ 10%
water /
B: ammonium
90% acetonitrile
10 mM
acetate/ 10% DI water /
10
mM
ammonium
acetate
Gradient:
time (min.) %B
time (min.) %B
Gradient:
%B
(min.) %B
0 time (min.)
40
6 time100
1
7
0 100 40
640
100
Flow Rate:
0.3 mL/min
1
100
7
40
Detection:
UV
248
nm
Flow Rate:
0.3 mL/min
Sample:
40 mg strength Lipitor® tablet was ground and
Detection:
UV 248 nm
added to a 50 mL volumetric flask
with a por®
tabletwas
was ground and
Sample:
40solvent
mg strength
Lipitor
tion of
B diluent.
The solution
added
a 50
volumetric
flasksolwith a porsonicated
10to
min
andmL
diluted
to mark with
tion
of solvent
B diluent.
The
solution
vent B.
It was
then filtered
through
a 0.45
µm was
min and
dilutedCorp.
to mark with solnylonsonicated
membrane10
(MicroSolv
Technology
Eatontown,
The
filtrate
was diluted
4xa 0.45 µm
vent NJ,
B. USA).
It was
then
filtered
through
in a diluent
50/50 solvent
B/ 1N HCl.
It was Corp.
nylon of
membrane
(MicroSolv
Technology
heated
in a dry bath
for 10The
min filtrate
at 85°C.was diluted 4x
Eatontown,
NJ, USA).
Peaks:
1. Atorvastatin
in a diluent of 50/50 solvent B/ 1N HCl. It was
2. Atorvastatin lactone
Method Conditions
Column:
Column:
Peaks:
Discussion
APP A-308
For more information
www.MTC-USA.com or
technical@hichrom.co.uk
Peak 1
Peak
1
Peak 2
Peak 2
heated in a dry bath for 10 min at 85°C.
1. Atorvastatin
2. Atorvastatin lactone
This application note demonstrates how data obtained on a 4um
Bidentate
C18™ column (Fig. B) can be adapted for a 2.2um
Discussion
phase (Fig. A). The retention times of both analytes are very
comparable.
The method
excellent
of the on a 4um
This application
noteproduces
demonstrates
howseparation
data obtained
API and its main acid degradant. It is also LC-MS compatible
Bidentate C18™ column (Fig. B) can be adapted for a 2.2um
and so could be used in clinical applications involving plasma
phase (Fig. A). The retention times of both analytes are very
samples.
APP A-183
For more information
comparable. The method produces excellent separation of the
API and its main acid degradant. It is also LC-MS compatiblewww.MTC-USA.com or
technical@hichrom.co.uk
and so could be used in clinical applications involving plasma
samples.
www.MTC-USA.com
Column: Cogent Cogent
Column:
DiamondDiamond
Hydride™,Hydride™,
4µm, 100A 4µm, 100A
Catalog
70000-7.5P
Catalog
No.: No.:
70000-7.5P
Dimensions:
Dimensions:
4.6 x 754.6
mmx 75 mm
Solvent:
O/ DI
0.1%
formic
acidformic
(v/v) acid (v/v)
Solvent: A: DI H2A:
H2O/
0.1%
B: Acetonitrile/
0.1% formic
acidformic
(v/v) acid (v/v)
B: Acetonitrile/
0.1%
Gradient:
time
(min.)
%B
Gradient:
time (min.) %B
0
95
0 95
95
1
1 50
95
6
6 95
50
7
7
95
Post Time:
3 min
injection
1 µL 3 min
PostVol.:
Time:
Flowinjection
Rate:
1.0 mL/min
Vol.:
1 µL
Peaks:
1. Triazolam
(internal standard)
Flow Rate:
1.0 mL/min
2. Alprazolam (API)
Peaks:
1. Triazolam (internal standard)
3, 4. Impurities
Detection:
254 nm2. Alprazolam (API)
t0:
0.9 min3, 4. Impurities
Detection:
t0:
Discussion
254 nm
0.9 min
The USP assay method for Alprazolam uses a bare silica
Discussion
column
and a complex mobile phase consisting of acetonitrile,
chloroform, butyl alcohol, and acetic acid. In this method a
The
USP
assay method
for Alprazolam
uses a bare
simple LC-MS
compatible
mobile phase
is used and produces
column
a complex
of aceto
excellent
peakand
shapes
for both mobile
the APIphase
and itsconsisting
USP internal
chloroform, butyl alcohol, and acetic acid. In this me
standard.
Furthermore,
a resolution
of 4.3 mobile
was obtained
simple LC-MS
compatible
phasebetween
is usedthe
and pro
two peaks,
whichpeak
meetsshapes
the USPfor
system
Rs>its
2.0.USP i
excellent
both suitability
the API ofand
Two impurity
peaks
are
also
observed,
which
further
illustrates
standard.
the resolution
capabilities
of the column.
Furthermore,
a resolution
of 4.3 was obtained betwe
two peaks, which meets the USP system suitability of R
Two impurity peaks are also observed, which further illu
the resolution capabilities of the column.
HPLC Columns
A P P L I C AT I O N N O T E S
Vardenafil (Levitra®)
LC-MS Compatible Assay Method
Cefpodoxime Proxetil
USP Assay Method
Method Conditions
Method Conditions
Method Conditions
Method Conditions
Column:
Cogent
4µm, 100A
Column:
Cogent UDA™,
4µm,UDA™,
100A
Catalog
No.: 40031-7.5P
Catalog
No.: 40031-7.5P
Dimensions:
4.6 x 75 mm
Dimensions:
4.6 x 75 mm
Solvents:
A: DI H2O / 0.1% formic acid (v/v)
Solvents:
A: DI H2O / 0.1% formic acid (v/v)
B: Acetonitrile/ 0.1% formic acid (v/v)
Acetonitrile/
0.1%%B
formic acid (v/v)
Gradient:
time (min.)B: %B
time (min.)
Gradient:0
time
90 (min.) 6 %B
40time (min.) %B
1
90
790
90
0
6
40
Post Time:
3 min
1
90
7
90
Injection Vol.: 2 microL
Post Time:
Flow rate:
1.0 mL/min3 min
Injection
Detection:
UVVol.:
210 nm2 microL
Sample:
20mg strength
Flow rate:
1.0 Levitra®
mL/mintablet was ground
and addedUV
to a 210
25mLnm
volumetric flask. A
Detection:
portion of 50/50 solvent A/solvent B diluent
Sample:
tablet was ground
was added20mg
and thestrength
flask was Levitra®
sonicated 10
tomark
a 25mL
volumetric
flask. A
min. Then itand
wasadded
diluted to
and mixed.
A
portion wasportion
filtered through
a 0.45
μm nylon
of 50/50
solvent
A/solvent B diluent
syringe filter
(MicroSolv
Tech
Corp).
was added and the flask was sonicated 10
Peak:
1. Vardenafil
min. Then it was diluted to mark and mixed. A
t0:
0.7 min
portion was filtered through a 0.45 μm nylon
syringe filter (MicroSolv Tech Corp).
Discussion
Peak:
1. Vardenafil
Vardenafil in a tablet formulation can be readily assayed with
t0: compatible gradient
0.7 min
Overlay of five runs
this LC-MS
method. The peak tailing factor
APP A-266
For more information
www.MTC-USA.com or
technical@hichrom.co.uk
was close to unity. The compound has several amine groups
which can produce tailing with ordinary HPLC columns that
Discussion
have a number of surface silanols. The MS-compatible mobile
APPofA-149
Overlay
five runs
phase means that the method can be adapted to more complex
Vardenafil in a tablet formulation can be readily assayed
Forwith
more information
samples such as plasma. Five runs are shown in the figure to
this
LC-MS
compatible
gradient
method.
The
peak
tailing
factor
illustrate the repeatability of the data.
www.MTC-USA.com or
was close to unity. The compound has several amine groups
technical@hichrom.co.uk
which can produce tailing with ordinary HPLC columns that
have a number of surface silanols. The MS-compatible mobile
phase means that the method can be adapted to more complex
samples such as plasma. Five runs are shown in the figure to
illustrate the repeatability of the data.
Forced Degradation of Clopidogrel
Separation of API and Degradants
Method
Conditions
Method
Method
Method
Conditions
Conditions
Conditions
Method
Conditions
APP A-258
For more information
www.MTC-USA.com or
technical@hichrom.co.uk
Discussion
t:
0
2. Cefpodoxime Proxetil, R-epimer
1.9 min
The USP assay method for cefpodoxime proxetil specifies a resolution
of not less than 2.5 must be obtained between the two epimers of the
Discussion
prodrug.
Following the method using a Cogent Bidentate C18™
column, the average resolution was calculated to be 2.8. In addition,
The USP assay method for cefpodoxime proxetil specifies a resol
the R epimer tailing factor must be not more than 1.5. Again, this data
of not
than 2.5with
must
obtained
between
the two
epimers o
meets
thisless
requirement
a be
tailing
factor of
1.2. Finally,
the data
prodrug.
Following with
the a method
using
a Cogent
C
shows
good repeatability
retention time
%RSD
from five Bidentate
runs of
column, the average resolution was calculated to be 2.8. In add
0.2%.
the R epimer tailing factor must be not more than 1.5. Again, this
meets this requirement with a tailing factor of 1.2. Finally, the
shows good repeatability with a retention time %RSD from five ru
0.2%.
Hormone Replacement Capsule
Separation of Estriol, Estradiol and Progesterone
Column:
Cogent
Diamond
Hydride™,
4µm,
100A
Column:
Column:
Column: Cogent
Cogent
Cogent
Cogent
Diamond
Diamond
Diamond
Hydride™,
Hydride™,
Hydride™,
4µm,
4µm,
4µm,
100A
100A
100A
Column:
Diamond
Hydride™,
4µm, 100A
Catalog
No.:
70000-15P-2
Catalog
Catalog
Catalog
No.:
No.:
No.:
70000-15P-2
70000-15P-2
70000-15P-2
Catalog
No.:
70000-15P-2
Dimensions:
x 2.1
150
mm
Dimensions:
2.12.1
x 150
mm
Dimensions:
Dimensions:
Dimensions:
2.1
2.1
x x150
x150
150
mm
mm
mm
Solvents:
A: DI H
/ 0.1%
formic
acid
(v/v)
2H
Solvents:
O
/O0.1%
acid
(v/v)
2A:
/2O
0.1%
/formic
0.1%
/ 0.1%
formic
formic
formic
acid
acid
acid
(v/v)
(v/v)
(v/v)
Solvents:
Solvents:
Solvents:A: DIA:H
A:
DI
DI
DI
HOH
2
2O
Acetonitrile/
0.1%
formic
acid
(v/v)
B:B:
Acetonitrile/
0.1%
formic
acid
(v/v)
B:B:
Acetonitrile/
B:Acetonitrile/
Acetonitrile/
0.1%
0.1%
0.1%
formic
formic
formic
acid
acid
acid
(v/v)
(v/v)
(v/v)
Gradient:
time
(min.)%B%B
time
(min.)
Gradient:
time
(min.)
time
(min.)
%B %B
Gradient:
Gradient:
Gradient: time
time
time
(min.)
(min.)
(min.)
%B
%B
%B
time
time
time
(min.)
(min.)
(min.)
%B
%B
%B
0 0
95 95
7 7
60 60
0 0 0 95 95959595 8 8 795
7 795 606060
2 2
2 22
959595
8 88
959595
Temperature: 2525
Temperature:
°C°C
Temperature:
Temperature:
Temperature:
25°C
25°C°C
Post
Time: 3 min
3 25
min
Post
Time:
Post
Post
Post
Time:
Time:
Time:
3min
3min
min
Injection
Vol.:
Injection
Vol.:1 microL
1 3microL
Flow
Rate:
0.40.4
mL/min
Flow
Rate:Vol.:
mL/min
Injection
Injection
Injection
Vol.:
Vol.:
1
1microL
1microL
microL
Detection:
ESI
–
POS
-mL/min
Agilent
6210
MSD
TOFTOF
mass
6210
MSD
mass
Detection:
ESI
–
POS
- Agilent
Flow
Flow
Flow
Rate:
Rate:
Rate: 0.4
0.4
0.4
mL/min
mL/min
spectrometer.
spectrometer.
6210
6210
6210
MSD
MSD
MSD
TOF
TOF
TOF
mass
mass
mass
Detection:
Detection:
Detection:
ESI
ESI
ESI
– –POS
–POS
POS
- Agilent
- Agilent
- Agilent
+
Peaks:
1. 1.
API:
Clopidogrel,
m/zm/z
322.0663
[M+H]
Peaks:
API:
Clopidogrel,
322.0663
[M+H]+
spectrometer.
spectrometer.
spectrometer.
2. 2.
Degradant:
Clopidogrel
Acid,
m/z
308.0507
Clopidogrel
Acid,
m/z 308.0507
+ + +
Peaks:
Peaks:
Peaks:
1.Degradant:
1.
API:
1.API:
Clopidogrel,
Clopidogrel,
Clopidogrel,
m/z
m/z
m/z
322.0663
322.0663
322.0663
[M+H]
[M+H]
[M+H]
+ API:
+
[M+H]
2.[M+H]
2.
Degradant:
2.Degradant:
Degradant:
Clopidogrel
Clopidogrel
Clopidogrel
Acid,
Acid,
Acid,
m/z
m/z
m/z
308.0507
308.0507
308.0507
3. 3.
Degradant:
Clopidogrel
N-oxide,
m/z
Degradant:
Clopidogrel
N-oxide,
m/z
+ ++ +
[M+H]
[M+H]
[M+H]
338.02
[M+H]
338.02
[M+H]+
3.
3.
Degradant:
3.Degradant:
Degradant:
Clopidogrel
Clopidogrel
Clopidogrel
N-oxide,
N-oxide,
N-oxide,
m/z
m/z
m/z
t0t: :
0.90.9
min
min
0
+ + +
Discussion
t0:t0t:0:
Discussion
Column:
Cogent
Bidentate
4µm, 100Å
Column:
Cogent
Bidentate
C18™, C18™,
4µm, 100Å
Catalog
40018-25P
Catalog
No.:No.:40018-25P
Dimensions:4.6 x4.6
250 mm
Dimensions:
250xmm
Mobile
Phase:
60% 60%
20 mM
acetate/ acetate/
Mobile
Phase:
20ammonium
mM ammonium
40% 40%
acetonitrile
acetonitrile
Temperature:
30
°C
Temperature: 30 °C
Flow rate:
1.0 mL/min
Flow rate:
1.0 mL/min
Injection Vol.: 20 µL
Injection Vol.:
20 nm
µL
Detection:
UV 235
Detection:
UV
235 nm
Sample:
Stock Solution:
A 200 mg strength
Sample:
Stock Solution:
A 200
strength
cefpodoxime
proxetil tablet
wasmg
ground
and
added
to a 100 mL volumetric
flask. was
The ground a
cefpodoxime
proxetil tablet
flask added
was diluted
mark
the mobileflask. The
to ato100
mLwith
volumetric
phase
and was
sonicated.
A portion
was
then
flask
diluted
to mark
with
the mobile
filtered with a 0.45 micron nylon syringe
phase and sonicated. A portion was then
filter (MicroSolv Tech Corp).
filtered
with 100
a 0.45
nylon syringe
Working
Solution:
µL ofmicron
the stock
filter
solution
was(MicroSolv
diluted Tech
with Corp).
900 µL of the
Working
mobile
phase. Solution: 100 µL of the stock
Peaks:
1. Cefpodoxime
Proxetil,
S-epimer
solution was
diluted
with 900 µL of the
2. Cefpodoxime
Proxetil, R-epimer
mobile phase.
t0: Peaks:
1.9 min
1. Cefpodoxime Proxetil, S-epimer
338.02
338.02
338.02
[M+H]
[M+H]
[M+H]
0.9
0.9
0.9
min
min
min
A sensitive, selective, and rapid ANP HPLC–MS method was
A
sensitive,
selective, and rapid ANP HPLC–MS method was
Discussion
Discussion
Discussion
developed
for the simultaneous quantification of clopidogrel
developed
simultaneous
quantification
of and
clopidogrel
(Plavix®)
andfor
its the
degradants.
Separation
of the drug
the
(Plavix®)
and
its
degradants.
Separation
ofHPLC–MS
the
drug method
and
the was
A
A
sensitive,
A
sensitive,
sensitive,
selective,
selective,
selective,
and
and
rapid
rapid
rapid
ANP
ANP
ANP
HPLC–MS
HPLC–MS
method
method
was
was
degradation products
underand
stress
conditions
was
successfully
degradation
products
under
stress
conditions
was
developed
developed
developed
forforfor
the
the
the
simultaneous
simultaneous
simultaneous
quantification
quantification
quantification
ofofclopidogrel
ofclopidogrel
clopidogrel
achieved
on a Cogent
Diamond
Hydride™
column.
Thesuccessfully
method
achieved
onand
aand
Cogent
Diamond
Hydride™
column.
The
method
(Plavix®)
(Plavix®)
(Plavix®)
and
itsits
degradants.
itsdegradants.
degradants.
Separation
Separation
Separation
ofofthe
of
the
the
drug
drug
drug
and
and
and
the
the
the
was
transferred
from
UV-HPLC
method.
The
developed
method
was
transferred
from
UV-HPLC
method.
The
developed
method
can
be
used for products
the
determination
ofstress
clopidogrel
in commercial
under
under
under
stress
stress
conditions
conditions
conditions
was
was
was
successfully
successfully
successfully
degradation
degradation
degradation
products
products
can
be
used
for
the
determination
of
clopidogrel
in
commercial
tablets
for
quality
control
with
an
application
to
a
content
uniforachieved
achieved
achieved
onon
aona
Cogent
aCogent
Cogent
Diamond
Diamond
Diamond
Hydride™
Hydride™
Hydride™
column.
column.
column.
The
The
The
method
method
method
APP A-175
tablets
for
quality
control
with
an application to
aitcontent
uniformity
test.
The
method
isfrom
also
stability-indicating
as
isdeveloped
suitable
was
was
was
transferred
transferred
transferred
from
from
UV-HPLC
UV-HPLC
UV-HPLC
method.
method.
method.
The
The
The
developed
developed
method
method
method
mity
The method
is also stability-indicating
asits
it degrais suitable
For
more
information
for
the test.
determination
of clopidogrel
in the presence of
can
can
can
bebeused
beused
used
forforthe
forthe
the
determination
determination
determination
ofofclopidogrel
ofclopidogrel
clopidogrel
inincommercial
incommercial
commercial
its degrafor theproducts
determination
of clopidogrel
in the presence
dation
under all
stress conditions
using HCl,ofNaOH,
www.MTC-USA.com or
tablets
tablets
tablets
forfor
quality
forquality
quality
control
control
control
with
with
with
an
an
application
anapplication
application
toto
aHCl,
toa
content
acontent
content
uniforuniforunifordation
all stress
conditions
using
NaOH,
light
and products
hydrogenunder
peroxide.
mity
mity
mity
test.
test.
test.
The
The
The
method
method
method
is isalso
isalso
also
stability-indicating
stability-indicating
stability-indicating
asasitasitisitissuitable
issuitable
suitable
technical@hichrom.co.uk
light
and
hydrogen
peroxide.
presence
presence
presence
ofof
its
ofits
degraitsdegradegraforfor
the
forthe
the
determination
determination
determination
ofof
clopidogrel
ofclopidogrel
clopidogrel
inin
the
inthe
the
dation
dation
dation
products
products
products
under
under
under
allallstress
allstress
stress
conditions
conditions
conditions
using
using
using
HCl,
HCl,
HCl,
NaOH,
NaOH,
NaOH,
light
light
light
and
and
and
hydrogen
hydrogen
hydrogen
peroxide.
peroxide.
peroxide.
www.MTC-USA.com
Method Conditions
MethodConditions
Conditions
MethodMethod
Conditions
Column:
Cogent
UDC
Cholesterol™
4µm
Column:
Cogent UDC
Cholesterol™
4µm,
100A
Column:
Cogent
UDC
Cholesterol™
Column:
Cogent
UDC
Cholesterol™
Catalog
No.:Catalog
69069-7.5P
Catalog
No.:
69069-7.5P
No.:
69069-7.5P
Catalog
No.:
69069-7.5P
Dimensions: 4.6 x 75 mm
Dimensions:
4.64.6
x4.6
75
Dimensions:
xmm
75
mm
Dimensions:
x acid
75
mm
Solvents:
A: DI H2O/ 0.1%
formic
(v/v)
O/
0.1%
formic
acid
(v/v)
Solvents:
A:0.1%
DI
Hformic
0.1%
formic
acid
Solvents:
B: Acetonitrile/
acid
(v/v)
O/
0.1%
formic
acid
(v
Solvents:
A:A:
DI
H2H
2DI
2O/
Gradient:
time (min.) B:
%B
Acetonitrile/
0.1%
formic
acid
(v
B:
Acetonitrile/
0.1%
formic
a
B:
Acetonitrile/
0.1%
formic
aci
0
33
Gradient:
time
(min.)
%B%B
Gradient:
time
(min.)
%B
Gradient:
time
(min.)
2
33
33 3333
11
65 0 0 0
12
33 2
2 2 33 3333
Post Time:
3 min
11 1111 65 6565
Flow rate:
1.0 mL/min
12 1212 33 3333
Detection:
UV 210 nm
Injection
Vol.:
10 Time:
µL
Post
Time:
3 min
Post
min
Post
Time:
33
min
Peaks:
1. Estriol
Flow
rate:
mL/min
Flow
rate: 1.01.0
1.0
mL/min
Flow
mL/min
2.rate:
Estradiol
Detection:
UVUV
210
nm
3. Progesterone
Detection:
UV
210
nm
Detection:
210
nm
Injection
Vol.:
10 10
µL
Injection
Vol.:
10
Injection
Vol.:
µLµL
Peaks:
1. Estriol
Peaks:
Estriol
Peaks:
1.1.
Estriol
This gradient method features
a Estradiol
separation of the three
2. Estradiol
2.2.
Estradiol
components of a hormone replacement formulation. Excellent
3. Progesterone
Progesterone
3.
Progesterone
separation is obtained between
the3.
three
compounds using the
Discussion
Cogent UDC-Cholesterol™ column.
Figure A shows a five run overlay of the formulation extract
Discussion
Discussion
injections,Discussion
demonstrating the excellent run-to-run repeatability
of the method.
This
gradient
method
features
a aseparation
This
gradient
method
features
aand
separ
gradient
method
separat
Figure This
B shows
a zoomed-in
view so features
that the estriol
components
of present
a of
hormone
formul
components
replacement
fo
estradiol
peaks,
which are
inhormone
much replacement
lower
concentration
components
of
a ahormone
replacement
form
than progesterone,
can
beis
seen
clearly.
Figure
B also
shows
separation
is obtained
between
the
three
compo
separation
obtained
between
the
three
c
separation
is
obtained
between
the
three
com
separation of an impurity from the progesterone peak.
Cogent
UDC-Cholesterol™
column.
Cogent
UDC-Cholesterol™
column.
Cogent
UDC-Cholesterol™
column.
Figure
A shows
a five
run
overlay
of of
the
form
Figure
shows
fiverun
runoverlay
overlay
ofthe
thef
Figure
A Ashows
a afive
injections,
demonstrating
thethe
excellent
run-to-r
injections,
demonstrating
theexcellent
excellent
run
injections,
demonstrating
run-t
of the
method.
the
method.
ofof
the
method.
Figure
B shows
a zoomed-in
view
so so
that
Figure
shows
zoomed-in
view
soth
Figure
B Bshows
a azoomed-in
view
estradiol
peaks,
which
areare
present
in much
lowe
estradiol
peaks,
which
are
present
much
estradiol
peaks,
which
present
in in
much
lo
than
progesterone,
cancan
be
seen
clearly.
Figure
than
progesterone,
canbe
beseen
seen
clearly.
F
than
progesterone,
clearly.
Fig
separation
of an
impurity
from
thethe
progesterone
separation
an
impurity
from
the
progeste
separation
ofof
an
impurity
from
progester
HPLC Columns
TM
CogentTM TYPE-C
silica LC phases
These columns
CogentTM TYPE-CTM silica
canLC
bephases
usedhave
for the ability
to retain polar solutes
at high
concentrations
reverse
(RP)
or normal of organic
solvent by aqueousphase
normal-phase
(ONP)(ANP)
modeand non-polar
compounds under reversed-phase
(RP)
as well as ANP.conditions.
These revolutionary columns use patented bonding
technology to create a surface populated by siliconUse typical
hydride functional groups instead of silanols. The lack
method
development
of surface silanols leads to fast
equilibration times,
strategy
when
using lifetimes
excellent peak shape and extended column
these
columns
for a wide range of analytes. These application notes
RP or
ONPofmodes.
demonstrate the in
unique
abilities
Cogent TYPE-C
silica LC columns for a range of clinical analysis
applications. Further application notes are available
at www.MTC-USA.com or from Hichrom Limited at
technical@hichrom.co.uk
Cogent TYPE-C columns can be operated in 3
modes of chromatography: reversed-phase (RP),
normal-phase (NP) and aqueous normal-phase.
The surface silanols that are present in all Type
A and B silicas, even after bonding and extensive
endcapping, form a strong association with water
resulting in a ‘hydration shell’ surrounding the silica.
However, the silica hydride particles of TYPE-C silica
are only slightly hydrophobic and therefore have a
weak attraction for water allowing them to be used
in aqueous normal-phase (ANP) mode, which unlike
HILIC, does not require a ‘water-rich’ environment
in order to operate.
com
retention begins depends on the solute as well as
the stationary phase selected.
3. If su
obta
suc
90,
sep
Because of the attached organic group that is part
of the stationary phase, the Type-C columns can
also retain nonpolar compounds based on the
typical reversed phase mechanism. The diagram
below illustrates how this dual retention capability
works for both polar (metformin) and nonpolar
(glyburide) compounds:
4. In a
rete
colu
pha
com
acet
rete
Aqueous Normal Phase (ANP) and
Reversed-Phase (RP) Separations
25
Retention Time (mins)
RETENTION TIME
Using Aqueous
Normal Phase (ANP)
20
REVERSED
PHASE
ANP
5. If th
con
tech
15
10
5
0
40
50
60
70
80
90
100
% Acetonitrile in eluent
MOBILE PHASE
Figure 1. Dual RP and ANP retention capability
For
Cogent TYPE-C silica based phases (Bidentate C18,
Bidentate C8, UDC-Cholesterol, Diamond Hydride,
Phenyl Hydride, UDA, Diol and Silica-C) have the
ability to operate in ANP mode which enables the
retention of polar solutes at high concentrations
of the organic component whilst maintaining an
aqueous component in the eluent. The exact point
in the composition of the eluent where ANP
retention begins depends on the solute as well as
the stationary phase. In addition, TYPE-C columns
can also retain non-polar compounds based on a
typical reversed-phase mechanism. Figure 1 illustrates
the dual retention capability for both polar (metformin)
and non-polar (glyburide) compounds. In this case,
with an eluent composition of less than 70%
acetonitrile, glyburide and metformin are both
retained by a reversed-phase mechanism, with the
metformin eluting first. With increasing percentages
of acetonitrile, the retention of metformin increases
significantly due to ANP mechanisms and now elutes
after glyburide.
For further technical advice and additional application
notes on Cogent TYPE-C Silica LC columns, contact
MicroSolv Technologies, USA, www.MTC-USA.com or
global distributor Hichrom Limited, UK
www.hichrom.co.uk, technical@hichrom.co.uk
www.MTC-USA.com
www.hichrom.co.uk
P2089-10-14-003-3000