Simultaneous Determination of Pharmaceutical
Molecules and Counterions on a New Stationary
Phase with Anion-Exchange, Cation-Exchange,
and Reversed-Phase Functionalities
Xiaodong Liu, Andy Woodruff, and Christopher Pohl, Dionex Corporation, Sunnyvale, CA USA
ABSTRACT
A new stationary phase column, Acclaim® Trinity™ P1, based on
Nano-polymer Silica Hybrid (NSH) technology, provides three retention
mechanisms simultaneously—anion-exchange interaction, cationexchange interaction and reversed-phase interaction. It is based on
high-purity, spherical, porous silica particles and is designed for
pharmaceutical applications such as the determination of both anionic
and cationic counter-ions, simultaneous separation of drug molecules
and counter-ions, as well as separation of acidic, neutral, and basic
ingredients in pharmaceutical formulations.
INTRODUCTION
Pharmaceuticals consist of a diversity of acidic, basic, and neutral
molecules. It is important and desirable that pharmaceuticals with different
charges and hydrophilicity can be analyzed in the same separation. One
example is the determination of drug substances and counterions, a
crucial assay in drug development.
Reversed-phase liquid chromatography (RPLC) is most commonly
used for analyzing drug molecules with intermediate or higher hydrophilicity, but it often fails to retain highly hydrophilic analytes, such as
catecholamines and counterions (e.g., Na+ and Cl– ions). Although ionpairing chromatography improves the retention and selectivity of highly
hydrophilic ionizable analytes, it requires long equilibration times, a
dedicated column, and the mobile phase is MS-incompatible. For hydrophilic ionic analyte (e.g., counterions) determination, ion chromatography (IC) provides a reliable, selective, and sensitive solution. Hydrophilic
Interaction Chromatography (HILIC) complements the aforementioned
techniques and is suitable for analyzing highly polar analytes. However,
challenges include solubility of the analytes in the mobile phase and
greater solvent consumption compared to RP methods.
Mixed-mode chromatography combines both ion-exchange and RPLC
properties, and can retain and separate both drug molecules and
counterions. Because of the complexity and versatility of pharmaceutical analytes in terms of hydrophility and ionization, it is required that
the stationary phase provide cation-exchange, anion-exchange, and
reversed-phase interactions simultaneously. Despite the fact that packing materials having amphoteric or zwitterionic functionality have been
developed, anion-exchange and cation-exchange functional groups
on these materials neutralize one another due to their proximity if both
functional groups are ionized. Consequently, they behave as salt-exchange materials rather than an anion-exchanger and a cation-exchanger
when both functional groups are ionized.
In this study, we will report a new stationary phase designed for pharmaceutical applications, including analysis of drug molecules, determination
of both anionic and cationic counterions, simultaneous separation of drug
molecules and counterions, and simultaneous separation of acidic, neutral,
and basic ingredients in pharmaceutical formulations. The new stationary
phase is based on high-purity, spherical, porous silica particles. The innerpore area is functionalized with an organic layer that provides both reversedphase and anion-exchange properties. The outer-pore area, conversely, is
modified with cation-exchange functionality using proprietary nanoparticle
technology. As a result, this column simultaneously provides reversedphase, anion-exchange, and cation-exchange retention so that neutral,
acidic, and basic analytes can be separated in the same chromatographic
analysis. Column chemistry, chromatographic features, and demonstration
of key applications will be presented.
COLUMN CHEMISTRY
Nano-Polymer Silica Hybrid (NSH) Technology
• Silica particles coated with nano-polymer beads
• Inner-pore: reversed-phase/weak anion-exchange
• Outer surface: strong cation-exchange
FEATURES, VALUES, and APPLICATIONS
2. Simultaneous Determination of API and Counterion
Features
• Multi-mode retention mechanism: anion-exchange, cation-exchange,
and reversed-phase
• Adjustable selectivity
Values
500
2
t0
1
2
mV
• Optimal selectivity and greater flexibility in method development
• Retain ionic and ionizable compounds without ion-pairing reagents
• A broad range of applications
0.5 mL/min
t0
ONa
0
0
1
2
Minutes
3
500
2
Peaks:
mV
34
5
6
7
1
8 9 10 11
0
0
3
6
Minutes
9
12
15
(100 to 150 ppm)
1. Procaine
2. Choline
3. Tromethamine
4. Sodium
5. Potassium
6. Meglumine
7. Mesylate
8. Nitrate
9. Chloride
10. Bromide
11. Iodide
O
NH
H S
CH3
CH3
– +
2
mV
1
0
1
2
Minutes
3
4
25943
Figure 4. Hydrophilic acidic drug and counterion – Penicillin G, K.
25859
300
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: 30/70 v/v CH3CN/
60 mM (total) NH4OAc, pH 5
Temperature: 30 °C
Flow Rate:
0.5 and 1.8 mL/min
Inj. Volume: 2 µL
Detection:
UV, 254 nm
Sample:
Trimipramine maleate
(0.5 mg/mL in mobile phase)
1.8 mL/min
4
mV
2
1
0
0
35
7
6
3
10
9
111213 1415
8
6
Minutes
9
16
12
15
10
0
90
1. K+
2. Penicillin G
Peaks:
1
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phases: (A) CH3CN, (B) D.I. H2O
(C) 0.2 M NH4OAc, pH4
Gradient:
Time (min) –10 0
2 7
%A
60 60 60 10
%B
35 35 35 0
%C
5
5 5 90
Temperature: 30 °C
Flow Rate:
0.5 mL/min
Inj. Volume: 5 µL
Detection:
ELS detector
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: 60/40 CH3CN/
20 mM (total) NH4OAc, pH 5.2
Temperature: 30 °C
Flow Rate:
0.6 mL/min
Inj. Volume: 2.0 µL
Detection:
ELS detector
Sample:
Penicillin G, Potassium Salt
(0.5 mg/mL in mobile phase)
O K
O
t0
Figure 1. Isocratic separation of pharmaceutical counterions.
110
H3CO
4
25868
N
400
O
Figure 3. Hydrophobic acidic API and counterion – Na, Naproxen.
1. Simultaneous Separation of Pharmaceutical Counterions
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 100 mm
Mobile Phase: 60/40 v/v CH3CN/20 mM (total)
NH4OAc, pH 5
Temperature: 30 °C
Flow Rate:
0.5 mL/min
Inj. Volume: 5 µL
Detection:
ELS detector
1. Na+
2. Naproxen
Peaks:
1
Applications
• Pharmaceutical counterions (both anions and cations)
• Drug substances and respective counterions
• Mixtures of basic and acidic drugs with respective counterions
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: 75/25 v/v CH3CN/
30 mM (total) NH4OAc buffer
Temperature: 30 °C
Flow Rate:
0.5 and 1.5 mL/min
Inj. Volume: 2.5 µL
Detection:
ELS detector
Sample:
Na, Naproxen
(0.5 mg/mL in mobile phase)
1.5 mL/min
Peaks: 80 to 150 ppm
1. Procaine
2. Choline
3. Tromethamine
4. Sodium
5. Potassium
6. Meglumine
7. Mesylate
8. Maleate
9. Chloride
10. Bromide
11. Iodide
12. Phosphate
13. Malate
14. Tartrate
15. Citrate
16. Oxalate
t0
2
mAU
1
0.5 mL/min
Peaks:
1. Trimipramine
2. Maleate
t0
O
2
0
1
2
Minutes
3
N
4
CH2 CH CH2 N
CH3
• HO C CH
O
CH
C OH
CH3
25869
Figure 5. Hydrophobic basic API and counterion – trimipramine maleate.
15
25860
Figure 2. Gradient separation of pharmaceutical counterions.
2
CH3
Simultaneous Determination of Pharmaceutical Molecules and Counterions on a New Stationary Phase
with Anion-Exchange, Cation-Exchange, and Reversed-Phase Functionalities
4. Method Ruggedness
800
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 x 50 mm
Mobile Phase: 20/80 v/v CH3CN/
30 mM NH4OAc, pH 5.2
Temperature: 30 °C
Flow Rate:
0.5 mL/min
Inj. Volume: 2.5 µL
Detection:
ELS detector
Sample:
1,1-Dimethylbiguanide•HCl
(0.2 mg/mL in mobile phase)
1
mV
t0
Peaks:
2
0
0
1
2
Minutes
3
4
1. 1,1-Dimethylbiguanide
2. Chloride
400
2
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: CH3CN/
30 mM (total) NH4OAc buffer
1
76% CH3CN (v/v)
Temperature: 30 °C
Flow Rate:
0.6 mL/min
2
Inj. Volume: 2.0 µL
Detection:
ELS detector
Rs = 7.6
Sample:
Na, Naproxen
1
(0.5 mg/mL in mobile phase)
75% CH3CN (v/v)
Peaks:
1. Na+
2
2. Naproxen
Rs = 7.2
mV
5
t0
Figure 6. Hydrophilic basic API and counterion – 1,1-dimethylbiguanide•HCl.
3. Simultaneous Analysis of APIs and Respective Counterions in
Over-the-Counter Medicines
Rs = 8.6
1
25944
0
1
2
Minutes
3
2
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: (A) CH3CN
(B) D.I. H2O
(C) 0.2 M NH4OAc, pH 5.2
mV
Na+
0
–100
Cl–
mAU
–10
ELSD
Gradient:
Time (min) –6
0
1 2 8
A
20
20 20 80 80
B
72.5 72.5 72.5 0 0
C
7.5 7.5 7.5 20 20
4
200
OH
t0
0
H
N
Temperature:
Flow Rate:
Inj. Volume:
UV, 254 nm Detection:
CH3
CH3
1
2
3
Rs = 7.8
CO2H
O
4
5
Minutes
6
7
8
1
2
mV
1 Rs = 8.2
2
t0
30 °C
0.5 mL/min
3 µL
UV, 254 nm and
ELS detector in series
1
0
1
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: 75/25 v/v CH3CN/
NH4OAc buffer
32 mM NH4OAc Temperature: 30 °C
Flow Rate:
0.6 mL/min
Inj. Volume: 2.0 µL
Detection:
ELS detector
Sample:
Na, Naproxen
(0.5 mg/mL in mobile phase)
30 mM NH4OAc
Peaks:
1. Na+
2. Naproxen
ONa
Rs = 8.6
28 mM NH4OAc
2
Minutes
* Background subtraction applied
25950
O
H3CO
25945
400
H3C
4
Figure 9. Method ruggedness – variation in solvent.
H CH3
800
ONa
74% CH3CN (v/v)
3
O
H3CO
4
25946
Figure 10. Method ruggedness – variation in buffer concentration.
Figure 7. Separation of mixture of acidic and basic APIs and counterions –
Aleve™ Sinus and Headache (OTC).
400
2
600
O
OH
4
mAU
OH
t0
H
N
1
0
0
CH3
CH3
1
2
3
1. Pseudo-ephedrine
2. Chlorpheniramine
3. Maleate
4
4. Ibuprofen
* Background subtraction applied
Rs = 7.4
1
pH 5.0
mV
2
t0
25951
1. Na+
2. Naproxen
1
ONa
pH 5.2
0
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: 75/25 v/v CH3CN/
30 mM NH4OAc buffer
Temperature: 30 °C
Flow Rate:
0.6 mL/min
Inj. Volume: 2.0 µL
Detection:
ELS detector
Sample:
Na, Naproxen
(0.5 mg/mL in mobile phase)
Peaks:
Rs = 8.2
Peaks:
3
2
Minutes
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: A: CH3CN, B: D.I. H2O
C: 0.2 M NH4OAc, pH 4.1
Gradient:
Time (min)
–4
0
0.1 1 4
A:
25 25 25 80 80
B:
65 65 65 0 0
C:
10 10 10 20 20
Temperature: 30 °C
Flow Rate:
0.5 mL/min
Inj. Volume: 2 µL
Detection:
UV, 254 nm
1
2
Minutes
3
H3CO
O
4
25947
Figure 11. Method ruggedness – variation in pH.
Figure 8. Over-the-counter medicine – Advil® Allergy and Sinus (OTC).
3
CONCLUSIONS
Acclaim Trinity P1 Column
• High-efficiency silica column designed for pharmaceutical applications
• Concurrent reversed-phase, anion-exchange, and cation-exchange properties
• Optimized selectivity for separating APIs and counterions
NSH Technology
• Optimal selectivity
• Maximum flexibility in method development
Key Pharmaceutical Applications
• Pharmaceutical counterions (both anions and cations)
• Drug substances and respective counterions
• Mixtures of basic and acidic drugs with respective counterions
Acclaim is a registered trademark and Trinity is a trademark of Dionex Corporation.
Aleve is a trademark of Bayer HealthCare LLC. Advil is a registered trademark of Wyeth.
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