Determination of Common Counterions and Impurity Anions in Pharmaceuticals Using a Capillary HPIC System with Suppressed Conductivity and Charge Detection Recently, identification and quantification of ions in early stage drug development has gained increasing attention, because the APIs maybe contaminated with different counter ions from synthesis steps, and because selecting the counter ion to enhance APIs’ solubility and stability is becoming a key step in formulation development. This presentation demonstrates the identification and quantification of 22 commonly found anions in pharmaceuticals in a single run using a high-pressure capillary IC system (HPIC) with 4-μm particle ion –exchange column, and CD-QD dual detectors.

1. 1
Determination of Common Counterions
and Impurity Anions in
Pharmaceuticals Using a Capillary HPIC
System with Suppressed Conductivity
and Charge Detection
Hua Yang
Application Chemist
Thermo Fisher Scientific
The world leader in serving science

2. Outline
2
• Why ion analysis is important for the pharmaceutical
industry?
• The instrument used for the ion analysis: Why HPIC, capillary
and two detectors?
• Method of identified and quantified 22 anions in a single run
and its application

3. Drug Development is Lengthy and Costly
3
Drug R&D
~6 Years
~ 7 Years
1-2 Years
Drug discovery
~10,000 Compounds
Pre-clinical
~250
Clinical trials
~5
FDA review
<2
$1-5 billion and ~15 years to develop a new drug

4. Why is Ion Analysis Needed?
Fact: More than 50% of all pharmaceutical active ingredients
(APIs) are administered as salts
4
• Late stage: Compliance with FDA regulations
• Pharmaceutical products must be tested fro composition to verify their
identity, strength, quality, and purity
• Early stage: Development and selection of the best
formulation for late stage drug development
• Raw material quality control (counterion identity, stoichiometry
confirmation)
• Counterion screening to improve API properties such as solubility,
stability, and processiblity

5. Capillary HPIC System with Dual Detectors
Deionized water
18 MΩ-cm resistivity
Anion Trap Column
5
Data
Management
Waste
H20
Pump*
EGC*
CR-ATC*
Degas
Module*
ACES CRD
* High-pressure module up to 5000 psi
ASTC*
Non-Metallic Pump
Eluent Generator
Cartridge
Continuously-
Regenerated
Anion Trap Column
Auto sampler
Electrolytic
Eluent
Suppressor
Columns
Injection Valve
with internal
sample loop
Conductivity
Detector (CD)
Carbonate
Removal
Device
Charge
Detector (QD)

6. Why HPIC?
6
• Remember UHPLC?
• As the particle size decreases from 8 μm to 4 μm, the column
efficiency doubles
• This drop in particle size increases the column pressure by
4x
• Like HPLC, IC is moving towards smaller particle column
technology
• HPIC instrumentation can now handle the pressure of these
smaller particle columns, even at higher flow rates

7. HPIC Theory
7
Influence of the particle diameter on pressure and efficiency
100
0
0 2 4 6 8 10
Linear Velocity u [mm/s]
1200
1000
Column pressure [bar]
800
600
400
200
0
0 2 4 6 8 10
Linear Velocity u [mm/s]
10 μm particles
5 μm particles
3 μm particles
2 μm particles
Optimal flow rate for
maximum separation
efficiency/resolution
Theoretical Plate Height [μm]
Faster flows for faster separations generate higher pressure
Smaller particles for higher efficiency generate higher pressure

8. 8
High Efficiency Dionex IonPac 4 μm Particle IC Columns
Ion-exchange columns with 4 μm particle-size
Benefits
• Smaller particles provide better performance
• Faster run times with higher flow rates using 150 mm
columns
• Better resolution with standard flow rates using 250 mm
columns
High resolution using the
Dionex IonPac AS11-HC-4μm
column
Fast run using the
5.5
μS
Thermo Scientific™ Dionex™
IonPac™ AS18-4μm column
10
1 0 40
Minutes
μS
0 3
-0.5
5
μS
0
Minutes 0 40
Minutes
Applications
• Anions in environmental
waters
• Organic acids in foods and
beverages
• Amines in chemical process
solutions
High resolution using the
Dionex IonPac CS19-4μm
column
Improved resolution finds more ions in a single run

9. The Dimension of Scale
9
Parameter Analytical IC Capillary IC
Column diameter 4 mm 0.4 mm
Flow rate 1.0 mL/min 10 μL/min
Injection volume 25 μL 0.4 μL
Eluent consumption 43.2 L/month 0.432 L/month
EGC Lifetime
(@75 mmol/L)
28 days 18 months
EG Current (50 mM KOH) 80.4 mA 0.804 mA
K+ Consumption/Year
26.3 Moles (50 mM
KOH)
0.263 Moles (50
mM KOH)
H2O Consumption/Year 525.6 L 5.25 L

10. The Dimension of Scale – The Concentration Factor
10
Overlay of chromatograms with 4 mm, 2 mm, and 0.4 mm
columns – all with equal injection volume (0.4 μL)
16
-2
1
Sodium
Lithium
Ammonium
Potassium
Magnesium Calcium
16
-2
Sodium
Lithium
Ammonium
Sodium
Potassium
0 2 4 6 8 10 12 14
16
-20
Capillary IC with 0.4 μL injection volume
Conductivity [μS]
Retention time [min]
Microbore IC with 0.4 μL injection volume
Standardbore IC with 0.4 μL injection volume
Potassium
Magnesium Calcium
Lithium
Ammonium
Magnesium Calcium

11. Why Capillary?
• Capillary IC separates ions at mL/min flow rates on 0.4 mm
ID columns with 0.4 μL sample injection
• Lower consumption of eluent (5.2 L water/year)
• Long life time of consumable parts (EGC/18 months)
• Higher mass sensitivity and less sample needed
• Better results and lower cost of ownership
System can be always on and always ready for your samples
11

12. Capillary IC Dionex IC Cube Module and Dual
CD/QD Detectors
12
Guard and Separation Columns
4-Port Injection Valve
Analysis with confidence
Thermo Scientific
Dionex CRD 180
Carbonate Removal
Device
Thermo Scientific™
Dionex™ ACES™ Anion
Capillary Electrolytic
Suppressor
Conductivity Detector
(CD)
Cap IC Degas
Charge Detector (QD)

13. Suppressed Conductivity Detection
13
Time
F -
Cl - SO
2-
4
F - Cl - SO4
2-
Time
μS
μS
Without suppression
With suppression
Eluent (KOH)
Sample F-, Cl-, SO4
2-
Ion-Exchange
Separation Column
Anion Electrolytically
Regenerated
Suppressor
in H2O
KF, KCI, K2SO4
in KOH
Injection valve
Counter ions
HF, HCI, H2SO4

14. Electrodialytic Charge Detection
14
- +
Cation-exchange
Membrane
Anion-exchange
Membrane
H20
DC
H2O 2H+ + ½ O2 + 2e– 2H2O + 2e– 2OH– + H2
20
A+ Y-Y-H
A+
H20
-
NO2 -
0 5 10 15 20 Time/ min
180
160
140
120
Capacitance / fF
IO3
-
BrO3
NO3
-
I-Br-
-
NO2 -
0 5 10 15 20 25
Time/ min
180
160
140
120
Capacitance / fF
IO3
-
BrO3
NO3
-
I-Br-
Signal is proportional to charge

15. Capillary HPIC System with Dual Detectors
Deionized water
18 MΩ-cm resistivity
Anion Trap Column
15
Data
Management
Waste
H20
Pump*
EGC*
CR-ATC*
Degas
Module*
ACES CRD
* High-pressure module up to 5000 psi
ASTC*
Non-Metallic Pump
Eluent Generator
Cartridge
Continuously-
Regenerated
Anion Trap Column
Auto sampler
Electrolytic
Eluent
Suppressor
Columns
Injection Valve
with internal
sample loop
Conductivity
Detector (CD)
Carbonate
Removal
Device
Charge
Detector (QD)
System is always ready
No manual eluent preparation
Minimal method development
Analysis with confidence

16. Most Commonly Found Anions in Pharmaceuticals
• Counterions
Chloride Gluconate Acteate Glycolate
Formate Pyruvate Glucuronate Nitrate
Bromide Glutarate Succinate Malate
Tartrate Malonate Benzoate Maleate
Sulfate Fumarate Phosphate Citrate
Tosylate Benzenesulfonate Lactate
• Impurity ions
Fluoride Nitrite Trifluoroacetate
16

17. 22 Anions Mixed Standard for Pharmaceutical Analysis
10
17
Column: Dionex IonPac AS11-HC-4μm, 0.4 mm i.d.
Instrument: Thermo Scientific™ Dionex™ ICS-5000+ HPIC™
system
Eluent Source: Thermo Scientific Dionex EGC-KOH capillary
cartridge
Gradient: 1.52 mM KOH (05 min); 28 mM (58 min),
86 mM (826 min), 1670 mM (2632 min),
70 mM (3238 min)
Flow Rate: 0.0150 mL/min
Inj. Volume: 0.40 μL
Column Temp.: 30 °C
IC Cube Temp.: 15 °C
Detection: CD: Suppressed Conductivity Detector
QD: Charge Detector, 6V
Suppressor: Dionex ACES 300 suppressor, AutoSuppression,
recycle mode
Sample: Mixed Standard
Peaks: mg/L mg/L
1.Gluconate 7.5 12. Glutarate 8.6
2. Lactate 6.8 13. Succinate 8.5
3. Acetate 9.6 14. Carbonate -
4. Formate 8.5 15. Tartrate 10.3
5. Pyruvate 9.1 16. Benzoate 18.6
6. Galacturonate 11.1 17. Maleate 6.6
7. Chloride 2.1 18. Sulfate 3.4
8. Nitrite 2.1 19. Fumarate 7.5
9. TFA 8.5 20. Benzenesulfonate 17.7
10. Bromide 4.2 21. Phosphate 7.8
11. Nitrate 4.8 22. Citrate 11.3
23. Tosylate 13.3
μA
CD
10
2
10
15
14
17
22
0 10 20 30 40
Minutes
-1
QD
μS
-1
1
3
4
5
6
7
8 9
11
12
13
16
18
19
20
21
23

18. Retention Time and Responses at the Concentration
10
18
Peaks Ret. CD QD
(Min) (μS Min) (μA Min)
1.Gluconate 5.45 0.324 0.210
2. Lactate 5.75 0.667 0.324
3. Acetate 6.08 0.657 0.345
4. Formate 7.62 0.792 0.238
5. Pyruvate 9.12 0.823 0.327
6. Galacturonate 10.60 0.487 0.298
7. Chloride 11.86 0.670 0.134
8. Nitrite 13.20 0.458 0.096
9. TFA 17.12 0.774 0.313
10. Bromide 18.19 0.599 0.110
11. Nitrate 19.97 0.975 0.199
12. Glutarate 20.50 1.003 0.481
13. Succinate 21.25 0.779 0.346
14. Carbonate - - -
15. Tartrate 22.86 1.979 0.565
16. Benzoate 23.64 1.203 0.612
17. Maleate 24.99 0.631 0.249
18. Sulfate 26.01 0.829 0.191
19. Fumarate 28.82 1.587 0.489
20. Benzenesulfonate 30.05 1.040 0.469
21. Phosphate 32.29 0.660 0.289
22. Citrate 33.09 0.999 0.358
23. Tosylate 35.80 0.700 0.355
Coelution: Gluconate/Fluoride, Acetate/Glycolate,
Succinate/Malate, and Tartrate/Malonate
μA
CD
10
2
10
15
14
17
22
0 10 20 30 40
Minutes
-1
QD
μS
-1
1
3
4
5
6
7
8 9
11
12
13
16
18
19
20
21
23

19. Ion Identification and Quantification by CD and QD
A
μA μS
QD
-1 -2
4
19
Column: Dionex IonPac AS11-HC-4μm, 0.4 mm i.d.
Instrument: Dionex ICS-5000+ HPIC system
Eluent Source: Dionex EGC-KOH capillary cartridge
Gradient: 1.52 mM KOH (05 min); 28 mM (58 min),
86 mM (826 min), 1670 mM (2632 min),
70 mM (3238 min)
Flow Rate: 0.0150 mL/min
Inj. Volume: 0.40 μL
Column Temp.: 30 ºC
IC Cube Temp.: 15 ºC
Detection: CD: Suppressed Conductivity Detector
QD: Charge Detector, 6V
Suppressor: Dionex ACES 300 suppressor,
AutoSuppression, recycle mode
Samples A and B are two mixtures each with
three anions
Peak Ret. (Min) Concentration (mg/L)
CD QD Diff. (%)
A (Pass) 1. Acetate 6.08 15.1 15.6 3
2. Chloride 11.86 2.9 2.9 0
3. Tartrate 22.86 12.9 13.2 2
B (Fail) 1. Acetate 6.08 24.0 20.4 15
2. Chloride 11.86 2.9 2.9 0
3. Tartrate 22.86 10.2 11.5 13
Retention time suggests both A and B can be mixtures of
Acetate, Chloride and Tartrate. With <5% acceptance criteria ,
mixture A passes and confirmed as the mixture; mixture B fails.
14
QD
0 5 10 15 20 25
Min
4
-1
CD
B
1
3
1
3
2
14
-2
μA
μS
CD
2

20. Chloride in an Allergy Drug Tablet
9
20
Column: Dionex IonPac AS11-HC-4μm, 0.4 mm i.d.
Instrument: Dionex ICS-5000+ HPIC system
Eluent Source: Dionex EGC-KOH capillary cartridge
Gradient: 1.52 mM KOH (05 min); 2-8 mM (58 min),
816 mM (826 min), 16  70 mM (2632 min),
70 mM (3238 min).
Flow Rate: 0.0150 mL/min
Inj. Volume: 0.40 μL
Column Temp.: 30 °C
IC Cube Temp.: 15 °C
Detection: CD: Suppressed Conductivity Detector
Suppressor: Dionex ACES 300 suppressor,
AutoSuppression, recycle mode
Samples A: One tablet dissolved in 1000 mL water
B: 5-fold dilution of A by water
C: Water blank
Peaks Ret. Concentration
(Min) (mg/L)
A B C
1. Acetate 6.08 0.26  na
2. Chloride 11.86 8.50 1.70 na
3. Nitrite 13.20 0.11  na
4. Nitrate 19.97 0.18  na
5. Carbonate    
6. Sulfate 26.01 0.05  na
μS
A
0 10 20 30
Minutes
-1
B
C
2
1 3 4 5 6

21. CD vs. QD Detections for an Allergy Drug Tablet
1 2
21
Column: Dionex IonPac AS11-HC-4μm, 0.4 mm i.d.
Instrument: Dionex ICS-5000+ HPIC system
Eluent Source: Dionex EGC-KOH capillary cartridge
Gradient: 1.52 mM KOH (05 min); 2-8 mM (58 min),
8-16 mM (826 min), 16  70 mM (2632 min),
70 mM (3238 min).
Flow Rate: 0.0150 mL/min
Inj. Volume: 0.40 μL
Column Temp.: 30 °C
IC Cube Temp.: 15 °C
Detection: CD: Suppressed Conductivity Detector
QD: Charge Detector, 6V
Suppressor: Dionex ACES 300 suppressor,
AutoSuppression, recycle mode
Samples One tablet dissolved in 1000 mL water
Peaks Ret. Concentration
(Min) (mg/L)
CD QD
1. Acetate 6.08 0.3 0.4
2. Chloride 11.86 8.5 8.5
3. Nitrite 13.20 0.1 
4. Nitrate 19.97 0.2 <LOQ
5. Carbonate  (from eluent)
6. Sulfate 26.01 0.1 0.1
7. Unknown 12.70  
μS
CD
0 10 20 30
Minutes
0
0
7
6
5
1
QD
3
4
2

22. CD Calibration Curve of Chloride from 0.1 to 500 mg/L
22
0 200 400 600
Chloride (mg/L)
200
Area (μS*min)
0
r2 = 0.9999 %RSD = 2.08
LOQ = 0.004 mg/L

23. Chloride Concentration in Allergy Drug Tablets
23
Tablet No.
Weight
(g/tablet)
Chloride
(mg/tablet)
CD QD Label
1 0.710 8.50 8.47
2 0.726 8.45 8.29
3 0.692 7.93 8.02
4 0.710 8.30 8.42
5 0.730 8.39 8.61
Average 0.714 8.32 8.36 8.32
% RSD 2.14 2.76 2.66

24. Tartrate in a Supplement Tablet
9
24
Column: Dionex IonPac AS11-HC-4μm, 0.4 mm i.d.
Instrument: Dionex ICS-5000+ HPIC system
Eluent Source: Dionex EGC-KOH capillary cartridge
Gradient: 1.52 mM KOH (05 min); 2-8 mM (58 min),
8-16 mM (826 min), 1670 mM (2632 min),
70 mM (3238 min).
Flow Rate: 0.0150 mL/min
Inj. Volume: 0.40 μL
Column Temp.: 30 ºC
IC Cube Temp.: 15 ºC
Detection: CD: Suppressed Conductivity Detector
Suppressor: Dionex ACES 300 suppressor,
AutoSuppression, recycle mode
Samples A: One tablet dissolved in 1000 mL water
B: 20 - fold dilution of A by water
C: Water blank
Peaks Ret. Concentration
(Min) (mg/L)
A B C
1. Acetate 6.08 0.01  na
2. Formate 7.62 0.39  na
3. Chloride 11.86 0.11  na
4. Nitrite 13.20 0.23  na
5. Nitrate 19.97 0.44  na
6. Carbonate    
A
0 10 20 30 7. Tartrate 22.86  12.37 na
-1
B
C
1
2 3 4
6
5

25. Contaminants in a Supplement Tablet
1
25
Column: Dionex IonPac AS11-HC-4μm, 0.4 mm i.d.
Instrument: Dionex ICS-5000+ HPIC system
Eluent Source: Dionex EGC-KOH capillary cartridge
Gradient: 1.52 mM KOH (05 min); 2-8 mM (58 min),
8-16 mM (826 min), 1670 mM (2632 min),
70 mM (3238 min).
Flow Rate: 0.0150 mL/min
Inj. Volume: 0.40 μL
Column Temp.: 30 ºC
IC Cube Temp.: 15 ºC
Detection: CD: Suppressed Conductivity Detector
QD: Charge Detector, 6V
Suppressor: Dionex ACES 300 suppressor,
AutoSuppression, recycle mode
Samples: One tablet dissolved in 1000 mL water
Peaks Ret. Concentration
(Min) (mg/L)
CD QD
1. Acetate 6.08 0.01 0.01
2. Formate 7.62 0.39 0.36
3. Chloride 11.86 0.11 0.14
4. Nitrite 13.20 0.23 0.32
5. Nitrate 19.97 0.44 0.36
6. Carbonate It is from eluent and under tartrate peak
7. Tartrate 22.86  
8. Unknown 27.90  
μA
CD
μS
0 10 20 30
Minutes
0
QD
1 2
3
4
6
5
2
0
8

26. CD Calibration Curve of Tartrate from 1.25 to 50 mg/L
26
0 20 40 60
Tartrate (mg/L)
8
Area (μS*min)
0
r2 = 0.9998 %RSD = 1.50
LOQ = 0.06 mg/L

27. Tartrate Concentration in Supplement Tablets
27
Tablet No.
Weight
(g/tablet)
Tartrate
(mg/tablet)
CD QD Label
1 1.283 241.5 234.1
2 1.293 244.0 235.5
3 1.267 242.3 233.1
4 1.300 251.7 229.1
5 1.320 257.6 233.9
Average 1.293 247.4 233.1 251
% RSD 1.53 2.8 1.0

28. Conclusions
• IC is better suited for ionic analytes analysis. IC separates
and directly detects ionic analytes, even without UV
chromophores.
• Using an HPIC system with suppressed conductivity and
charge detectors:
• 22 common pharmaceutical anions were separated in a single analytical
28
run using a Dionex IonPac AS11HC-4μm capillary column
• Multiple counterions in drug products were easily identified and
quantified with confidence

29. Thank you!
29
OT71427-EN 1114S