1

The world leader in serving science

Hua Yang

Application Chemist

Thermo Fisher Scientific

Determination of Common Counterionsand Impurity Anions in Pharmaceuticals Using a Capillary HPIC System with Suppressed Conductivity and Charge Detection

2

Outline

• 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

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?

• 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

Fact: More than 50% of all pharmaceutical active ingredients

(APIs) are administered as salts

5

Capillary HPIC System with Dual Detectors

Data Management

Waste

H20

Degas

Module*

CR-ATC*

Pump*

EGC*

CRDACES

* High-pressure module up to 5000 psi

ASTC*

Non-Metallic Pump

Deionized water

18 MΩ-cm resistivity

Eluent Generator Cartridge

Anion Trap Column

Continuously-Regenerated Anion Trap Column

Auto sampler

Electrolytic Eluent Suppressor

Columns

Injection Valve

with internal

sample loop

Carbonate

Removal

Device

ConductivityDetector (CD)

Charge

Detector (QD)

6

Why HPIC?

• 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

Influence of the particle diameter on pressure and efficiency100

0

0 2 4 6 8 10

Linear Velocity u [mm/s]

Co

lum

n p

res

su

re [

ba

r]

0

200

400

600

800

1000

1200

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

Th

eo

reti

cal P

late

He

igh

t [µ

m]

Faster flows for faster separations generate higher pressure

Smaller particles for higher efficiency generate higher pressure

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

Thermo Scientific™ Dionex™

IonPac™ AS18-4µm column

0 401

10

Minutes

µS

0 3-0.5

5.5

µS

Minutes 0 400

5

µS

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

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/Year26.3 Moles (50 mM

KOH)

0.263 Moles (50

mM KOH)

H2O Consumption/Year 525.6 L 5.25 L

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The Dimension of Scale – The Concentration Factor

Overlay of chromatograms with 4 mm, 2 mm, and 0.4 mm

columns – all with equal injection volume (0.4 µL)

-2

161

LithiumSodium

Ammonium

PotassiumMagnesium Calcium

-2

16

0 2 4 6 8 10 12 14

-20

16

Capillary IC with 0.4 µL injection volume

Co

nd

uctivity [

µS

]

Retention time [min]

Microbore IC with 0.4 µL injection volume

Standardbore IC with 0.4 µL injection volume

Lithium

Sodium

Ammonium

Potassium

Magnesium Calcium

Lithium

Sodium

Ammonium

Potassium

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

12

Capillary IC Dionex IC Cube Module and Dual CD/QD Detectors

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)

Charge Detector (QD)

Cap IC Degas

13

Suppressed Conductivity Detection

Time

F-

Cl - SO42-

F -Cl - SO4

2-

Time

µS

µS

Without suppression

With suppression

Eluent (KOH)

Sample F-, Cl-, SO42-

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

Cation-exchange

Membrane

Anion-exchange

Membrane

H20

- +DC

H2O 2H+

+ ½ O2 + 2e–2H2O + 2e

–2OH

–+ H2

A+ Y-

Y-

H20

A+

H200 5 10 15 20 25

Time/ min

120

140

160

180

Ca

pa

cit

an

ce

/ f

F

IO3-

BrO3-

NO3-NO2

-

I-

Br-

0 5 10 15 20 25Time/ min

120

140

160

180

Ca

pa

cit

an

ce

/ f

F

IO3-

BrO3-

NO3-NO2

-

I-

Br-

Signal is proportional to charge

15

Capillary HPIC System with Dual Detectors

Data Management

Waste

H20

Degas

Module*

CR-ATC*

Pump*

EGC*

CRDACES

* High-pressure module up to 5000 psi

ASTC*

Non-Metallic Pump

Deionized water

18 MΩ-cm resistivity

Eluent Generator Cartridge

Anion Trap Column

Continuously-Regenerated Anion Trap Column

Auto sampler

Electrolytic Eluent Suppressor

Columns

Injection Valve

with internal

sample loop

Carbonate

Removal

Device

ConductivityDetector (CD)

Charge

Detector (QD)

System is always ready

No manual eluent preparation

Minimal method development

Analysis with confidence

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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

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22 Anions Mixed Standard for Pharmaceutical Analysis

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: 1.52 mM KOH (05 min); 28 mM (58 min),

86 mM (826 min), 1670 mM (2632 min),

70 mM (3238 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

Minutes

3010 200 40

-1

10

QD

CD

µS

10

-1

1

2

3

4

5

6

7

8 9

10

11

12

13

14

15

1617

18

19

20

21

22

23

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Retention Time and Responses at the Concentration

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

Minutes

3010 200 40

-1

10

QD

CD

µS

10

-1

1

2

3

4

5

6

7

8 9

10

11

12

13

14

15

1617

18

19

20

21

22

23

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Ion Identification and Quantification by CD and QD

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: 1.52 mM KOH (05 min); 28 mM (58 min),

86 mM (826 min), 1670 mM (2632 min),

70 mM (3238 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

0 5 10 15 20 25

Min

µA µS

4

-1

CD

B

A

1

3

1

3

-2-14

2

14

-2

µAµS

CD

QD

QD

2

20

Chloride in an Allergy Drug Tablet

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: 1.52 mM KOH (05 min); 2-8 mM (58 min),

816 mM (826 min), 16 70 mM (2632 min),

70 mM (3238 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

Minutes

3010 200

-1

9

B

A

C

2

3 4 651

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CD vs. QD Detections for an Allergy Drug Tablet

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: 1.52 mM KOH (05 min); 2-8 mM (58 min),

8-16 mM (826 min), 16 70 mM (2632 min),

70 mM (3238 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

Minutes

3010 200

0

1 2

0

7

6

5

1

QD

CD

34

2

22

CD Calibration Curve of Chloride from 0.1 to 500 mg/L

0 200 400 600

Chloride (mg/L)

0

200A

rea (

µS

*min

)

r2 = 0.9999 %RSD = 2.08

LOQ = 0.004 mg/L

23

Chloride Concentration in Allergy Drug Tablets

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

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Tartrate in a Supplement Tablet

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: 1.52 mM KOH (05 min); 2-8 mM (58 min),

8-16 mM (826 min), 1670 mM (2632 min),

70 mM (3238 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

7. Tartrate 22.86 12.37 na3010 200

-1

9

B

A

C

12 3 4

65

25

Contaminants in a Supplement Tablet

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: 1.52 mM KOH (05 min); 2-8 mM (58 min),

8-16 mM (826 min), 1670 mM (2632 min),

70 mM (3238 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

Minutes

3010 200

0

1

QD

CD

1 2

34

6

5

2

0

8

µS

26

CD Calibration Curve of Tartrate from 1.25 to 50 mg/L

0 20 40 60

Tartrate (mg/L)

0

8A

rea (

µS

*min

)

r2 = 0.9998 %RSD = 1.50

LOQ = 0.06 mg/L

27

Tartrate Concentration in Supplement Tablets

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

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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

run using a Dionex IonPac AS11HC-4µm capillary column

• Multiple counterions in drug products were easily identified and

quantified with confidence

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Thank you!

OT71427-EN 1114S