analysis of pharmaceutically relevant non-chromophoric ... · b) 1:10 dilution of sample a a% b%...

Analysis of Pharmaceutically

Relevant Non-Chromophoric

Compounds by Ion-Exchange

Chromatography

Joachim Weiss, Ph.D.

Thermo Fisher Scientific GmbH, Dreieich, Germany

Proprietary & Confidential 2 Dionex Confidential Dionex Confidential Dionex Confidential 2

Outline

• Introduction

• Application areas of IC in the pharmaceutical industry

• Structural identification of counter ions

• Organic acid analysis

• Analysis of amines

• Sulfur-containing antibiotics

• Direct determination of ionic drugs

• Carbohydrates, amino acids, proteins, nucleotides

• USP monographs using Thermo Scientific Dionex

columns

• Conclusions


INTRODUCTION


Ion-Exchange Chromatography Offers Simple, Direct, and Interference-Free Analyses

• Sensitive Detection

Samples can be diluted to decrease the

concentration of matrix components

• Specific Detection

Low abundant analytes can be detected in

presence of large concentration of matrix

components

• Analyte-Specific Separations

The selectivity of the separator columns is

tailored for the specific requirements of the

various analyte classes


Sample Preparation for Ion-Exchange Chromatography

Problem:

Analytes often have to be determined in lipophilic matrices (drugs,

intermediates); however, IC is usually carried out in an aqueous

environment.

Problem solution:

Addition of organic solvents to the mobile phase

Sample preparation: Liquid-liquid extraction

Sample preparation: Solid-phase extraction

Proprietary & Confidential 6 Dionex Confidential

Reagent-Free IC (RFIC) Technology

• Combination of three techniques

• Electrolytic eluent generation

• Electrolytic eluent purification

• Electrolytic eluent suppression

• All techniques depend on the electrolysis of water

Anode H2O 2 H+ + ½ O2 (g) + 2e– +

–

OXIDATION

REDUCTION

Cathode 2e– + 2H2O 2 OH– + H2 (g)

Proprietary & Confidential 7

Reagent-Free IC System

Pump

Waste

Eluent Generator

Electrolytic Suppressor

H20

Analytical Column

Injector

Guard Column

Chromatography Data System

Conductivity Cell

Recycle Mode


Advantages of Electrolytic Eluent Preparation

• Only de-ionized water is used as the carrier

• Generation of high-purity, contaminant-free acid and base

eluents online

• Electrical control of eluent concentration with minimal delay

• Capable of generating acids or bases at concentrations up to

100 (200) mmol/L at 1 mL/min (10 µL/min)

• Control via chromatography data system


Hydroxide Eluent Generation for Anion Analysis

Vent [KOH] Current

Flow rate

Degas

Unit

Pt anode

(H2O 2H+ + ½O2 + 2e-)

Cation

Exchange

Connector

KOH

Generation

Chamber Pump

H2O

Pt cathode

(2H2O + 2e- 2OH- + H2)

[ + ]

[ - ]

KOH + H2 KOH

H2

K+ Electrolyte Reservoir

K+

EluGen KOH Cartridge

CR-ATC Anion Trap


Separation of Common Anions Using Electrolytically Generated KOH

0

30

µS

1

2

4

5

3

A

Column

re-equilibration

Minutes

0 2 4 6 8 10

µS

0

35

1 2 3

4

5

B

Column: IonPac AG11, AS11 (4 mm)

Eluent: A: 15.5 mmol/L KOH

B: 0.5 to 25 mmol/L KOH in 8 min

Flow rate: 2 mL/min

Inj. volume: 25 µL

Detection: Suppressed conductivity

AutoSuppression, Recycle Mode

Peaks: 1. Fluoride 2 mg/L

2. Chloride 3

3. Nitrate 10

4. Sulfate 15

5. Phosphate 15


STRUCTURAL IDENTIFICATION OF COUNTER IONS


Analysis of Counter Ions

• Type of analytes:

• Inorganic anions such as chloride and bromide

• Simple organic acids such as acetate

• Less common organic counter ions such as methylsulfate,

methanesulfonate, and trifluoroacetate

• IEX methodology:

• Anion exchange chromatography

• Conductivity detection

• Eluent desalting techniques for enhanced sensitivity and

specificity


Analysis of Trifluoroacetate in a Peptide Sample

Minutes

0 2 4 6 8 10 12 14 18 16 20

0

µS

0.4

1

2

3

Column: IonPac AS14/AG14

Eluent: 3.5 mmol/L Na2CO3

0.8 mmol/L NaHCO3

Flow rate: 1.2 mL/min

Inj. volume: 10 µL

Detection: Conductivity after desalting

AutoSuppression, Recycle Mode

Sample: 40 µg/mL of a peptide

Peaks: 1. Chloride 0.3 mg/L

2. Sulfate 0.9

3. Trifluoracetate 6.5


TFA Analysis in a Phosphate-Containing Saline Matrix

1

Pe

ak A

rea

Re

sp

on

se

0.05

0.45

0 5 10 15 20

2

3

Minutes

Column: IonPac AS11-HC + guard, 4 mm Eluent: KOH (EG) Gradient: 15 mmol/L for 13 min isocratic, then to 80 mmol/L (3 min isocratic) Flow rate: 1 mL/min Inj. volume: 100 μL Temperature: 30°C Detection: Suppressed conductivity, AutoSuppression, recycle mode Peaks: 1. Chloride – 2. Trifluoroacetate 1 µg/mL 3. Orthophosphate –


Separation of Sulfate Counter Ion and Anionic Impurities in Humatin®

0.4

4.0

µS

1

2 3

4

5

6

7

8 9

A

0 10 20 30 0

70

Minutes

µS

6

B

Column: IonPac AS11-HC + guard, 2 mm

Eluent: KOH (EG)

Gradient: 1 mmol/L 0-5 min, 1-5 mmol/L 5-9 min,

5-38 mmol/L 9-20 min, 38-60 mmol/L

20-25 min, 60 mmol/L 25-30 min


Temperature: 30ºC

Inj. volume: 5 µL

Detection: Conductivity after desalting,

Recycle mode

Sample: A) 2.5 mg/mL Paromomycin (Humatin)

B) 1:10 dilution of sample A

A% B%

Peaks: 1. Unknown

2. Acetate 0.08

3. Unknown

4. Chloride 0.03

5. Carbonate

6. Sulfate n. d. 24.7

7. Orthophosphate 0.23

8. Pyrophosphate 0.04

9. Unknown


Surface Chemistry and Specifications of Acclaim Multi-Mode WAX-1

Surface Chemistry Physical Data

Base silica: Spherical, high purity

Particle size: 3, 5 μm

Pore size: 120 Å

Surface area: 300 m2/g

Elemental analysis: C – 15.4% N – 1.8%

Surface coverage: 2.6 µmol/m2

Anion exchange capacity: 790 µequiv/column

Functional group: Alkylamine

Ion-Exchange

Function

Hydrophobic

Alkyl Chain

O N H

N Me Me

Silica


Column: Acclaim Mixed-Mode WAX-1

Eluent: 50/50 v/v MeCN/buffer (2.68 g

potassium monophosphate and 0.2 g

pyrophosphate in 1000 g DI H2O, pH

is adjusted with NaOH and HCl)

Temperature: 30 °C

Flow rate: 1 mL/min

Inj. volume: 5 µL

Detection: UV, 210 nm

Peaks: 1. Lactate

2. Acetate

3. Nitrate

4. Bromide

5. Maleate

6. Benzoate

7. Benzene sulfonate

8. Tosylate

9. Succinate

10. Malate

11. Fumarate

AU

0 10 20

Minutes

1 2

3

4

5

6

7 8

9 10 11

Analysis of Pharmaceutically Relevant Anions on a Mixed-Mode Column


ORGANIC ACID ANALYSIS


• Ion-Exclusion Chromatography

• Ion-Exchange Chromatography

• Ion-Pair Chromatography

• Reversed-Phase Chromatography

(Ion Suppression)

• HILIC

• Mixed-Mode Liquid Chromatography

LC Methods for Organic Acid Analysis


Typical Formulation of a Cough Suppressant

Dextromethorphan hydrobromide Active

Citric acid Inactive

FD&C Red 40 Inactive

Flavors Inactive

Glycerol Inactive

Propylene glycol Inactive

Saccharin sodium Inactive

Sodium benzoate Inactive

Sorbitol Inactive

Water Inactive


Gradient Elution of Anionic Components in a Cough Suppressant

0

µS

4

5

6 7 8 10 9 12 1 2 3

14 0

Minutes

8 6 4 2 10 12 16 18

18

11

4

5

6

7 8 10

9

11

0

1

µS

Column: IonPac AS11

Eluent: KOH

Gradient: 0.5 mmol/L isocratically for 2.5 min,

then linearly to 5 mmol/L in 3.5 min,

then to 38 mmol/L in 12 min

Flow rate: 2 mL/min

Inj. volume: 10 µL


Sample: (1:10 (w/w) diluted formulation)

Peaks: 1. unknown --

[mg/L] 2. unknown --

3. Chloride 0.08

4. Bromide 30

5. Benzoate 80

6. unknown --

7. unknown --

8. Sulfate 0.2

9. Orthophosphate 0.09

10. unknown --

11. Citrate 130

12. Saccharin 120


0.1 g/L

P

OH

OHO

0 10 20

Minutes

Pro

panephosphonic

acid

Courtesy of Dr. Heiko Potgeter, Novartis Pharma AG, Basel, CH

Analysis of an Intermediate: Propanephosphonic Acid

Column: IonPac AS14A, 5 µm

Eluent: 8 mmol/L Na2CO3 +

1 mmol/L NaHCO3


Inj. volume: 25 µL


Sample: Aqueous wash solution of a

crystalline intermediate

Sample

preparation: Direct injection of the sample


Eluent: 50/50 v/v MeCN/50 mmol/L

phosphate buffer, pH 2.8

for Acclaim Mixed-Mode WAX-1

20/80 v/v MeCN/50 mmol/L

phosphate buffer, pH 2.8

for Acclaim 120 C18

Temperature: 30°C

Flow rate: 1 mL/min

Inj. volume: 2 µL


Peaks: 1. Benzoic acid (0.2 mg/mL)

2. Phthalic acid (0.2 mg/mL)

3. Benzene-1,2,3-tricarboxylic

acid (0.2 mg/mL)

Complementary Selectivity to Reversed-Phase Chromatography

0 10 20

Minutes

AU

Acclaim WAX-1 (5 μm, 150 mm x 4.6 mm ID)

Acclaim 120 C18 (5 μm, 150 mm × 4.6 mm i. d.)

2

1

3

2 1 3

C O 2 H

C O 2 H

C O 2 H

C O 2 H

C O 2 H

C O 2 H


Eluent: 20 mmol/L phosphate buffer, pH 6 for

Acclaim Mixed-Mode WAX-1

50 mmol/L phosphate buffer, pH 2.6

for Acclaim OA

Temperature: 30 °C

Flow rate: 1 mL/min

Inj. volume: 5 µL


Peaks: 1. Lactic acid (0.8 mg/mL)

2. Ascorbic acid (0.25 mg/mL)

3. Acetic acid (0.8 mg/mL)

0 4 8

Minutes

AU 1,2

3

Acclaim WAX-1 (5 μm, 150 mm × 4.6 mm i. d.)

Acclaim OA (5 μm, 150 mm × 4.6 mm i. d.)

2

3 1

Separation of Lactate, Acetate, and Ascorbic Acid

Ascorbic acid (Vitamin C)

O O

O H H O

H O

H O

Lactic acid

O H

O H

O

Acetic acid

O H

O


Acrylic Acid and Oligomers

Column: Acclaim HILIC-10, 3 µm

Dimensions: 150 mm × 4.6 mm i. d.

Eluent: 90/10 v/v MeCN/10 mmol/L (total) NH4OAc, pH5

Flow rate: 1 mL/min

Temperature: 30 °C

Inj. volume: 2 µL


Peaks: (5 mg/mL in eluent)

1. Acrylic acid (monomer)

2. Dimer

3. Trimer

4. Tetramer

5. Pentamer

6. Hexamer

0 6 12 9 3 15

Minutes

mA

U

0

30

1

2

3

4

5 6


ANALYSIS OF AMINES


Schematics of a Cation Exchanger for Hydrophobic and Polyvalent Amines

Grafted Carboxylic Acid Cation Exchange Polymer Layer

Boundary Layer

55% crosslinked 7 µm macroporous

450 m2/g

(EVB-DVB) particle core

(EVB-DVB) Particle Core Pore Structure


Separation of Biogenic Amines on IonPac CS19

Column: IonPac CS19 (250 mm 2 mm i. d.)

Eluent: MSA (EG)

Gradient: 10 mmol/L for 7 min, 10-40 mmol/L over

6 min, 40-60 mmol/L over 7 min


Inj. volume: 25 µL

Temperature: 30 °C


Peaks: 1. Lithium 0.1 mg/L 7. Putrescine 15

2. Sodium 0.1 8. Cadaverine 9

3. Ammonium 0.1 9. Histamine 13

4. Potassium 0.1 10. Agmatine 10

5. Magnesium 0.1 11. Spermidine 3

6. Calcium 0.1 12. Spermine 6

0 5 10 15 20 25 30 Minutes

1 2

3 4 5 6

8

9

10

11

12

7 4.0

-0.5

µS

29

Fast Separation of Common Cations and Biogenic Amines

Column: Dionex IonPac CS19-4µm + guard

(250 × 0.4 mm i. d,)

Eluent: 9 mmol/L MSA, gradient to 70 mmol/L MSA

at 7 min, isocratic to 7.7 min, back to 9 mmol/L

MSA at 7.8 min


Inj. volume: 0.4 µL

Temperature: 30°C

Detection: Suppressed conductivity,

CCES300, AutoSuppression

Recycle mode

Peaks: 1. Lithium 0.05 mg/L

2. Sodium 0.2

3. Ammonium 0.25

4. Potassium 0.5

5. Magnesium 0.25

6. Calcium 0.5

7. Impurity

8. Putrescine 7.5

9. Cadaverine 4.5

10. Histamine 6.5

11. Agmatine 5.0

12. Spermine 3.0

13. Spermidine 1.5

NOTE: Concentrations are only approximate

0 2 4 6 8 10

0

7

µS

Minutes

1 2 3 4

5

6

7

8

9

10

11

12

13

30

Separation of Carbachol, Choline, and Bethanechol together with Inorganic Cations

Column: IonPac CS17 + guard (4 mm)

Eluent: 5 mmol/L MSA (EG)

Temperature: 30ºC


Inj. volume: 25 µL


AutoSuppression, recycle mode

Peaks: 1. Lithium 0.1 mg/L

2. Sodium 0.4

3. Ammonium 0.5

4. Potassium 1.0

5. Dimethylamine 1.0

6. Choline 1.0

7. Carbachol 1.0

8. Bethanechol 1.0

9. Magnesium 0.5

10. Calcium 1.0 0 5 10 15 20

Minutes 25

1

2

3 4

5

6

7

9

8

10

1.6

µS

0

31

Alcon Lens Solution Spiked with Carbachol

Column: IonPac CG17, CS17 (4 mm)


Temperature: 30 ºC


Inj. volume: 25 µL


AutoSuppression

Sample: Alcon lens solution,

1:100 diluted

Peaks: 1. Sodium 2.37 mg/L

2. Carbachol 1.04

0

Minutes

25

0

0.75

µS

1

2

5 10 15 20

32

Separation of Bethanechol

Bethanechol chloride: 2-[(aminocarbonyl)oxy]-N,N,N-trimethyl-1-propanaminium

chloride (cholinergic)

Traditional

analytical method: Gravimetric assay acc. USP 24 NF 19 (page 230)

Goal: To replace with a more specific IC assay that also measures

stability [Pharmacopeial Forum (2001) 27(1), 155-157]

IC methodology: Separation by cation exchange and detection by suppressed

conductivity

Advantage: Analysis of the drug component and its degradation product

2-hydroxypropyltrimethylammonium chloride (2-HPTA)

33

Separation of Bethanechol and 2-Hydroxypropyltrimethyl Ammonium

Column: IonPac CS14 + guard


Flow rate: 1 mL/min

Inj. volume: 25 µL


AutoSuppression, recycle mode

Peaks: 1. Sodium

2. Unknown

3. Magnesium

4. 2-Hydroxypropyltri-

methylammonium 2 mg/L

5. Bethanechol 2 mg/L

Minutes

0 2 4 6 10 8

0

1

2

µS

1

2

3

4 5

H 2 N

C

O

CH

CH 3

CH 2 N

CH 3

CH 3

CH 3

O +

Cl -

34

HILIC Separation of Good’s Buffer Salts

Column: Acclaim HILIC-10, 3 µm

Dimensions: 150 × 4.6 mm i. d.

Eluent: 85/15 (v/v) MeCN/10 mmol/L (total) NH4OAc, pH 5

Temperature: 30 °C

Flow rate: 1 mL/min

Inj. volume: 10 µL

Detection: Corona CAD ultra

Peaks: (0.1 mg/mL each in eluent)

1. TAPS

2. CHES

3. MOPS

4. TES

5. HEPES

0 4 8 6 2 10 Minutes

mV

0

800

1

2

3

4

5

35

ANALYSIS OF SULFUR COMPOUNDS

36

0 5 10

Minutes

0.51

µC

0.46

1 2 3

4

5

IPAD

0.3

AU

-0.7

1 UV (215 nm)

Column: Vydac C8 (208TP5451)

Temperature: 30°C

Eluent: 0.1 mol/L NaOAc - MeCN

(91:9 v/v), pH 3.75

Flow rate: 1 mL/min

Detection: Integrated amperometry,

Au electrode

Peaks: 1.-3. unknown

4. Lincomycin 10 µg/mL

5. unknown

Analysis of Lincomycin Using Integrated Amperometry

O

N H

H

CH 3

SCH 3

OH

OH

HO

H OH

O

N

H 3 C

H 3 C

HCl

.

37

ANALYSIS OF IONIC DRUGS

38

Direct Determination of Ionic Drugs

Cl

Cl

P P

O O

O O

HO OH

Clodronatee

Example: Disodium salt of dichloromethylenebisphosphic acid

Drug against hypercalcaemia and osteoporosis

Possible impurities resulting from synthesis:

Chloride, chloro-substituted esters, methylenebisphosphonic acid

and monochloromethylenebisphosphonic acid

39

Separation of Clodronate and Potential Impurities on a Hydroxide-Selective Anion Exchanger

1

2

3

4

5 6

7

8 9

10

35

µS

0 0 20 10

Minutes

Column: IonPac AS5

Temperature: 45°C

Eluent: NaOH

Gradient: 20 to 100 mmol/L

in 20 min

Flow rate: 1 mL/min


Peaks: 1. Chloride

2. Nitrate

3. Diester of dichloro-

methylenebisphosphonic acid

4. Sulfate

5. Orthophosphate

6. Monoester of dichloro-

methylenebisphosphonic acid

7. Clodronate

8. Monochloromethylenebis-

phosphonic acid

9. Methylenebisphosphonic acid

10. Carbonylbisphosphonic acid

G.E. Taylor, J. Chromatogr. A 770 (1997) 261.


Gradient Elution of Bisphosphonates, Excipients and Anions

Column: Dionex IonPac AS18-Fast, 0.4 mm

Temperature: 40 ˚C

Eluent: KOH (EG)

Gradient: 40-50 mmol/L from 0-5 min, 50-100

mmol/L from 5-8 min Flow rate: 20 µL/min Inj. volume: 10 µL, 20-100 µg/L each


0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1

2

3

4

µS

Minutes

Concentration: 40.00 mM

100.00

40.00

1

2

3

4

5 6

7

8 9

10 11

12

13

50.00

Peaks: 20-100 µg/L each

1. Fluoride 8. Benzoate

2. Chloride 9. Hydroxybenzoate

3. Nitrite 10. Citrate

4. Sulfate 11. Etidronate

5. Bromide 12. Clodronate

6. Nitrate 13. Tiludronate

7. Orthophosphate


MIXED-MODE SEPARATIONS (ASSAYS)


Separation of Basic, Neutral, and Acidic Pharmaceuticals

Column: Acclaim Mixed-Mode WAX-1

Dimension: 150 mm × 4.6 mm i. d.


potassium monophosphate

and 0.5 g pyrophosphate in

1000 g D.I. H2O, pH is

adjusted to 6.0 with NaOH)

Temperature: 30 °C

Flow rate: 1 mL/min

Inj. volume: 5 µL


Peaks: 1. Acetaminophen

2. Amitriptyline

3. Hydrocortisone

4. Dexamethasone

5. Iodide

6. Benzoate

7. Phenanthrene

8. Ketoprofen

9. Naproxen, Na salt 0 10 20

Minutes

AU

5

1

2

3

4

6

7

8

9

O

O-

O

HN

O

OH

N

O- O

O

Me HO Me

H

H H

OH

O

OH

O

Me HO

Me

H

F H

OH

O HO

Me

OH

O

O


Analysis of a Pain Reliever Formulation (Painaid, Zee Medical, Inc.)

Column: Acclaim Mixed-Mode WAX-1, 5 µm

Dimensions: 150 mm × 4.6 mm i. d.


monophosphate and 0.5 g

diphosphate in 1000 g DI H2O, pH 6

with NaOH)

Temperature: 30 °C

Flow rate: 1 mL/min

Inj. volume: 1 µL


Peaks: 1. Caffeine

2. Acetaminophen

3. Salicylamide

4. Aspirin

5. Salicylic acid Sample Preparation:

1. A 500-mg tablet is ground into fine powder

2. 400 mg powder dissolved in 40 g of DI water

3. After sonicating for 5 min, the resulting suspension was

filtered with 0.45-μm membrane filter

4. Dilute the filtrate 4-times with DI water

0 10 20

Minutes

1

2

3

4 AU

O O

OH O N

N N

N

O

O

HN

O

OH

OH

NH2 O

OH

OH O

5


Acclaim Trinity Nanopolymer Silica Hybrid (NSH) Technology

Silica Bead

Nanopolymer Particle

Anion Exchange/Reversed-phase

Cation Exchange


Acclaim Trinity

Features:

• Mixed-mode retention mechanism: reversed-phase, anion exchange,

cation exchange, and HILIC

• Adjustable selectivity

• Simultaneous separation of basic, neutral, and acidic analytes

• Retains ionic and ionizable compounds without ion-pairing reagents

• Broad range of applications

• Key Pharmaceutical Applications:

• Determination of pharmaceutical counter ions (both anions and cations)

• Analysis of APIs and counter ions

• Separation of mixtures containing acidic, basic, and neutral analytes


Simultaneous Separation of Acidic and Basic APIs and Counter Ions

0 6 12 9 3

Minutes

1

2

3

4 5

6 7

8

9

10 11

12

13

14

15

16

mV

0

400

15

Column: Acclaim Trinity P1, 3 µm

Dimensions: 50 mm × 3 mm i. d.

Eluent: A : MeCN,

B : DI H2O,

C : 0.2 mol/L NH4OAc, pH 4

Gradient: Time -10 0 2 7 15

(min)

%A 60 60 60 10 10

%B 35 35 35 0 0

%C 5 5 5 90 90

Temperature: 30 °C


Inj. volume: 2 µL

Detection: Corona ultra (Gain = 100 pA;

Filter: med; Neb. Temp.: 30 °C)

Peaks: (50-100 mg/L)

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. Sulfate


Basic Drug and Counter Ion Analysis: Trimipramine Maleate



Eluent: 20/80 v/v MeCN/30 mmol/L

(total) NH4OAc, pH 5.2

Temperature: 30 °C


Inj. volume: 2 µL


Sample: Trimipramine maleate

(0.1 mg/mL in eluent)

Peaks: 1. Trimipramine

2. Maleate

0 2 4 3 1 5

Minutes

mA

U

1

2

0

200

t0


Analysis of APIs and Counter Ions in a Real Pharmaceutical Formulation



Eluent: A: MeCN

B: DI H2O

C: 0.2 mol/L NH4OAc, pH 4.1

Gradient: Time -4 0 0.1 1 4

(min)

A: 25 25 25 80 80

B: 65 65 65 0 0

C: 10 10 10 20 20

Temperature: 30 °C

Flow rate: 1 mL/min

Inj. volume: 2 µL


Sample: Advil Allergy and Sinus (OTC)

Peaks: 1. Pseudo-ephedrine

2. Chlorpheniramine

3. Maleate

4. Ibuprofen *Background subtraction applied Minutes

t0

0 2 4 3 1

0

600

mAU

3

4

1

2


Ionic Impurities in a Real Pharmaceutical Formulation

0.0 0.5 1.0 1.5 2.0 2.5

-2

0

2

4

6

8

10 pA Sodium

Chloride

Without

Spiking

0.1% Cl-

0.15% Cl-

3.0 3.5 4.0 4.5 5.0

min

Diclofenac

Column: Dionex Acclaim Trinity P1, 3 µm (50 mm × 3 mm i. d.)


Eluent: 75/25 (v/v) MeCN / 120 mmol/L ammonium acetate,

pH 4.8 (30 mmol/L total ionic strength)


CHARGED AEROSOL DETECTION (CAD)


Corona Charged Aerosol Detector

• Consistent response

• Response independent of chemical structure

• Excellent for relative measurement when

standards are unavailable (e.g.,

impurity/degradants)

• Excellent sensitivity

• Detects low to sub nanograms on column

• Mass sensitive (not concentration sensitive)

• Dynamic range

• Over 4 orders of magnitude from ng to µg

• Measure active ingredients and impurities

at <0.05% in a single analysis

• Broad applicability

• Universal detection of any non-

volatile molecule

• Superior reproducibility

• RSDs ~2%, even at low levels


1

2

3

4

5

6

7

89

101

2

3

4

5

6

7

89

10

1. Nebulization

2. N2 gas for Aerosol

3. Aerosol to Drying Tube

4. Large Drops to Waste

5. Evaporation

6. Charging of Nitrogen

7. Charges on Particle

8. Ion Trap

9. Electrometer Signal

10. Data Station

Corona Charged Aerosol Detectors



• Nebulization

The eluent from the HPLC column

enters the instrument where it is

nebulized with a gas, typically

nitrogen.

The spray is directed onto an

impactor which removes larger

droplets. Gas flow rate through

the whole system is about

4 L/min.

HPLC Eluent

Nebulizer & Impactor

Gas Inlet

Drain

Analyte Particles



• Nebulization

• Evaporation

The residual mobile phase is

evaporated resulting in analyte

particles. Particles size depends

on concentration in aerosol

droplets.

Drying Tube

Residual Mobile Phase

High analyte mass concentration with residual mobile phase

Low analyte mass concentration with residual mobile phase

High analyte mass concentration results in larger particles and lower

surface area to mass ratios

Low analyte mass concentration results in smaller particles and

higher surface area to mass ratios



• Nebulization

• Evaporation

• Particle Charging

Concurrent with sample

nebulisation, a second stream of

the nitrogen is split and this gas

becomes ionised by the high

voltage of the Corona charger.

Next the analyte particles and

ionised gas collide in a mixing

chamber where the charge is

transferred to the particle.

Corona Needle Secondary gas

stream positively charged by a high-voltage platinum corona

wire

Mixing Chamber Positive charge is

transferred to the analyte particles by

charged opposing secondary gas stream


Mixing Chamber

Dry Analyte Particles Charged Nitrogen Gas

Analyte Particles

with Charges

Charges remain on surface of analyte particles. Particles

remain intact and do not ionise.

The more surface area, the more charge that is carried by the

particle.



• Nebulization

• Evaporation

• Particle Charging

• Analyte Measurement

After passing by an ion trap to

remove excess ionised gas, the

charged particles impact with the

collector. The charge is then

transferred and measured by a

sensitive electrometer.

Collector Analyte particles

transfer their charge

Ion Trap Negatively charged ion trap

removes high mobility particles

Electrometer Charge is drawn off and measured by a

sensitive electrometer

Signal Out Signal is directly

proportional to quantity of analyte in sample


CAD – A Non-Linear Detection Method

• Charged Aerosol Detection is

not linear over the entire

dynamic range because of the

relationship between solute

concentration and resulting

particle size.

• Fundamental property of all

detection methods based on

nebulization (CAD and ELSD)

• Calibration behavior can be

regarded as linear over

1.5-2 orders of magnitude.

• A quadratic regression is

recommended.


CAD – A Non-Specific Detection Method for Non-Volatile Compounds


CAD – Broad Applicability

Any non-volatile compound responds on the Corona CAD independent of chemical characteristics.

High MW –

Neutral, non-polar –

Neutral, polar –

Acidic –

Basic –

Zwitterionic –

Albumin, Dextrin

Estradiol, Umbelliferone

Glucose, Fructose, Lactose, Sucrose

Salicylic Acid

Propranolol, Nortriptyline, Amitriptyline,

Caffeine, Homocysteine, Methionine

Analytes do not have to undergo ionization in order to be measured!


CAD – Comparison with RI and ELSD

150

200

250

300

350

400

450

500

550

600

650

700

750

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Charged Aerosol Detection

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Evaporative Light Scattering Detection

220

240

260

280

300

320

340

360

380

400

420

440

460

480

500

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

RI

ELSD

CAD

32 KDa

PEG Reagent Dimer

21.5%

9.1%

25.6%

RI

● Low sensitivity – large injection volume for detecting the dimer

● Sample overloading

● Poor separation and peak form

● Not compatible with gradient elution

ELSD

● Non-linear

● Lower sensitivity at low concentrations

● Significantly smaller value for the dimer

CAD

● More sensitiva than RI – better chromatography

● More linear than ELSD

● Slightly larger value for the dimer


System Setup For Inverse Gradients

• Requires exact timing of the inverse gradient (delay volume):

1 T. Górecki, F. Lynen, R. Scucs, P. Sandra, Universal response in liquid chromatography using charged aerosol detection, Anal. Chem. 2006, 78, 3186-3192.

COLUMN COMPARTMENT

Pump 1 Pump 2

AUTOSAMPLER

DETECTOR

CO

LU

MN

CO

LU

MN

COLUMN COMPARTMENT

Pump 1 Pump 2

AUTOSAMPLER

DETECTOR

CO

LU

MN

DGP


Inverse Gradient Effect on Chromatography

Minutes

1 2 3 4 0

Uniform response in charged aerosol detection

Gradient influence without compensation


Chromeleon Wizard for Inverse Gradients

http://www.youtube.com/watch?v=ytF-

kWz7cpY&feature=relmfu Video available on: Video available on:

http://www.youtube.com/watch?v=KvhuiCbniXc&list=UUsDLFLsTOAXIKf-

0bmFfGcg&index=1&feature=plcp

http://www.youtube.com/watch?v=ytF-kWz7cpY&feature=relmfu

http://www.youtube.com/watch?v=KvhuiCbniXc&list=UUsDLFLsTOAXIKf-0bmFfGcg&index=1&feature=plcp










USP Monographs Based on Thermo Scientific Dionex Columns

USP Column Packing

Designation

Thermo Scientific

Dionex Column

USP Monograph

L12 IonPac AS4A-SC Calcium gluconate (limit

of oxalate test)

L31 IonPac AS10 Irbesatan (testing limit of

azide)

L46 CarboPac PA1 Fluordeoxyglucose F18

Streptomycin sulfate

L46 IonPac AS14A Ademethionine disulfate

tosylate

L46 IonPac AS11 Topiramate (limit of

sulfamate and sulfate)

L46 OmniPac PAX100 Voriconazole (impurities)

L47 CarboPac MA1 Amikacine sulfate

Kanamycin sulfate


USP Monographs Based on Thermo Scientific Dionex Columns / 2

USP Column Packing

Designation

Thermo Scientific

Dionex Column

USP Monograph

L48 IonPac AS5 Ferumoxides (assay for

citrate)

L48 IonPac AS7 Risedronate sodium

L50 OmniPac PAX500 Erythromycin ointment

L50 OmniPac PAX100 Betadex sulfobutyl ether

sodium (limit of NaCL)

L53 IonPac CS14 Bethanechol chloride

L61 IonPac AS11 Magnesium carbonate

and citric acid for oral

solutions

Magnesium citrate for

oral solutions

Etidronate disodium

(limit of phosphite)


USP Monographs Based on Thermo Scientific Dionex Columns / 3

USP Column Packing

Designation

Thermo Scientific

Dionex Column

USP Monograph

L61 IonPac AS11 Enoxaparin sodium (free

sodium content)

L69 CarboPac PA20 Heparin sodium

L74 IonPac AS14A Ademethionine disulfate

tosylate (content of

sulfate)

L76 IonPac SCS-1 Cefepime hydrochloride

(limit of N-

methylpyrrolidone)

L77 IonPac CS17 Methacholine chloride

(test for acetylcholine)


ANALYSIS OF CARBOHYDRATES AND AMINO ACIDS


CarboPac™ Columns

Oligosaccharides CarboPac PA100

CarboPac PA200

Monosaccharides, including low-level

analyses

CarboPac PA10

CarboPac PA20

Monosaccharide alditols, mixtures of

mono- and di-saccharides and mono- and

di-saccharide alditols

CarboPac PA1 General purpose, wood sugars, Rhm/GalN

CarboPac MA1

Applications Column


• Produce and purify glycoprotein (therapeutic)

• Release and purify oligosaccharides for characterization

or QA

• Hydrolyze 5 h @ 100ºC (protein or oligosaccharide)

• Neutral sugars (Fuc, Gal, Glc, Man): 2 mol/L TFA

• Amino sugars (GalN, GlcN): 6 mol/L HCl

• Evaporate to dryness

• Re-suspend with internal standard

• Chromatograph (HPAE-PAD)

Therapeutic Protein Monosaccharide Analysis


Short Analysis Times with CarboPac PA20

Column: CarboPac PA20

Dimension: 150 mm × 3 mm i. d.

Eluent: 8-20 mmol/L NaOH


Detection: Pulsed amperometry,

Au electrode

Peaks: 1. Fucose

2. Galactosamine

3. Glucosamine

4. Galactose

5. Glucose

6. Mannose

0

100

0 2 4 6 8

Minutes

8 mmol/L

10 mmol/L

12 mmol/L

14 mmol/L

16 mmol/L

18 mmol/L

20 mmol/L

1 2 3 4 5 6

nA


Analysis of MAb Hydrolysates

5 µg Mab hydrolysate

+ AminoTrap

5 µg Mab hydrolysate;

No AminoTrap

Mix of 6, 100 pmol;

No AminoTrap

Sugar mix, 100 pmol

+ AminoTrap

200

0

375

0 5 10 15 20 25 30

nC

Minutes

3 4 1 2 5 6

1 2 3 4 5 6


Eluent: 16 mmol/L NaOH


Detection: IPAD, Au electrode

Peaks: 1. Fucose 2. Galactosamine 3. Glucosamine 4. Galactose 5. Glucose 6. Mannose


• Sialic Acids are a family of neuraminic acids

• “Sialic Acid” = N-acetylneuraminic acid = NANA = Neu5Ac

• Sialic acids are negatively charged at pH 7

• Separations are carried out on CarboPac PA10/PA20

• Hydrolysis conditions

• HCl - 0.1 mol/L, 80ºC, 1 h

• TFA - 0.1 mol/L, 80ºC, 1 h

• HOAc - 2.0 mol/L, 80ºC, 3 h

HPAE-PAD Sialic Acid Analysis


Separation of Sialic Acids


Eluent: A. 100 mmol/L NaOH

B. 100 mmol/L NaOH

1 mol/L NaOAc

0-10 min 7-30% B

Flow rate: 1 mL/min

Detection: Pulsed amperometry,

Au electrode

Inj. volume: 20 µL

Peaks: 1. Neu5Ac 200 pmol

2. KDN 200

3. Neu5Gc 200

0 2 4 6 8 10 12

Minutes

-5

30

nC

1

2

3


Sialic Acid Analysis of Bovine Fetuin


Dimension: 150 mm x 3 mm ID

Gradient: 20-200 mmol/L NaOAc in 0.1 mol/L

NaOH over 10 min


Detection: IPAD,

Disposable Au electrode

-150

0

250

Minutes

0 2 4 6 8 10 12 14 16 18 20

nC

NANA

NGNA

A

B

NANA

NGNA

B. NANA & NGNA standard

A. 0.1 mol/L HCl fetuin hydrolysate


• Separation of oligosaccharides

• CarboPac PA100 / PA200 columns, NaOAc gradient in NaOH

• Identification of separated oligosaccharides

• Comparison with known standards

• Collection for MALDI-TOF MS

• Treatment with exoglycosidases and observation of changes in

chromatographic behavior

Glycoprotein Oligosaccharide Analysis


Separation of Neutral and Sialylated Oligosaccharide Standards


Eluent: 0 to 250 mmol/L NaOAc over 110 min

in 100 mmol/L NaOH

Flow rate: 1 mL/min

Detector: PAD, Au electrode

Peaks: 1. Man3GlcNAc2, fucosylated

2. Man3GlcNAc2

3. Asialo, agalacto bi, core fuc

4. Asialo, agalacto bi

5. Asialo bi, core fuc

6. Asialo bi

7. Man9GlcNAc2

8, 9. Disialylated bi (reduced)

10, 11. Trisialylated tri (reduced)

12, 13. Tetrasialylated tri (reduced)

0 10 20 30 40 50 60 70

20

200

nA

Minutes

1

2

3

4

5

6

7

8 9

10

11

12 13


Separation of Tri- and Tetra-Sialylated Oligosaccharides

40 45 50 55 60 65 70

20

120

nA

Minutes

1

2 Column: CarboPac PA-100

Eluent: 0 to 250 mmol/L NaOAc over

110 min in 100 mmol/L NaOH

Flow rate: 1 mL/min Detector: PAD, Au electrode

NANA — Gal — GlcNAc — Man

14 1


14 1

Man — GlcNAc — GlcNAc

14 14

NANA — Gal — GlcNAc

3 14

1

13

14

Peak 1


3 14 1


14 1

Man — GlcNAc — GlcNAc

14 14

NANA — Gal — GlcNAc

3 14

1

13

14

Peak 2


N-Linked Oligosaccharides Derived from Human Polyclonal IgG – CarboPac PA100 vs. PA200

Column: CarboPac PA100/200 and guard

Eluent: NaOAc gradient in

100 mmol/L NaOH


Detection: Integrated pulsed amperometry,

Au electrode

Sample: PNGase-F digest of hIgG

nC

Minutes 0 5 10 15 20 25 30

20

150

PA100

PA200


Amino Acid Analysis with Direct Detection

• Separation on a pellicular anion exchanger

• Detection via IPAD

(Integrated Pulsed Amperometric Detection)


Waveform for Amino Acid Analysis

Time [s] Pot. [V] Integ.

0.00 – 0.20

0.04 – 0.20

0.05 – 0.05

0.11 – 0.05 Begin

0.12 + 0.28

0.41 + 0.28

0.42 – 0.05

0.56 – 0.05 End

0.57 – 2.00

0.58 – 2.00

0.59 + 0.60

0.60 – 0.20

E4 E3

0.0 0.1 0.2 0.3 0.4 0.5 0.6

Time (s)

0.8

0.4

0.0

- 0.4

- 0.8

- 1.2

- 1.6

- 2.0

Po

ten

tia

l (V

vs. A

g/A

gC

l)

E2

E5

E6

E1

Integration period


Chromatographic Conditions for Amino Acid Analysis (AAA-DirectTM)

Column: AminoPac PA10 with guard

55% crosslinked EVB/DVB substrate

(8.5 µm) agglomerated with 180 nm

nanobeads (30-40% crosslinked);

functional group: quaternary ammonium

Column dimensions: 250 mm × 2 mm i. d.

Exchange capacity: 60 µequiv (12 µequiv)

Eluent: NaOH / NaOAc gradient


Inj. volume: 1-25 µL

Solvent stability: 100%

pH stability: 0-14


A: 200 pmol Standard

B: Fetuin Hydrolysate

0

400

nC

5 10 15 20 25 30 35

Arg

OHLys

Lys

GalN

GlcN

Ala

Thr

Gly

Val

OHPro

Ser

Pro

Ile

Leu Met

NLe

His

Phe

Glu

Asp

(Cys)2

Tyr

0

0

300 GalN

GlcN

Arg Lys

Ala Thr

Val

Ser Pro

Ile

Leu

NLe

His

Phe

Glu Asp

Tyr

(Cys)2

Gly

Minutes

nC

5 10 15 20 25 30 35 0

40

40

Fetuin Hydrolysate


0 5 10 15 20 25 30

100

500

nC

1

2

3

4

5

6

7 12

8

9

10

11 13 14

15 16

18

17

19 20

Minutes

-2

-1.5

-1

-0.5

0

0.5

0 200 400 600

Time (ms)

Po

ten

tia

l (V

)

Integral

Peaks: 1. Ornithine

2. Lysine

3. Glutamine

4. Asparagine

5. Glucose

6. Alanine

7. Threonine

8. Glycine

9. Valine

10. Hydroxyproline

11. Serine

12. Proline

13. Isoleucine

14. Leucine

15. Methionine

16. Taurine

17. Histidine

18. Phenylalanine

19. Glutamate

20. Aspartate

Cell Culture Medium with Low Glucose Content (1:250 diluted)


• Analysis of counter ions on ion-exchange materials with high

chromatographic efficiencies

• Cation exchangers in combination with conductivity detection

as alternative to ODS phases and derivatization techniques for

amine analysis

• Direct determination of ionic drugs with their metabolites and

potential impurities

• High resolution anion exchange separations of carbohydrates

and related compounds derived from glycoproteins

• Innovative AAA by anion exchange chromatography with direct

detection

Conclusions


Do people

know about

it?

IEC has a great

future in the

pharmaceutical

industry

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