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The world leader in serving science

Matthew Neely

Pittcon™ Conference & Expo 2014

March 2-6, 2014

Pharmaceutical Analysis of API and Counter Ions in Complex Formulations in a Single Injection

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Outline

• Challenges in pharmaceutical analysis• Mixed-mode chromatography overview• Mixed-mode chromatography for pharmaceutical analysis

• API and counter ions

• Thermo Scientific™ Dionex ™ Corona™ Veo™ Charged Aerosol Detector• Near universal, mass sensitive detector for routine LC determinations of

any non-volatile and many semi-volatile analytes

• Ion-Count Solutions• Unmatched performance for counter ion analysis by dedicated column

technology, unique charged aerosol detection and established Thermo Scientific™ Dionex™ Ultimate™ 3000 HPLC technology

• Summary

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Challenges in Pharmaceutical Analysis

• Pharmaceutical molecules are highly diverse: hydrophobicity, charge, size, etc.

• Most drug candidates are highly polar; many have poor chromophores.• Reversed-phase columns (e.g. C18) – the “work horse”

• Not suited for highly hydrophilic analytes

• Hydrophilic interaction (HILIC)/normal phase (NP) – the solution?• Solubility challenge

• Sample matrix effect

• Method ruggedness

• Ion exchange chromatography – good for charged analytes• Lack of retention for neutral analytes

• Lack of selectivity for analytes with same charge

• Possible solution…

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Mixed-Mode Chromatography

• Definition• Hydrophobic interaction + ion-exchange interaction

• Benefits• Adjustable selectivity• Simplified mobile phase (no ion-pairing reagents)• Simultaneous separation of different types of analytes

• Types• Anion-exchange/reversed-phase (AEX/RP)• Cation-exchange/reversed-phase (CEX/RP)• Anion-exchange/cation-exchange/reversed-phase (AEX/CEX/RP)

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RP/IEX Bimodal Mixed-Mode Columns

Silica Gel Silica Gel Silica Gel

Ion-exchange Reversed-phase

Mixed beads Mixed ligands Single ligand(embedded)

Single ligand(tipped)

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RP/AEX/CEX Trimodal Mixed-Mode Columns

Cation-exchange

Anion-exchange

Mixed beads Single ligand(amphoteric)

Nanopolymer Silica Hybrid(NSH)

Silica Gel

CEX

RP/AEX

Silica GelSilica Gel

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Acclaim Trinity P1 and P2 Columns

Thermo Scientific™ Acclaim™ Trinity™ P1 Column• RP / WCX / WAX • Monovalent ions• Higher hydrophobicity

Acclaim Trinity P2 Column• HILIC / WCX / SAX• Multivalent ions• Higher hydrophilicity

Nanopolymer Silica Hybrid (NSH) technology

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Acclaim Trinity P1 vs. P2 Columns

Attributes Acclaim Trinity P1 Column Acclaim Trinity P2 Column

Column chemistry

Nanopolymer Silica Hybrid Technology (NSH)

Retention mechanism

RP/WAX/SCX HILIC/SAX/WCX

Counter ions High resolution for mono-valent ions. Some capability for multivalent ions

Most suitable for general screening of a broad range of ions (both mono- and multi-valents)

Simultaneous determination of API and counter ions

Generally good for both hydrophilic and hydrophobic APIs and respective counter ions

Good for hydrophilic APIs and counter ions

Other applications

Generally applicable to any applications that involve ionic analytes, including both hydrophobic and hydrophilic ionics, and hydrophobic neutrals

Potential solution for any hydrophilic molecules, neutral or ionic, such as sugars

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Challenge: API and Counter Ions

• Salt formation is important in drug development• 50% of all drugs are formulated as salt forms

• Challenges• RP: little or no retention for counter ion; little to adequate retention

for API• IEX: need both AEX and CEX columns• HILIC: limited use

• Solution• Acclaim Trinity P1 column– RP/AEX/CEX trimodal phase• Acclaim Trinity P2 column – HILIC/WCX/SAX trimodal phase• Designed for API and counter ion analysis

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Pharmaceutical-Related Anions and Cations – Trinity P1 Column

Column: Acclaim Trinity P1, 3 µmDimensions: 3.0 × 100 mm

Mobile Phase: 60/40 v/v CH3CN/20 mM (total) NH4OAc, pH5

Temperature: 30 °CFlow Rate: 0.5 mL/minInj. Volume: 2 µLDetection: Thermo Scientific™ Dionex™ Corona™ ultra™

(Gain = 100 pA; Filter = med; Neb Temp = 30°C)

Peaks: (50 to 100 ppm) 1. Choline 2. Tromethamine 3. Sodium 4. Potassium 5. Meglumine 6. Mesylate 7. Nitrate 8. Chloride 9. Bromide10. Iodide

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0 6 1293 15Minutes

mV

1

2

4

5 6

7 9 10

0

300

8

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Pharmaceutical-Related Anions and Cations – Trinity P2 Column

Column: Acclaim Trinity P2, 3 µmDimensions: 3.0 x 50 mmMobile Phase: D.I. water and 100 mM NH4OFm, pH 3.65Temperature: 30°CFlow Rate: 0.60 mL/minInj. Volume: 1 µLDetection: Corona VeoSamples: 0.02 – 0.10 mg/mL each in D.I. water

Peaks:1. Phosphate2. Sodium3. Potassium4. Chloride5. Malate6. Bromide7. Nitrate8. Citrate9. Fumarate10. Sulfate11. Magnesium12. Calcium

Gradient:

Time (min)

H2O 0.1 M Ammonium

formate, pH3.65

-8 90 10

0 90 10

1 90 10

11 0 100

15 0 1000 5 10 15

0.0

1.8pA

Minutes

1

2

3

4

5

6 7

8

9

10 11

12

12

t0

0.6 mL/min

2.0 mL/min

Column: Acclaim Trinity P1, 3 µmDimensions: 3.0 x 50 mm

Mobile Phase: 80/20 v/v CH3CN/20 mM (total) NH4OAc, pH5Temperature: 30 °CFlow Rate: 0.6 and 2.0 mL/minInj. Volume: 2.5 µLDetection: Corona ultra

(Gain = 100pA; Filter = med; Neb Temp = 30°C)

Sample: Na, Naproxen (0.2 mg/mL in mobile phase)Peaks:

1. Na+

2. Naproxen

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12

ONa

Minutes0 2 431

mV

1000

t0

1

0

Naproxen Sodium – Trinity P1 Column

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API and Counter-Ions in Adderall – Trinity P2 Column

Column: Acclaim Trinity P2, 3 µmDimensions: 3.0 x 50 mmLC System: UltiMate 3000 RSLC Mobile Phases: A: Acetonitrile

B: WaterC: 100 mM Ammonium formate, pH 3.65

Gradient: -8.0 0.0 0.5 5.0 10.0 12.0 %A 35 35 35 35 20 20 %B 59 59 59 0 0 0 %C 6 6 6 65 80 80Flow: 0.60 mL/minTemperature: 30 °CInjection: 5 µLDetector: Diode array, UV 254 nm

Corona Veo (evaporator 55 °C, data rate 5 Hz, filter 2 sec, power function 1.5)Blank subtracted baseline.

Sample: Standards in 100 mM acetic acid; equivalent to 200 µg/mL Adderall-XR

Peaks: 1. Aspartate 24 µg/mL2. Sodium

3. Saccharin 244. Amphetamine 1225. Sulfate26

0

20mAU

UV

0 2 4 6 8 10 12

0

20pA

Minutes

Charged aerosol

12

35

4

3

Adderall is indicated in ADHD treatment

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Why Use Mixed-Mode Columns?

• Retention• Highly hydrophilic analytes, e.g. pharmaceutical counterions

• Selectivity • Adjustable selectivity allows for easy method optimization

• Throughput• Determining analytes of various charges and hydrophobicity with one

single injection

• MS-compatibility • No need for ion-pairing reagent for hydrophilic charged analytes

• Method robustness• More resistant to sample matrix effect than HILIC

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Corona Veo Detector—The Next Generation Charged Aerosol Detector

• Uses the fastest growing and most rapidly adopted universal LC detection technology since diode array detection (aka. PDA or DAD)

• Provides unbiased detection for a wide variety of analyte classes

• New design incorporates unique, proprietary technologies only available from Thermo Fisher Scientific

A novel, near universal, mass sensitive detector for routine LC determinations of any non-volatile and many semi-volatile analytes

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Introduction to Charged Aerosol Detection

Comparison of Charged Aerosol Detection to UV and MS

• Used to quantitate any non-volatile and many semi-volatile analytes with LC

• Provides consistent analyte response independent of chemical structure and molecule size

• Neither a chromophore, nor the ability to ionize, is required for detection

• Dynamic range of over four orders of magnitude from a single injection (sub-ng to µg quantities on column)

• Mass sensitive detection – provides relative quantification without the need for reference standards

• Compatible with gradient conditions for HPLC, UHPLC and Micro LC

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Particle Charging for Charged Aerosol Detection

Mixing Chamber

Dry analyte particles

Charged nitrogen gas

Analyte particleswith charged

surface

• Charges remain on surface of analyte particles.

• Particles remain intact and do not ionize.

• The more surface area, the more charge is carried by the particle

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Corona Veo Detector – What's New ?

• Radically new FocusJet™ concentric nebulization system improves sensitivity and precision

• All new evaporation scheme widens the scope of applications to include low flow capabilities for capillary and micro LC, as well as UHPLC

• Usability and serviceability are enhanced by countless improvements, many of which came from our customers

This entirely new detector incorporates many design and performance improvements:

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Corona Veo Detector with FocusJet Nebulizer Technology• The advanced concentric

nebulizer design ensures a superior aerosol atomization pattern for uniform droplet formation

• Exchangeable design gives users direct access for improved serviceability and ease-of-use

• Accepts typical inlet fittings, including fingertight Thermo Scientific™ Dionex™ Viper™ capillary fingertight fittings

FocusJet Concentric Nebulizer

Viper Fingertight Fitting

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FocusJet Concentric Nebulization System

• The inlet liquid and nebulization gas streams coaxially form into a stable aerosol at the nebulizer tip

• Small droplets are transported upward into the heated evaporation sector

• Larger droplets fall and are expelled by a precision micro-pump

Coaxial N2 flow

Capillary Inlet

Aerosol

FocusJet Concentric Nebulizer Tip

Cross-flow Nebulizer Active drain pump

Aerosol

To Evaporation Sector

Gas In (N2) Inlet

Concentric Nebulizer

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Corona Veo SD/RS Detectors – Product Differences

Model / FeatureCorona Veo

DetectorCorona Veo RS Detector

Flow Rate Range 0.20 – 2.0 mL/min 0.01 – 2.0 mL/min

Evaporation Temp 35°C or 50°C Ambient +5°C to +100°C

Data Acquisition Rate Up to 100 Hz Up to 200 Hz with CM 7.x

Integrated Stream Switching Valve (SSV)

- 6-port, 2-position micro valve

Standalone Interface Integrated color LCD touch screen

Inlet Gas Pressure (Reqd) 4.8 - 5.5 bar (70-80 psig; 482 – 551 kPa)

Internal Gas Regulation Manual adjustment Electronic control

Liquid Waste Non-pressurized

Enhanced Linearity Via Power Function (User applied)

Analog Signal Output Optional Optional

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Electronic Gas Regulation – Corona Veo RS Detector

• Provides ultra-precise gas pressure to ensure stable particle flow

• Ensures optimum detector performance over the entire inlet flow range of the Corona Veo RS detector (0.01 – 2.0 mL/min)

• Analytical and Micro LC operation modes are selectable through remote control via the chromatography data system

Internal Electronic Gas Regulator

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Integrated Stream Switching– Corona Veo RS Detector

Stream Switching Valve (SSV)

• Externally accessible automated 6-port 2-position valve• Diverts flow to waste in event of gas or pump flow errors• Simplify system operations by enabling access to alternate devices

without the need to unplumb/replumb modules (UV, MS etc.)• Improve chromatography results for complex samples by allowing

elimination of harmful salts or collection of unretained peaks

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• User Selectable Power Function Value (PFV)• Increases the observed linear range of calibration data

• Acts to improve mass balance determinations

Power Function with Corona Veo Detectors

Extend the linear dynamic range using the Power Function

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Benefits of Charged Aerosol Detection

• Used to quantitate any non-volatile and many semi-volatile analytes at sub-nanogram levels with LC

• Consistent analyte response is independent of chemical structure and molecule size

• Neither a chromophore nor the ability to ionize is required for detection

• Dynamic range of over four orders of magnitude within a single injection (sub-ng to µg on column)

• Mass sensitive detection – provides relative quantification without the need for reference standards

• Compatible with gradient conditions for HPLC, UHPLC and Micro LC

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Corona Detector Summary

• Corona Veo Charged Aerosol detectors:• Provides consistent inter-analyte responses across a wide

range• Best frontline detector for weak and non-chromophore

analytes• Ideal replacement for older, less capable universal

detection technologies (RI, ELSD, etc.)• A complimentary detector to UV, Diode Array and MS• Expands applications potential for future assay

development• Improved linearity with power function

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What Do We Mean by IonCount Solution?

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Chromeleon 7.2: eWorkflows

1. Choose eWorkflow, select instrument, and click “Launch”

2. Enter number of samples and start position

3. Sequence is created and can be immediately run

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Predefined eWorflows for API and Counter Ion

• Purpose: API and counter ion analysis• Generic organic gradient with isocratic ion strength (can be

fine tuned for specific problems)• Resolving power for monovalent counter ions

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0

0,00

1,00

2,00

3,00

3,85

min

pA 1

2

34

5

6

Column: Acclaim Trinity P1, 3x50mm

System: UltiMate 3000 LPG SD

Mobile phase: A - H2O

B - ACN

C-200 mM NH4From, pH=3.65

Gradient: 0-14 min 5-85% B, 14-15 min 85% B, 15.0-15.5 min, 85-5% B, 15.5-22 min 5% B

0-22min 10% C

Flow rate: 700 L/min

Power Func.: 1.5

Temperature: 30 ºC

Injection: 1 µL

Detection: Corona Veo RS

Analytes: 1. Sodium

2. Potassium

3. Chloride

4. Bromide

5. Nitrate

6. Maleic Acid

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Predefined eWorflows for API and Counter Ion

• Purpose: API and counter ion analysis• Generic organic gradient with isocratic ion strength (can be

fine tuned for specific problems)• API (Cefazolin) with sodium as counter ion

Column: Acclaim Trinity P1, 3x50mm

System: UltiMate 3000 LPG SD

Mobile phase: A - H2O

B - ACN

C-200 mM NH4From, pH=3.65

Gradient: 0-14 min 5-85% B, 14-15 min 85% B, 15.0-15.5 min, 85-5% B, 15.5-22 min 5% B

0-22min 10% C

Flow rate: 700 L/min

Power Funct: 1.5

Temperature: 30 ºC

Injection: 1 µL

Detection: Corona Veo RS

Analytes: 1. Sodium

2. Cefazolin

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0

0,00

2,50

5,00

7,50

10,00

1

2

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Predefined eWorflows for Counter Ion Screening

• Purpose: Screening of a wide range of counter ions• Broad ionic strength gradient (10 to 100 mM)• Applicable to mono- and divalent ions

Column: Acclaim Trinity P2, 3x50mm

System: UltiMate 3000 LPG SD

Mobile phase: A - H2O

B - ACN

C-100 mM NH4From, pH=3.65

Gradient: 0-1 min 10% C, 1-11 min 10-100% C, 11-20 min 100% C, 20-21 100-10% C, 21-29 min 10% C

Flow rate: 600 L/min

Power Func.: 1.5

Temperature: 30 ºC

Injection: 1 µL

Detection: Corona Veo RS

Analytes: 1. Phosphate 8. Fumarate

2. Sodium 9. Sulfate

3. Potassium 10. Magnesium

4. Chloride 11. Calcium

5. Malae

6. Bromide

7. Nitrate

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0

0,00

1,00

2,00

3,00

4,00

min

pA

20131030-12ions-runs #8 12ions-PFV1.5 evap50degs CAD_1

1

2

3

4

56 7 8 9

10

11

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Summary

• Selectivity is the KEY• Mixed-mode chromatography addresses unmet challenges in

pharmaceutical analysis:• API and counter ion by RP/AEX/CEX trimodal columns (e.g. Acclaim

Trinity P1 and P2)• Unique Charged Aerosol Detector for consistent response of analytes

that are weak or non-chromophoric molecules• Unmatched performance for counter ion analysis by dedicated column

technology, unique charged aerosol detection and established UltiMate 3000 technology

• New IonCount complete solution for ease of use and quick methods development of new API and counter ion analysis

• Easy operation by predefined eWorkflows, Thermo Scientific™ Dionex™ Chromeleon™ 7.2 Chromatography Data System, and viper connection tubing

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Thank You!

OT70992_E 03/14S