Chromatography: Pharmaceutical Analysis of API and Counter Ions in Complex Formulations in a Single Injection
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The document discusses challenges in pharmaceutical analysis, specifically related to the detection and separation of active pharmaceutical ingredients (APIs) and counter ions using mixed-mode chromatography. It introduces the Thermo Scientific Dionex Corona Veo Charged Aerosol Detector, highlighting its advantages for quantifying various non-volatile analytes and its improvements over previous detection technologies. Additionally, it presents specific chromatography column setups, methodologies for method optimization, and predefined workflows for analyzing APIs and counter ions.
Chromatography: Pharmaceutical Analysis of API and Counter Ions in Complex Formulations in a Single Injection
- 1. 1 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
- 2. 2 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
- 3. 3 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…
- 4. 4 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)
- 5. 5 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)
- 6. 6 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
- 7. 7 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
- 8. 8 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
- 9. 9 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
- 10. 10 Pharmaceutical-Related Anions and Cations – Trinity P1 Column Column: Acclaim Trinity P1, 3 µm Dimensions: 3.0 × 100 mm Mobile Phase: 60/40 v/v CH3CN/20 mM (total) NH4OAc, pH5 Temperature: 30 °C Flow Rate: 0.5 mL/min Inj. Volume: 2 µL Detection: 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. Bromide 10. Iodide 3 0 6 1293 15 Minutes mV 1 2 4 5 6 7 9 10 0 300 8
- 11. 11 Pharmaceutical-Related Anions and Cations – Trinity P2 Column Column: Acclaim Trinity P2, 3 µm Dimensions: 3.0 x 50 mm Mobile Phase: D.I. water and 100 mM NH4OFm, pH 3.65 Temperature: 30°C Flow Rate: 0.60 mL/min Inj. Volume: 1 µL Detection: Corona Veo Samples: 0.02 – 0.10 mg/mL each in D.I. water Peaks: 1. Phosphate 2. Sodium 3. Potassium 4. Chloride 5. Malate 6. Bromide 7. Nitrate 8. Citrate 9. Fumarate 10. Sulfate 11. Magnesium 12. 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 0 100 0 5 10 15 0.0 1.8 pA Minutes 1 2 3 4 5 6 7 8 9 1011 12
- 12. 12 t0 0.6 mL/min 2.0 mL/min Column: Acclaim Trinity P1, 3 µm Dimensions: 3.0 x 50 mm Mobile Phase: 80/20 v/v CH3CN/20 mM (total) NH4OAc, pH5 Temperature: 30 °C Flow Rate: 0.6 and 2.0 mL/min Inj. Volume: 2.5 µL Detection: 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 21 1 2 ONa Minutes 0 2 431 mV 1000 t0 1 0 Naproxen Sodium – Trinity P1 Column
- 13. 13 API and Counter-Ions in Adderall – Trinity P2 Column Column: Acclaim Trinity P2, 3 µm Dimensions: 3.0 x 50 mm LC System: UltiMate 3000 RSLC Mobile Phases: A: Acetonitrile B: Water C: 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 80 Flow: 0.60 mL/min Temperature: 30 °C Injection: 5 µL Detector: 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/mL 2. Sodium 3. Saccharin 24 4. Amphetamine 122 5. Sulfate 26 0 20 mAU UV 0 2 4 6 8 10 12 0 20 pA Minutes Charged aerosol 1 2 3 5 4 3 Adderall is indicated in ADHD treatment
- 14. 14 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
- 15. 15 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
- 16. 16 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
- 17. 17 Particle Charging for Charged Aerosol Detection Mixing Chamber Dry analyte particles Charged nitrogen gas Analyte particles with 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
- 18. 18 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:
- 19. 19 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
- 20. 20 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 To Evaporation Sector Gas In (N2)Inlet Concentric Nebulizer
- 21. 21 Corona Veo SD/RS Detectors – Product Differences Model / Feature Corona Veo Detector Corona 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
- 22. 22 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
- 23. 23 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
- 24. 24 • 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
- 25. 25 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
- 26. 26 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
- 27. 27 What Do We Mean by IonCount Solution?
- 28. 28 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
- 29. 29 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 3 4 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
- 30. 30 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
- 31. 31 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 5 6 7 8 9 10 11
- 32. 32 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