HPLC FOR PHARMACEUTICALSCIENTISTS

Edited by

YURI KAZAKEVICHSeton Hall UniversitySouth Orange, New Jersey

ROSARIO LOBRUTTONovartis PharmaceuticalsEast Hanover, New Jersey

WILEY-INTERSCIENCE

A JOHN WILEY & SONS, INC., PUBLICATION

InnodataFile Attachment0470087943.jpg

HPLC FOR PHARMACEUTICAL SCIENTISTS

HPLC FOR PHARMACEUTICALSCIENTISTS

Edited by

YURI KAZAKEVICHSeton Hall UniversitySouth Orange, New Jersey

ROSARIO LOBRUTTONovartis PharmaceuticalsEast Hanover, New Jersey

WILEY-INTERSCIENCE

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2007 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

HPLC for pharmaceutical scientists / edited by Yuri Kazakevich, Rosario LoBrutto.p. cm.

Includes bibliographical references and index.ISBN-13: 978-0-471-68162-5 (cloth)ISBN-10: 0-471-68162-8 (cloth)1. High performance liquid chromatography. 2. Drugs–Analysis. 3. Clinical chemistry.

I. Kazekevich, Yuri. II. LoBrutto, Rosario.

RS189.5.H53H75 2007615′.1901–dc22

2006046395

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

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CONTENTS

PREFACE xxiCONTRIBUTORS xxv

PART I HPLC THEORY AND PRACTICE 1

1 Introduction 3Yuri Kazakevich and Rosario LoBrutto

1.1 Chromatography in the Pharmaceutical World, 31.2 Chromatographic Process, 41.3 Classification, 41.4 History of Discovery and Early Development

(1903–1933), 61.5 General Separation Process, 8

1.5.1 Modern HPLC Column, 91.5.2 HPLC System, 9

1.6 Types of HPLC, 101.6.1 Normal-Phase Chromatography (NP HPLC), 101.6.2 Reversed-Phase HPLC (RP HPLC or RPLC), 111.6.3 Ion-Exchange Chromatography (IEX), 131.6.4 Size-Exclusion Chromatography (SEC), 14

1.7 HPLC Descriptors (Vr, k, N, etc.), 151.7.1 Retention Volume, 151.7.2 Void Volume, 161.7.3 Retention Factor, 171.7.4 Selectivity, 18

v

1.7.5 Efficiency, 191.7.6 Resolution, 22

References, 23

2 HPLC Theory 25Yuri Kazakevich

2.1 Introduction, 252.2 Basic Chromatographic Descriptors, 262.3 Efficiency, 272.4 Resolution, 322.5 HPLC Retention, 342.6 Retention Mechanism, 352.7 General Column Mass Balance, 372.8 Partitioning Model, 392.9 Adsorption Model, 402.10 Total and Excess Adsorption, 412.11 Mass Balance in Adsorption Model, 422.12 Adsorption of the Eluent Components, 432.13 Void Volume Considerations, 472.14 Thermodynamic Relationships, 49

2.14.1 Effect of the Eluent Composition, 532.15 Adsorption-Partitioning Retention Mechanism, 542.16 Secondary Equilibria, 57

2.16.1 Inclusion of Secondary Equilibria in the Mass Balance, 58

2.16.2 Salt Effect, 622.17 Gradient Elution Principles, 672.18 Types of Analyte Interactions with the Stationary Phase, 692.19 Conclusion, 70References, 71

3 Stationary Phases 75Yuri Kazakevich and Rosario LoBrutto

3.1 Introduction, 753.2 Type of Packing Material (Porous, Nonporous, Monolithic), 773.3 Base Material (Silica, Zirconia, Alumina, Polymers), 773.4 Geometry, 80

3.4.1 Shape (Spherical/Irregular), 803.4.2 Particle Size Distribution, 803.4.3 Surface Area, 813.4.4 Pore Volume, 823.4.5 Surface Geometry, 84

3.5 Adsorbent Surface Chemistry, 853.5.1 Surface Chemistry of the Base Material, 85

vi CONTENTS

3.5.2 Silica, 863.5.3 Silica Hybrid, 883.5.4 Polymeric Packings, 893.5.5 Zirconia (Metal Oxides), 903.5.6 Porous Carbon (or Carbon-Coated Phases), 90

3.6 Surface of Chemically Modified Material, 913.6.1 Limits of Surface Modification, 933.6.2 Chemical Modification, 933.6.3 Types of Bonded Phases, 1013.6.4 Structure of the Bonded Layer, 1033.6.5 Density of Bonded Ligands, 1053.6.6 Residual Silanoles, 1103.6.7 Surface Area of Modified Adsorbent, 110

3.7 Polymer-Based Adsorbents, 1133.8 Stationary Phases for Chiral Separations, 115

3.8.1 Polysaccharide-Coated Phases, 1153.8.2 Pirkle-Type Phases, 1163.8.3 Protein Phases, 1163.8.4 Molecular Imprinted Polymers for Chiral

Separations, 1173.9 Columns, 118

3.9.1 Capillary/Monolithic/Packed Columns, 1183.9.2 Column Cleaning, 1263.9.3 Column Void Volume, 1283.9.4 Mass of Adsorbent in the Column, 130

References, 132

4 Reversed-Phase HPLC 139Rosario LoBrutto and Yuri Kazakevich

4.1 Introduction, 1394.2 Retention in Reversed-Phase HPLC, 1404.3 Stationary Phases for RPLC, 1424.4 Mobile Phases for RPLC, 145

4.4.1 Eluent Composition and Solvent Strength of the Mobile Phase, 146

4.4.2 Type of Organic Modifier, 1514.4.3 Selectivity as a Function of Type and Concentration

of Organic Composition, 1534.5 pH Effect on HPLC Separations, 158

4.5.1 Mobile-Phase pH. Practical Considerations, 1584.5.2 Analyte Ionization (Acids, Bases, Zwitterions), 1604.5.3 pKa and pKb Relationship, 1614.5.4 Retention of Ionizible Analytes in Reversed-Phase

HPLC, 161

CONTENTS vii

4.5.5 Case Studies: Effects of pH on Ionizable Analyte Retention, 166

4.5.6 Mobile-Phase pH, 1714.5.7 Analyte Dissociation Constants, 1794.5.8 Determination of Chromatographic pKa, 180

4.6 Effect of Organic Eluent Composition on Analyte Ionization, 1824.6.1 Effect of Organic Modifier on Basic Analyte pKa

Shift, 1824.6.2 Effect of Organic Modifier on Acidic Analyte pKa

Shift, 1864.7 Synergistic Effect of pH, Organic Eluent, and Temperature on

Ionizable Analyte Retention and Selectivity, 1894.8 Examples of Applying pH Shift and Analyte pKa Shift Rules, 1914.9 Effect of Temperature on Analyte Ionization, 1954.10 Ion-Interaction Chromatography, 197

4.10.1 Introduction, 1974.10.2 Double Layer Theory, 1984.10.3 Ion Pairs, 2004.10.4 Chaotropic Effect, 206

4.11 Concluding Remarks, 227References, 228

5 Normal-Phase HPLC 241Yong Liu and Anant Vailaya

5.1 Introduction, 2415.2 Theory of Retention in Normal-Phase Chromatography, 2415.3 Effect of Mobile Phase on Retention, 2455.4 Selectivity, 248

5.4.1 Effect of Analyte Structure, 2485.4.2 Types of Stationary Phases, 249

5.5 Applications, 2515.5.1 Analytes Prone to Hydrolysis, 2515.5.2 Extremely Hydrophobic Compounds, 2525.5.3 Separation of Isomers, 2535.5.4 Carbohydrates, 2565.5.5 Separation of Saturated/Unsaturated Compounds, 257

5.6 Conclusions, 257References, 257

6 Size-Exclusion Chromatography 263Yuri Kazakevich and Rosario LoBrutto

6.1 Separation of the Analyte Molecules by Their Size, 2636.2 Molecular Size and Molecular Weight, 266

viii CONTENTS

6.3 Separation Mechanism, 2676.4 Calibration, 2686.5 Columns, 2716.6 Molecular Weight Distribution, 2736.7 Effect of Eluent, 2746.8 Effect of Temperature, 2746.9 Detectors, 2756.10 Solving Mass Balance Issues, 2756.11 Aqueous SEC Applications, 276References, 278

7 LC/MS: Theory, Instrumentation, and Applications to Small Molecules 281Guodong Chen, Li-Kang Zhang, and Birendra N. Pramanik

7.1 Introduction, 2817.2 Ionization Methods and LC/MS Interfaces, 282

7.2.1 Ionization Methods, 2827.2.2 Historical View of Interfaces, 2867.2.3 Common Interfaces, 2887.2.4 Special Interfaces, 290

7.3 Mass Analyzers, 2917.3.1 Magnetic Sector, 2917.3.2 Quadrupole, 2927.3.3 Ion Trap, 2937.3.4 Time-of-Flight, 2947.3.5 FT-ICR, 2957.3.6 Tandem MS, 296

7.4 Role of Instrumental Parameters on Ionization Efficiency in LC/MS, 2997.4.1 Optimization of Ionization Settings, 2997.4.2 Effect of Flow Rate, 302

7.5 Effect of Mobile-Phase Composition on Ionization Efficiency in LC/MS, 3037.5.1 Choice of Solvents, 3037.5.2 Choice of Mobile-Phase Additives, 3037.5.3 Adduct Formation, 3047.5.4 Effect of Analyte Concentration, 3047.5.5 Selected Ion Monitoring and Multiple Reaction

Monitoring, 3057.6 MS Interpretation, 305

7.6.1 Molecular Weight and Empirical Formula Determination, 305

7.6.2 Fragmentation Pattern, 3137.7 Practical Applications, 315

CONTENTS ix

7.7.1 High-Throughput LC/MS for Combinatorial Chemistry, 315

7.7.2 Characterization of Impurities and Decomposition Products in Bulk Drug Substances, 317

7.7.3 Pharmacokinetic Studies of Drugs, 3257.7.4 Identification of Drug Metabolites, 332

7.8 Conclusions, 336Acknowledgment, 338References, 338

8 Method Development 347Rosario LoBrutto

8.1 Introduction, 3478.2 Types of Methods, 348

8.2.1 Key Raw Materials, 3488.2.2 Drug Substance (Active Pharmaceutical Ingredient), 3528.2.3 Drug Product, 3558.2.4 Achiral Versus Chiral Methods, 359

8.3 Defining the Method, 3608.4 Method Development Considerations, 361

8.4.1 Sample Properties, 3618.4.2 Detector Considerations, 3678.4.3 Solution Stability and Sample Preparation, 3698.4.4 Choice of Stationary Phase, 3738.4.5 Mobile-Phase Considerations, 3758.4.6 Gradient Separations, 381

8.5 Method Development Approaches, 3858.5.1 If Analyte Structure Is Known, 3858.5.2 If Method Is Being Developed for Separation of Active

and Unknown Component, 3878.5.3 Defining System Suitability, 3898.5.4 Case Study 1: Method Development for a Zwitterionic

Compound, 3918.5.5 Case Study 2: Influence of pH, Temperature, and Type

and Concentration of Solvent on the Retention and Selectivity of Acidic (Phenolic) Compounds, 396

8.5.6 Case Study 3: Method Development for a Diprotic Basic Compound, 405

8.5.7 Case Study 4: Structural Elucidation Employing a Deuterated Eluent, 426

8.6 Effect of pH on UV Absorbance, 4298.7 Analyte pKa—From an Analytical Chemist’s Perspective, 432

8.7.1 Aromatic Acids, 4328.7.2 Amines, 434

x CONTENTS

8.8 Reversed-Phase Versus Normal-Phase Separations, 4358.9 Instrument/System Considerations, 438

8.9.1 Column/System Backpressure, 4388.9.2 Column Inlet and Outlet Frits, 4398.9.3 Seals, 4408.9.4 Mobile-Phase Preparation, 4408.9.5 Guard Columns, 4418.9.6 Instrument/System Considerations (Concluding

Remarks), 4428.10 Column Testing (Stability and Selectivity), 442

8.10.1 Column Selectivity Testing, 4428.10.2 Column Stability Testing, 4458.10.3 Choice of Buffer Related to Bonded-Phase

Stability, 4488.11 Concluding Remarks, 451References, 452

9 Method Validation 455Rosario LoBrutto and Tarun Patel

9.1 Introduction, 4559.2 Validation Report, 4579.3 Revalidation, 4589.4 Assignment of Validation Parameters, 459

9.4.1 Accuracy, 4609.4.2 Precision, 4709.4.3 Linearity, 4719.4.4 LOD/LOQ, 4819.4.5 Relative Response Factors, 4849.4.6 Stability of Solution, 4859.4.7 Ruggedness/Robustness, 4869.4.8 Specificity, 4909.4.9 Forced Degradation Studies (Solid State and Solution)—

Drug Substance and Drug Product, 4919.5 Distinguishing Drug-Related and Non-Drug-Related

Degradation Products, 4959.5.1 Drug Product Stress, 497

9.6 Concluding Remarks, 499References, 499

10 Computer-Assisted HPLC and Knowledge Management 503Yuri Kazakevich, Michael McBrien, and Rosario LoBrutto

10.1 Introduction, 50310.2 Prediction of Retention and Simulation of Profiles, 504

CONTENTS xi

10.2.1 General Thermodynamic Basis, 50510.2.2 Structure–Retention Relationships, 506

10.3 Optimization of HPLC Methods, 50710.3.1 Off-Line Optimization, 50710.3.2 On-Line Optimization, 51010.3.3 Method Screening, 51110.3.4 Method Optimization, 512

10.4 Structure-Based Tools, 51710.4.1 Knowledge Management, 51710.4.2 Applications Databases, 51910.4.3 Structure-Based Prediction, 521

10.5 Conclusion, 528References, 529

PART II HPLC IN THE PHARMACEUTICAL INDUSTRY 533

11 The Expanding Role of HPLC in Drug Discovery 535Daniel B. Kassel

11.1 Introduction, 53511.2 Applications of HPLC/MS for Protein Identification and

Characterization, 53611.3 Applications of HPLC/MS/MS in Support of Protein

Chemistry, 53811.4 Applications of HPLC/MS/MS in Support of Assay

Development and Screening, 53911.5 Sources of Compounds for Biological Screening, 54011.6 HPLC/MS Analysis to Support Compound Characterization, 542

11.6.1 Purity Assessment of Compound Libraries, 54411.7 Purification Technologies for Drug Discovery, 547

11.7.1 UV-Directed Purification, 54811.7.2 Mass-Directed Preparative Purification, 549

11.8 Higher-Throughput Purification Strategies, 55211.8.1 Fluorous Split-Mix Library Synthesis and Prepartive

LC/MS De-Mixing, 55211.8.2 Parallel Analysis and Parallel Purification, 55311.8.3 Streamlining the Purification Process, 558

11.9 ADME Applications, 55911.10 Fast Serial ADME Analyses Incorporating LC-MS and

LC-MS/MS, 56111.10.1 Automated Data Processing Is Instrumental to

Achieving High-Throughput ADME, 56111.10.2 Enhancing Throughput by Incorporating Pooling

Strategies, 563

xii CONTENTS

11.11 Parallel Approaches to Speeding ADME Analyses, 56311.11.1 Nonindexed Parallel Mass Spectrometry, 56311.11.2 Indexed (“MUX”) Parallel Mass Spectrometry, 564

11.12 Automated “Intelligent” Metabolic Stability and Metabolite ID, 565

11.13 Conclusions, 568References, 569

12 Role of HPLC in Preformulation 577Irina Kazakevich

12.1 Introduction, 57712.2 Initial Physicochemical Characterization (Discovery

Support), 57912.2.1 Ionization Constant, pKa, 58012.2.2 Partition and Distribution Coefficients, 58212.2.3 Solubility and Solubilization, 586

12.3 Chemical Stability, 59012.4 Salt Selection, 59412.5 Polymorphism, 59412.6 Preformulation Late Stage (Development Support), 59612.7 Conclusions, 599References, 600

13 The Role of Liquid Chromatography–Mass Spectrometry in Pharmacokinetics and Drug Metabolism 605Ray Bakhtiar, Tapan K. Majumdar, and Francis L. S. Tse

13.1 Introduction, 60513.2 Ionization Processes, 60613.3 Tandem-Mass Spectrometry (MS/MS), 61013.4 Sample Preparation Using an Off-Line Approach, 611

13.4.1 SPE, 61213.4.2 PPT, 61313.4.3 LLE, 615

13.5 Automated Sample Transfer, 61513.6 Sample Processing Using an On-Line Approach, 61613.7 Matrix Effect and Ion Suppression, 61913.8 Regulatory Requirements for LC/MS Method Validation, 62013.9 Ritalin®: An Application of Enantioselective LC-MS/MS, 62413.10 GleevecTM (STI571), 62613.11 Biomarkers, 62913.12 Conclusions, 633Acknowledgments, 633References, 633

CONTENTS xiii

14 Role of HPLC in Process Development 641Richard Thompson and Rosario LoBrutto

14.1 Responsibilities of the Analytical Chemist During Process Development, 641

14.2 HPLC Separation Modes, 64314.2.1 Reversed-Phase Liquid Chromatography, 64314.2.2 Normal-Phase Chromatography, 64414.2.3 Sub-/Supercritical Chromatography, 64514.2.4 Hydrophilic Interaction Chromatography, 64714.2.5 Ion-Exchange Chromatography, 64914.2.6 Chiral Chromatography, 650

14.3 Sample Preparation, 65314.4 HPLC Detectors, 65414.5 Method Development, 65714.6 In-Process Monitoring, 66014.7 Impurity Identification, 66314.8 Establishment of HPLC Selectivity by Stress Studies, 665

14.8.1 Stability in Solution and Forced Degradation Studies (Process Intermediate Compound A), 666

14.9 HPLC Method Validation, 67014.9.1 Prevalidation and System Suitability, 67114.9.2 Validation, 672

14.10 Technology Transfer, 67314.11 Concluding Remarks, 674References, 674

15 Role of HPLC During Formulation Development 679Tarun S. Patel and Rosario LoBrutto

15.1 Introduction, 67915.2 Prerequisite for Analytical Chemists During Formulation

Development, 68115.2.1 Major Degradation Pathways in Pharmaceuticals, 681

15.3 Properties of Drug Substance, 68215.3.1 Solubility of Drug Substance in Presence of

Formulation, 68215.3.2 Solution Stability, 683

15.4 Properties of Excipients, 68315.5 Impact of Excipients on Degradation of API(s), 68415.6 Test Methods for Most Common Dosage Forms in which

HPLC Is the Primary Technique, 68615.6.1 Assay and Related Substances, 68715.6.2 Stability-Indicating Method (SIM), 688

15.7 Forced Decomposition, 69115.8 Compatibility of Excipients with API(s) (Type and Ratio), 695

xiv CONTENTS

15.9 Mass Balance, 69815.9.1 Case Study 1, 69815.9.2 Case Study 2, 70215.9.3 Detection Considerations, 70615.9.4 Mass Balance Concluding Remarks, 707

15.10 Summary of Assay and Related Substances, 70715.11 Uniformity of Dosage Units, 70715.12 Blend Uniformity (BU), 70815.13 Cleaning Verification, 70915.14 Extractables/Leachables, 71015.15 Dissolution, 71315.16 Method Development, 713

15.16.1 Sample Preparation Solvent, 71415.17 Method Validation, 714

15.17.1 Completeness of Extraction, 71415.18 Testing of Samples, 715

15.18.1 Clinical Release, 71515.18.2 Stability, 715

15.19 Automation Opportunities, 71815.20 Implementation of Alternative Technologies, 71915.21 Challenges and Future Trends, 720References, 720A15.1 Addendum (Common Functional Groups), 723

A15.1.1 Carbonyls, 724A15.1.2 Nitrogen Functional Groups, 728A15.1.3 Ethers, Thioethers, 730A15.1.4 Alkyl/Aryl Halides, 730A15.1.5 Hydroxyls, 731A15.1.6 Thiols, 731A15.1.7 Phenols, 731A15.1.8 Olefins, 731A15.1.9 Dimerization, 732A15.1.10 Ring Transformations, 733

Addendum References, 733

16 The Role of HPLC in Technical Transfer and Manufacturing 735Joseph Etse

16.1 Introduction, 73516.2 Prerequisites for Transfer of HPLC Methods, 736

16.2.1 Availability of Either Fully or Partially Validated Methods, 736

16.2.2 Availability of the Finalized Pharmaceutical Active Ingredient (API), Known Degradation Products,By-products and Reference Standards, 738

CONTENTS xv

16.2.3 Availability of Drug Products Made by the Definitive Manufacturing Process, 739

16.2.4 Availability of Suitable Instruments and Personnel, 73916.2.5 Availability of a Protocol Containing Predetermined

Acceptance Criteria, 74016.3 Types of Technical Transfer, 745

16.3.1 From Analytical Research and Development (AR&D) to Quality Control (QC) Lab of the Commercial Organization, 745

16.3.2 Transfer from AR&D to Another AR&D Organization, 747

16.3.3 Transfer from AR&D to Contract Research Organization (CRO), 748

16.4 Different Approaches for Technical Transfer and Manufacturing, 74816.4.1 Comparative Testing, 74816.4.2 Co-validation of Methods, 75016.4.3 Revalidation of Methods, 75016.4.4 Waiver of Transfer, 752

16.5 Potential Pitfalls During Technical Transfer and Manufacturing, 75316.5.1 Sample Handling, 75316.5.2 Sample Type and Number of Replicate

Determination, 75516.5.3 Time of Testing, 75616.5.4 Instrumental Issues, 75716.5.5 Column and Instrumental Issues, 75716.5.6 Differences in Chromatographic Data Acquisition

Systems, 75916.6 Conclusion, 760References, 760

PART III HYPHENATED TECHNIQUES AND SPECIALIZEDHPLC SEPARATIONS 763

17 Development of Fast HPLC Methods 765Anton D. Jerkovich and Richard V. Vivilecchia

17.1 Introduction, 76517.2 Basic Theory, 766

17.2.1 Resolution and Analysis Time, 76717.2.2 Plate Height and Band-Broadening, 76917.2.3 Flow Velocity and Column Backpressure, 773

17.3 Monolithic Columns, 77517.3.1 Physical Properties and Preparation of Monolithic

Columns, 775

xvi CONTENTS

17.3.2 Chromatographic Properties and Applications of Monolithic Columns, 776

17.4 Ultra-High-Pressure Liquid Chromatography, 77717.4.1 Instrument Considerations when Using Ultra-High

Pressures, 77917.4.2 Chromatographic Effects of Ultra-High Pressures, 78117.4.3 UHPLC Applications, 78317.4.4 Method Transfer Considerations, 785

17.5 Separations on Chips, 78617.6 Optimizing Gradient Separations for Speed, 788

17.6.1 Advantages of Gradient Chromatography, 78817.6.2 Optimizing Instrumental Factors, 78817.6.3 Basic Parameters Controlling Speed and Resolution, 790

17.7 Instrumental Requirements for Operating High-Efficiency Columns, 79817.7.1 Extra-column Band-Broadening, 79817.7.2 Detector Requirements, 80217.7.3 Injection Considerations, 80417.7.4 Geometric Scaling Relationships, 806

17.8 Conclusions, 807References, 807

18 Temperature as a Variable in Pharmaceutical Applications 811Roger M. Smith

18.1 The Influence of Temperature on Chromatography, 81118.2 Effects on Method Transferability and Reproducibility, 81218.3 Elevated Temperature and Pharmaceutical Separations, 813

18.3.1 Effect of Temperature on Selectivity, 81418.3.2 Effect of Temperature on Separation Efficiency, 81518.3.3 Other Temperature Effects, 81718.3.4 Applications of Elevated Temperatures, 817

18.4 Superheated Water Chromatography, 82118.4.1 Columns for Superheated Water Chromatography, 82318.4.2 Detectors in Superheated Water Chromatography, 82418.4.3 Pharmaceutical Applications of Superheated Water

Chromatography, 82418.6 Subambient Separations, 82618.7 Conclusion, 830References, 830

19 LC/MS Analysis of Proteins and Peptides in Drug Discovery 837Guodong Chen, Yan-Hui Liu, and Birendra N. Pramanik

19.1 Introduction, 83719.2 General Strategies for Analysis of Proteins/Peptides, 838

CONTENTS xvii

19.2.1 HPLC Methods in Proteins/Peptides, 83819.2.2 MS Methods for Protein Characterization, 843

19.3 Applications for Biotechnology Products and Drug Targets, 84519.3.1 Biotechnology Products Development, 84519.3.2 Protein Glycosylation and Phosphorylation, 86019.3.3 Microwave-Assisted Methods for Proteins/Peptides, 87119.3.4 Drug–Protein Interaction by Affinity-Based

HPLC/MS, 87719.3.5 Multidimensional HPLC in Proteomics, 87919.3.6 Characterization of Adenovirus Structural Proteins for

Gene Therapy, 88419.4 Conclusions, 890Acknowledgment, 890References, 890

20 LC-NMR Overview and Pharmaceutical Applications 901Maria Victoria Silva Elipe

20.1 Introduction, 90120.2 Historical Background of NMR, 902

20.2.1 Historical Development of NMR, 90220.2.2 Historical Development of LC-NMR, 904

20.3 LC-NMR, 90520.3.1 Introduction, 90520.3.2 Modes of Operation for LC-NMR, 90820.3.3 Other Analytical Separation Techniques Hyphenated

with NMR, 91420.3.4 Applications of LC-NMR, 916

20.4 LC-MS-NMR (or LC-NMR-MS or LC-NMR/MS), 91620.4.1 Introduction, 91620.4.2 Modes of Operation for LC-MS-NMR, 91720.4.3 Applications of LC-MS-NMR, 926

20.5 Conclusions, 926Acknowledgments, 927References, 927

21 Trends in Preparative HPLC 937Ernst Kuesters

21.1 Introduction, 93721.2 Method Development in Preparative HPLC, 939

21.2.1 Optimization of Selectivity, 94021.2.2 Scale-Up of Analytical Methods, 94121.2.3 Adsorption Isotherms and Their Determination, 946

21.3 Columns and Stationary Phases, 95121.3.1 Stationary Phases, 951

xviii CONTENTS

21.3.2 Particle Size, Shape, and Distribution, 95421.3.3 Columns and Packing Procedures, 954

21.4 Choice of Preparative LC Technology, 95521.4.1 Classical Batch Elution, 95621.4.2 Recycling Chromatography, 95621.4.3 Displacement Chromatography, 95921.4.4 Simulated Moving Bed Chromatography, 962

21.5 Detection Tools, 97521.5.1 On-Line HPLC Detection, 97521.5.2 Preparative HPLC-MS, 977

21.6 Conclusion, 978Acknowledgments, 980References, 980

22 Chiral Separations 987Nelu Grinberg, Thomas Burakowski, and Apryll M. Stalcup

22.1 Introduction, 98722.1.1 Enantiomers, Diastereomers, Racemates, 988

22.2 Separation of Enantiomers Through the Formation of Diastereomers, 98922.2.1 Mechanism of Separation, 99022.2.2 General Concepts for Derivatization of Functional

Groups, 99122.3 Molecular Interactions, 992

22.3.1 The Probability of Molecular Interactions, 99222.3.2 The Types of Molecular Interactions, 99522.3.3 Chiral Separation Through Hydrogen Bonding, 99522.3.4 Chiral Separation Through Inclusion Compounds, 100222.3.5 Charge Transfer, 1011

22.4 Mixed Types of Interaction, 101822.4.1 Polysaccharide Phases, 101922.4.2 Antibiotic Phases, 102222.4.3 Protein Phases, 1026

22.5 Ligand Exchange, 103022.6 Chiral Mobile Phases, 1032

22.6.1 Chiral Mobile-Phase Retention Mechanisms, 103222.6.2 Selectivity with Chiral Mobile-Phase Additives, 103522.6.3 Chiral Additives with Chiral Stationary Phases, 103522.6.4 Interactions with Chiral Mobile Phases, 1037

22.7 Method Development for Chiral Separation, 103822.8 Concluding Remarks, 1040References, 1041

CHEMICAL AND DRUG COMPOUND INDEX 1053SUBJECT INDEX 1061

CONTENTS xix

PREFACE

In the modern pharmaceutical industry, HPLC is a major analytical toolapplied at all stages of drug discovery, development and production. Fast andeffective development of rugged analytical HPLC methods is more efficientlyundertaken with a thorough understanding of HPLC principles, theory andinstrumentation. The main focus of this book is reversed-phase analysis of small molecules, although there is some attention given to LC-MS of proteins, LC-NMR, ion-exchange, size exclusion, and normal phase chromatography.

The drug discovery and development process has undergone dramaticchanges particularly in the last decade. The process continues to evolve inresponse to new discoveries, new technologies, and increasing demand to get more drugs to the market more efficiently. Progress in drug discovery has been fueled by improvements in methodologies and technologies in-cluding automated high performance liquid chromatography (HPLC), fastHPLC, automated method development, HPLC-MS (mass spectrometry),HPLC-NMR (nuclear magnetic resonance) and high-throughput purificationmethods.

This book is unique in the sense that it elucidates the role of HPLC throughout the entire drug development process from drug candidate incep-tion to marketed drug product. It is written in a manner that scientists at alllevels of experience with HPLC will be able to find utility while maintaininga reasonable and manageable volume. The book covers the main theoreticaland practical aspects of modern HPLC at a level that is suitable for graduatestudents and chromatography practitioners in industry. In addition, for themore seasoned chromatographer, a description of the specifics of HPLC appli-cations at different stages of drug development and the latest advancements

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in fast, preparative, chiral and other modern LC techniques are included. Theinformation and discussions in this book are meant to increase the chro-matographer’s awareness of trends in HPLC technology with emphasis ontheir utilization in the various aspects of drug development. Researchers areprovided with the opportunity to better understand the use of HPLC not onlyin their respective “development silos,” but also throughout their organization.Theoretical background as well as practical and pragmatic approaches andactual examples of effective development of selective and rugged HPLCmethods from a physico-chemical point of view are provided throughout this book.

The contents, format and organization herein were inspired by the HPLCshort courses and graduate classes we have taught on separation science to adiverse population of pharmaceutical chemist drawn from all areas of drugdevelopment. We have observed a desire for a better understanding of work-flows in the various areas of drug development and how HPLC is integratedand embedded into these processes. The book is formatted to address themajor functions and tasks in which HPLC is applied.

Even though, there is no “cookbook” for HPLC method development thisbook provides several strategies that the reader could use when presented with a particular situation. These strategies could be stored as tools in the scientists’ “method development arsenal,” and drawn from when needed totackle a particular separation. Moreover, some novel approaches for imple-menting HPLC, fast HPLC, and hyphenated HPLC techniques towards phar-maceutical analysis are discussed. This book has the potential to serve as auseful resource for the chromatographic community. It can be used as a hand-book for the novice as well as the more experienced pharmaceutical chemistwho utilizes HPLC as an analytical tool to solve challenging problems regu-larly in the pharmaceutical industry.

The completion of this book could not have been possible without the help,inspiration and encouragement from many people.We are very grateful to ourfamilies for their understanding and support throughout the entire process of writing and editing. Also, we would like to thank our colleagues, students,friends, and peers for their helpful discussions and contributions to this work.Of special note, we would like to thank Alan Jones, Alexey Makarov, EvanO’Neill, Li Pan, Rajinder Singh, Fred Chan and Richard Vivilecchia. Weexpress our special gratitude to Dr. Harold McNair for his kind support, guid-ance and mentorship over the years and his continued inspiration in our on-going endeavors. We would also like to give special thanks to our wives,Ginevra LoBrutto and Irina Kazakevich, for all their support, patience, under-standing and encouragement. They have been an integral factor in allowing usto accomplish this contribution to the chromatographic and pharmaceuticalcommunity. Also, we would like to acknowledge all contributing authors whohave done an excellent job in writing their respective chapters, thus allowingfor facile integration of their topics into the framework of this book. We really

xxii PREFACE

enjoyed the many fruitful discussions with the contributors and duly acknowl-edge their dedication, efforts and commitment to this work.This has been trulya team effort and we believe the chromatographic community will appreciatethe contents and discussions provided within this book.

PREFACE xxiii

CONTRIBUTORS

Ray Bakhtiar, Department of Drug Metabolism, Merck Research Laborato-ries, Rahway, NJ 07065

Thomas Burakowski, R & D Chemical Development, Process DevelopmentLaboratory, Boehringer Ingelheim, Ridgefield, CT 06877

Guodong Chen, Schering-Plough Research Institute, Kenilworth, NJ 07033

Maria Victoria Silva Elipe, Department of Analytical Sciences, Amgen, Inc.,Thousand Oaks, CA 91320

Joseph Etse, Pharmaceutical and Analytical Research and Development,Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936

Nelu Grinberg, Chemical Development, Boehringer Ingelheim Pharmaceuti-cal, Inc., Ridgefield, CT 06877

Anton D. Jerkovich, Novartis Pharmaceuticals Corporation, East Hanover,NJ 07936

Daniel B. Kassel, Takeda Inc., San Diego, CA 92121

Irina Kazakevich, Schering-Plough Research Institute, Kenilworth, NJ 07033

Yuri Kazakevich, Department of Chemistry and Biochemistry, Seton HallUniversity, South Orange, NJ 07079

Ernst Kuesters, Chemical and Analytical Development, Novartis Pharma AG,CH-4002 Basel, Switzerland

xxv

Yan-Hui Liu, Schering-Plough Research Institute, Kenilworth, NJ 07033

Yong Liu, Analytical Research, Merck Research Laboratories, Rahway, NJ07065

Rosario LoBrutto, Group Head, Pharmaceutical and Analytical Develop-ment, Novartis Pharmaceuticals, East Hanover, NJ 07936

Tapan K. Majumdar, Novartis Institute for Biomedical Research, One HealthPlaza, East Hanover, NJ 07936

Michael McBrien, Advanced Chemistry Development, Inc. (ACD/Labs),Toronto, Ontario M5H 3V9 Canada

Tarun S. Patel, Pharmaceutical and Analytical Development, Novartis Phar-maceuticals, East Hanover, NJ 07936

Birendra N. Pramanik, Schering-Plough Research Institute, Kenilworth, NJ07033

Roger M. Smith, Department of Chemistry, Loughborough University,Loughborough, Leics LE11 3TU, UK

Apryll M. Stalcup, Department of Chemistry, University of Cincinnati,Cincinnati, OH 45221

Richard Thompson, Analytical Research, Merck Research Laboratories,Rahway, NJ 07065

Francis L. S. Tse, Bioanalytics and Pharmacokinetics, Novartis Pharmaceuti-cals Corporation, East Hanover, NJ 07936

Anant Vailaya, Analytical Research, Merck Research Laboratories, Rahway,NJ 07065

Richard V. Vivilecchia, Novartis Pharmaceuticals Corporation, East Hanover,NJ 07936

Li-Kang Zhang, Schering-Plough Research Institute, Kenilworth, NJ 07033

xxvi CONTRIBUTORS

PART I

HPLC THEORY AND PRACTICE