HANDBOOK OF LC-MS BIOANALYSIS

HANDBOOK OF LC-MS BIOANALYSIS

Best Practices, Experimental Protocols,and Regulations

Edited by

WENKUI LI, PhDNovartis Institutes for BioMedical Research,East Hanover, NJ, USA

JIE ZHANG, PhDNovartis Institutes for BioMedical Research,East Hanover, NJ, USA

FRANCIS L.S. TSE, PhDNovartis Institutes for BioMedical Research,East Hanover, NJ, USA

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Library of Congress Cataloging-in-Publication Data:

Handbook of LC-MS bioanalysis : best practices, experimental protocols, and regulations / edited by Wenkui Li, Ph.D., Novartis Institutes forBioMedical Research, Jie Zhang, Ph.D., Novartis Institutes for BioMedical Research, Francis L.S. Tse, Ph.D., Novartis Institutes for BioMedicalResearch.

pages cmIncludes bibliographical references and index.ISBN 978-1-118-15924-8 (cloth)

1. Drugs–Spectra–Handbooks, manuals, etc. 2. Drugs–Analysis–Handbooks, manuals, etc. 3. Mass spectrometry–Handbooks, manuals, etc.4. Liquid chromatography–Handbooks, manuals, etc. I. Li, Wenkui, 1964- editor of compilation. II. Zhang, Jie, 1962- editor of compilation.III. Tse, Francis L. S., editor of compilation. IV. Title: Handbook of liquid chromatography-mass spectrometry bioanalysis.

RS189.5.S65H36 2013615.1′901–dc23

2013004581

Printed in the United States of America

ISBN: 9781118159248

10 9 8 7 6 5 4 3 2 1

CONTENTS

PREFACE xi

CONTRIBUTORS xiii

ABBREVIATIONS xvii

PART I OVERVIEW OF LC-MS BIOANALYSIS

1 Roles of LC-MS Bioanalysis in Drug Discovery, Development, andTherapeutic Drug Monitoring 3Steve Unger, Wenkui Li, Jimmy Flarakos, and Francis L.S. Tse

2 Overview: Fundamentals of a Bioanalytical Laboratory 15Shefali Patel, Qiangtao (Mike) Huang, Wenying Jian, Richard Edom, and Naidong Weng

3 International Regulations and Quality Standards of Bioanalysis 29Surendra K. Bansal

PART II CURRENT UNDERSTANDING OF LC-MSBIOANALYSIS-RELATED REGULATIONS

4 Current Regulations for Bioanalytical Method Validations 39Mark E. Arnold, Rafael E. Barrientos-Astigarraga, Fabio Garofolo, Shinobu Kudoh,Shrinivas S. Savale, Daniel Tang, Philip Timmerman, and Peter van Amsterdam

5 Current Understanding of Bioanalytical Assay Reproducibility:Incurred Sample Reanalysis, Incurred Sample Stability, and IncurredSample Accuracy 47Manish S. Yadav, Pranav S. Shrivastav, Theo de Boer, Jaap Wieling, and Puran Singhal

6 LC-MS Bioanalytical Method Transfer 65Zhongping (John) Lin, Wenkui Li, and Naidong Weng

v

vi CONTENTS

7 Metabolites in Safety Testing 71Ragu Ramanathan and Dil M. Ramanathan

8 A Comparison of FDA, EMA, ANVISA, and Others on Bioanalysis inSupport of Bioequivalence/ Bioavailability Studies 83Bradley Nash

9 A Comparison of the Guidance of FDA, OECD, EPA, and Others onGood Laboratory Practice 89J. Kirk Smith

10 Current Understanding of Bioanalysis Data Management and Trend ofRegulations on Data Management 97Zhongping (John) Lin, Michael Moyer, Jianing Zeng, Joe Rajarao, and Michael Hayes

11 Regulatory Inspection Trends and Findings of BioanalyticalLaboratories 109Frank Chow, Martin Yau, and Leon Lachman

PART III BEST PRACTICE IN LC-MS BIOANALYSIS

12 Assessment of Whole Blood Stability and Blood/Plasma Distributionof Drugs 129Iain Love, Graeme T. Smith, and Howard M. Hill

13 Best Practice in Biological Sample Collection, Processing, and Storagefor LC-MS in Bioanalysis of Drugs 139Maria Pawula, Glen Hawthorne, Graeme T. Smith, and Howard M. Hill

14 Best Practices in Biological Sample Preparation for LC-MS Bioanalysis 165Guowen Liu and Anne-Francoise Aubry

15 Best Practice in Liquid Chromatography for LC-MS Bioanalysis 185Steve Unger and Naidong Weng

16 Best Practice in Mass Spectrometry for LC-MS 205Richard B. van Breemen and Elizabeth M. Martinez

17 Use of Internal Standards in LC-MS Bioanalysis 217Aimin Tan and Kayode Awaiye

18 System Suitability in LC-MS Bioanalysis 229Chad Briscoe

19 Derivatization in LC-MS Bioanalysis 239Tomofumi Santa

20 Evaluation and Elimination of Matrix Effects in LC-MS Bioanalysis 249Bernd A. Bruenner and Christopher A. James

21 Evaluation and Elimination of Carryover and/or Contaminationin LC-MS Bioanalysis 259Howard M. Hill and Graeme T. Smith

CONTENTS vii

22 Automation in LC-MS Bioanalysis 275Joseph A. Tweed

23 LC-MS Bioanalysis of Drugs in Tissue Samples 297Hong Gao, Stacy Ho, and John Williams

24 LC-MS Bioanalysis of Drugs in Urine 307Allena J. Ji

25 LC-MS Bioanalysis of Unbound Drugs in Plasma and Serum 317Theo de Boer and Jaap Wieling

26 LC-MS Bioanalysis of Drugs in Bile 327Hong Gao and John Williams

27 LC-MS Bioanalysis of Intracellular Drugs 337Fagen Zhang and Michael J. Bartels

28 LC-MS Bioanalysis of Endogenous Compounds as Biomarkers 353Wenying Jian, Richard Edom, and Naidong Weng

29 LC-MS Bioanalysis of Drugs in Hemolyzed and Lipemic Samples 369Min Meng, Spencer Carter, and Patrick Bennett

30 Best Practices in LC-MS Method Development and Validation for DriedBlood Spots 379Jie Zhang, Tapan K. Majumdar, Jimmy Flarakos, and Francis L.S. Tse

31 LC-MS Method Development Strategies for Enhancing MassSpectrometric Detection 391Yuan-Qing Xia and Jeffrey D. Miller

32 LC-MS Bioanalysis-Related Statistics 403David Hoffman

33 Simultaneous LC-MS Quantitation and Metabolite Identificationin Drug Metabolism and Pharmacokinetics 417Patrick J. Rudewicz

PART IV REPRESENTATIVE GUIDELINES AND/OR EXPERIMENTALPROTOCOLS OF LC-MS BIOANALYSIS

34 LC-MS Bioanalysis of Ester Prodrugs and Other EsteraseLabile Molecules 431Wenkui Li, Yunlin Fu, Jimmy Flarakos, and Duxi Zhang

35 LC-MS Bioanalysis of Acyl Glucuronides 447Jin Zhou, Feng (Frank) Li, and Jeffery X. Duggan

36 Regulated Bioassay of N-Oxide Metabolites Using LC-MS: Dealingwith Potential Instability Issues 461Tapan K. Majumdar

viii CONTENTS

37 Hydrolysis of Phase II Conjugates for LC-MS Bioanalysis of TotalParent Drugs 471Laixin Wang, Weiwei Yuan, Scott Reuschel, and Min Meng

38 LC-MS Bioanalysis of Reactive Compounds 479Hermes Licea-Perez, Christopher A. Evans, and Yi (Eric) Yang

39 LC-MS Bioanalysis of Photosensitive and Oxidatively Labile Compounds 491Corey M. Ohnmacht

40 LC-MS Bioanalysis of Interconvertible Compounds 505Nico van de Merbel

41 LC-MS Bioanalysis of Chiral Compounds 519Naidong Weng

42 LC-MS Bioanalysis of Peptides and Polypeptides 535Hongyan Li and Christopher A. James

43 LC-MS Bioanalysis of Nucleosides 551Laixin Wang and Min Meng

44 LC-MS Bioanalysis of Nucleotides 559Sabine Cohen, Marie-Claude Gagnieu, Isabelle Lefebvre, and Jerome Guitton

45 LC-MS Bioanalysis of Steroids 573Jie Zhang and Frank Z. Stanczyk

46 LC-MS Bioanalysis of Liposomal Drugs and Lipids 591Troy Voelker and Roger Demers

47 LC-MS Bioanalysis of Proteins 601Ziping Yang, Wenkui Li, Harold T. Smith, and Francis L.S. Tse

48 LC-MS Bioanalysis of Oligonucleotides 607Michael G. Bartlett, Buyun Chen, and A. Cary McGinnis

49 LC-MS Bioanalysis of Platinum Drugs 629Troy Voelker and Min Meng

50 Microflow LC-MS for Quantitative Analysis of Drugs in Supportof Microsampling 639Heather Skor and Sadayappan V. Rahavendran

51 Quantification of Endogenous Analytes in Biofluids by a Combination ofLC-MS and Construction of Calibration Curves Using Stable-Isotopes asSurrogate Analytes with True Biological Control Matrices 649Wenlin Li, Lucinda Cohen, and Erick Kindt

Appendix 1 Body and Organ Weights and Physiological Parameters inLaboratory Animals and Humans 659

CONTENTS ix

Appendix 2 Anticoagulants Commonly Used in Blood Sample Collection 661

Appendix 3 Solvents and Reagents Commonly Used in LC-MS Bioanalysis 663

Appendix 4 Glossary of Terms Used in LC-MS Bioanalysis 665

INDEX 673

PREFACE

Bioanalysis is the most heavily regulated area within thediscipline of drug metabolism and pharmacokinetics, whichsupports a large sector of drug development. The healthauthorities have very specific requirements regarding qualityand integrity of bioanalytical results, and different customersusually have additional expectations on the performance ofbioanalytical assays.

Much has happened in recent years toward faster, cheaper,and better ways of providing quality bioanalytical results.Both the European Medicines Agency (EMA) and the USFood and Drug Administration (FDA) have renewed or arerenewing their guidances on bioanalytical method validation(e.g., 21July2011/EMEA/CHMP/EWP/192217/2009) withthe ultimate goal of improving the quality of bioanalyti-cal results. Novel approaches to bioanalytical method devel-opment as well as advents of new liquid chromatographic(LC) techniques and mass spectrometric (MS) instrumentshave been reported. Various automatic laboratory procedures,electronic laboratory notebooks, and data management sys-tems are now available. All of these culminate in a remarkableimprovement in the quality, speed, and cost-effectiveness ofbioanalytical work and contribute to the value we deliver tothe patient.

Given the rapid changes within the field of bioanalysisand also in the larger area of drug development in which weoperate, it is timely to conduct a broad overview of our dis-cipline. This book is the first comprehensive handbook forLC-MS bioanalysis and provides an update on all importantaspects of LC-MS bioanalysis of both small molecules andmacromolecules. It not only addresses the needs of the bio-analytical scientists on the pivotal projects but also featuresperspectives on some advanced and emerging technologiesincluding high-resolution mass spectrometry and dried bloodspot (DBS) microsampling.

The 51 chapters of the book are divided into four parts.Part I provides a comprehensive overview on the role ofLC-MS bioanalysis in drug discovery and development andtherapeutic drug monitoring (Chapter 1), the key elementsof a regulated bioanalytical laboratory (Chapter 2), and thecurrent international regulations and quality standards of bio-analysis (Chapter 3).

In Part II, the global regulations and quality standardsrelated to LC-MS bioanalysis are reviewed and compared.Chapter 4 highlights the current regulations governingbioanalytical method validations from a number of countriesand regions including Brazil (ANVISA), Canada, China,the European Union (EMA), India, Japan, and the UnitedStates (FDA). This is followed by two in-depth reviews onthe topics of assay reproducibility (Chapter 5) and methodtransfer (Chapter 6). Chapter 7 presents the current practicesand regulatory requirements on Metabolites in Safety Testing(MIST). The guidances of regulatory bodies worldwideon bioanalysis for bioequivalence (BE)/bioavailability(BA) studies are compared in Chapter 8, whereas Chapter 9concerns the specific topic of good laboratory practice (GLP)and its interpretation and application by different agencies,countries, and regions. Of special interest is the rapid evolve-ment of regulations on bioanalytical data management,which is discussed in Chapter 10. Chapter 11 concludes PartII by giving a detailed analysis of regulatory inspectionsincluding health authority expectation, reported inspectionaltrends, citations, and regulatory followup letters. RecentFDA 483 observations as well as other “hot topics” inbioanalysis compliance that have raised concerns about dataintegrity are reviewed. Applicable best practices in LC-MSbioanalysis are portrayed in Part III. From this section ofthe book, the reader will find sound scientific rationale andhelpful practical instructions on the assessment of whole

xi

xii PREFACE

blood stability and blood/plasma distribution (Chapter 12),and on biological sample collection, processing and storage(Chapter 13). Chapter 14 introduces various sample prepa-ration techniques for LC-MS bioanalysis, while the bestpractices in LC separation and MS detection are discussedin Chapters 15 and 16, respectively. A good bioanalyticalmethod must be sensitive, specific, selective, reproducible,high-throughput, and fundamentally robust. Many factorsthat can contribute to the success of an assay are reviewedincluding the choice of internal standard (Chapter 17),evaluation of system suitability (Chapter 18), sensitivityenhancement via derivatization of analyte(s) of interest(Chapter 19), evaluation and elimination of matrix effect(Chapter 20), evaluation and elimination of carryover and/orcontamination (Chapter 21), and robotic automation (Chap-ter 22). Chapters 23–29 describe the bioanalysis of drugs,biomarkers, and other analytes of interest in various body flu-ids and tissues, and Chapter 30 is devoted to DBS samplingand related bioanalytical issues. Chapter 31 offers someuseful strategies for enhancing MS detection, and Chapter32 shows the proper use of statistics as a tool for ensuringadequate method performance in LC-MS bioanalysis. Thesimultaneous quantitative and qualitative LC-MS bioanalysisof drugs and metabolites are discussed in Chapter 33.

Part IV aims to provide detailed instructions with repre-sentative experimental protocols for the LC-MS bioanalysis

of various types of drug molecules commonly encountered inthe bioanalytical laboratory today (Chapters 34–49). Chap-ter 50 describes a typical procedure using microflow LC-MSfor the quantitative analysis of drugs in support of microsam-pling. Finally, a protocol on the quantification of endogenousanalytes in biofluids without a true blank matrix is given inChapter 51.

Our purpose in committing to this project was to providescientists in industry, academia, and regulatory agencies withnot only all the “important points to consider” but also all“practical tricks to implement” in LC-MS bioanalysis of var-ious molecules according to current health authority regula-tions and industry practices. In this book, we are confidentthat we have accomplished our goal. The book representsa major undertaking, which would not have been possiblewithout the contributions of all the authors and the patienceof their families. We also thank the terrific editorial staff atJohn Wiley & Sons and give a special acknowledgment toMichael Leventhal, Associate Editor, and Robert Esposito,Associate Publisher, at John Wiley & Sons for their premiersupport of this project.

Wenkui Li, PhDJie Zhang, PhD

Francis L.S. Tse, PhD

CONTRIBUTORS

Arnold, Mark E., Bioanalytical Sciences, Bristol-MyersSquibb Co., Princeton, NJ, USA

Aubry, Anne-Francoise, Bioanalytical Sciences, Bristol-Myers Squibb Co., Princeton, NJ, USA

Awaiye, Kayode, Bioanalytical, BioPharma Services Inc.,Toronto, ON, Canada

Bansal, Surendra K., Bioanalytical Research & Develop-ment, Non-Clinical Safety, Hoffmann-La Roche Inc., Nut-ley, NJ, USA

Barrientos-Astigarraga, Rafael E., Magabi PesquisasClınicas e Farmaceuticas Ltda, Itaqui Itapevi, Sao Paulo,Brazil

Bartels, Michael J., Toxicology and EnvironmentalResearch & Consulting, The Dow Chemical Company,Midland, MI, USA

Bartlett, Michael G., Department of Pharmaceutical &Biomedical Sciences, College of Pharmacy, Universityof Georgia, Athens, GA, USA

Bennett, Patrick, Thermo Fisher Scientific, San Jose, CA,USA

Briscoe, Chad, US Bioanalysis, PRA International, Lenexa,KS, USA

Bruenner, Bernd A. Bioanalytical Sciences, Pharmacoki-netics & Drug Metabolism, Amgen Inc., Thousand Oaks,CA, USA

Carter, Spencer, Tandem Labs, a Labcorp Company, SaltLake City, UT, USA

Chen, Buyun, Department of Pharmaceutical & BiomedicalSciences, College of Pharmacy, University of Georgia,Athens, GA, USA

Chow, Frank, Lachman Consultant Services Inc., Westbury,NY, USA

Cohen, Lucinda, NJ Discovery Bioanalytical Group, Merck,Rahway, NJ, USA

Cohen, Sabine, Hospices Civils de Lyon, Laboratoirede biochimie-toxicologie, Centre Hospitalier Lyon-Sud,Pierre Benite, Cedex, France

de Boer, Theo, Analytical Biochemical Laboratory (ABL)B.V., W.A. Scholtenstraat 7, Assen, The Netherlands

Demers, Roger, Tandem Labs, a Labcorp Company, WestTrenton, NJ, USA

Duggan, Jeffrey X., Bioanalysis & Metabolic Profil-ing, DMPK, Boehringer-Ingelheim Pharmaceuticals Inc.,Ridgefield, CT, USA

Edom, Richard, Bioanalysis, Janssen Research & Develop-ment, Global Development Operations, Raritan, NJ, USA

Evans, Christopher A., PTS-DMPK Bioanalytical Sci-ence & Toxicokinetics (BST), GlaxoSmithKline, King ofPrussia, PA, USA

Flarakos, Jimmy, Drug Metabolism & Pharmacokinet-ics, Novartis Institutes for BioMedical Research, EastHanover, NJ, USA

Fu, Yunlin, Drug Metabolism & Pharmacokinetics, NovartisInstitutes for BioMedical Research, East Hanover, NJ,USA

xiii

xiv CONTRIBUTORS

Gagnieu, Marie-Claude, Hospices Civils de Lyon, Lab-oratoire de pharmacologie, Hopital E. Herriot, Lyon,France

Gao, Hong, Drug Metabolism & Pharmacokinetics, VertexPharmaceuticals Inc., Cambridge, MA, USA

Garofolo, Fabio, Bioanalytical, Algorithme Pharma Inc.,Laval (Montreal), Quebec, Canada

Guitton, Jerome, Hospices Civils de Lyon, Laboratoirede ciblage therapeutique en cancerologie, Centre Hos-pitalier Lyon-Sud, Pierre Benite, Cedex, France; Univer-site Claude Bernard Lyon I, Laboratoire de Toxicologie,ISPBL, Lyon, France

Hawthorne, Glen, Department of Bioanalysis, HuntingdonLife Sciences, Alconbury, Cambridgeshire, UK

Hayes, Michael, Drug Metabolism & Pharmacokinet-ics, Novartis Institutes for BioMedical Research, EastHanover, NJ, USA

Hill, Howard M., Pharmaceutical Development, Hunting-don Life Sciences, Alconbury, Cambridgeshire, UK

Ho, Stacy, Drug Metabolism & Pharmacokinetics, Preclini-cal Biosciences, Sanofi, Waltham, MA, USA

Hoffman, David, Early Development Biostatistics, Sanofi,Bridgewater, NJ, USA

Huang, Qingtao (Mike), Bioanalysis, Janssen Research &Development, Global Development Operations, Raritan,NJ, USA

James, Christopher A., Bioanalytical Sciences, Pharma-cokinetics & Drug Metabolism, Amgen Inc., ThousandOaks, CA, USA

Ji, Allena J., Clinical Specialty Lab, Genzyme, A SanofiCompany, Framingham, MA, USA

Jian, Wenying, Bioanalysis, Janssen Research & Develop-ment, Global Development Operations, Raritan, NJ, USA

Kindt, Erick, Pharmacokinetics, Dynamics & Metabolism,Pfizer Inc., San Diego, CA, USA

Kudoh, Shinobu, Pharmaceuticals & Life-sciences Divi-sion, Shimadzu Techno-Research, Inc., Nakagyo-ku,Kyoto-shi, Kyoto-fu, Japan

Lachman, Leon, Lachman Consultant Services, Inc., West-bury, NY, USA

Lefebvre, Isabelle, Institut des Biomolecules MaxMousseron, UMR 5247 CNRS-UM1-UM2, UniversiteMontpellier 2, Montpellier, Cedex, France

Li, Feng (Frank), Alliance Pharma Inc., Malvern, PA, USA

Li, Hongyan, Bioanalytical Sciences, Pharmacokinetics &Drug Metabolism, Amgen Inc, Thousand Oaks, CA, USA

Li, Wenkui, Drug Metabolism & Pharmacokinetics, Novar-tis Institutes for BioMedical Research, East Hanover, NJ,USA

Li, Wenlin (Wendy), Pharmacokinetics, Dynamics &Metabolism, Pfizer Inc., San Diego, CA, USA

Licea-Perez, Hermes, PTS-DMPK Bioanalytical Science &Toxicokinetics (BST), GlaxoSmithKline, King of Prussia,PA, USA

Lin, Zhongping (John), Bioanalytical Services, FrontageLaboratories Inc., Malvern, PA, USA

Liu, Guowen, Bioanalytical Sciences, Bristol-Myers SquibbCo., Princeton, NJ, USA

Love, Iain, Department of Bioanalysis, Huntingdon Life Sci-ences, Alconbury, Cambridgeshire, UK

Majumdar, Tapan K., Drug Metabolism & Pharmacoki-netics, Novartis Institutes for BioMedical Research, EastHanover, NJ, USA

Martinez, Elizabeth M., Department of Medicinal Chem-istry & Pharmacognosy, University of Illinois College ofPharmacy, Chicago, IL, USA

McGinnis, A. Cary, Department of Pharmaceutical &Biomedical Sciences, College of Pharmacy, Universityof Georgia, Athens, GA, USA

Meng, Min, Tandem Labs, a Labcorp Company, Salt LakeCity, UT, USA

Miller, Jeffrey D., Product Applications, AB Sciex, Fram-ingham, MA, USA

Moyer, Michael, Bioanalytical Services, Frontage Labora-tories Inc., Malvern, PA, USA

Nash, Bradley, Global Quality & Compliance, PPD, Rich-mond, VA, USA

Ohnmacht, Corey M., US Bioanalysis, PRA International,Lenexa, KS, USA

Patel, Shefali, Bioanalysis, Janssen Research & Develop-ment, Global Development Operations, Raritan, NJ, USA

Pawula, Maria, Department of Bioanalysis, HuntingdonLife Sciences, Alconbury, Cambridgeshire, UK

Rahavendran, Sadayappan V., Pharmacokinetics, Dynam-ics & Metabolism, Pfizer Inc., San Diego, CA, USA

Rajarao, Joe, Global Professional Services, IDBS, Bridge-water, NJ, USA

Ramanathan, Dil, Kean University, New Jersey Center forScience, Technology & Mathematics, Union, NJ, USA

CONTRIBUTORS xv

Ramanathan, Ragu, Drug Metabolism and Pharmacokinec-tics, QPS LLC, Newark, DE, USA

Reuschel, Scott, Tandem Labs, a Labcorp Company, SaltLake City, UT, USA

Rudewicz, Patrick J., Metabolism & Pharmacokinectics,Novartis Institutes for BioMedical Research, Emeryville,CA, USA

Santa, Tomofumi, Graduate School of Pharmaceutical Sci-ences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Savale, Shrinivas S., Bioevaluation Centre, Torrent Pharma-ceuticals Limited, Gandhinagar, Gujarat, India

Shrivastav, Pranav S., Department of Chemistry, School ofSciences, Gujarat University School of Science, Navrang-pura, Ahmedabad, Gujarat, India

Singhal, Puran, Bioanalytical, Alkem Laboratories Ltd,Navi Mumbai, Maharashtra, India

Skor, Heather, Applied Proteomics, Inc., San Diego, CA,USA

Smith, Graeme T., Department of Bioanalysis, HuntingdonLife Sciences, Alconbury, Cambridgeshire, UK

Smith, Harold T., Drug Metabolism & Pharmacokinet-ics, Novartis Institutes for BioMedical Research, EastHanover, NJ, USA

Smith, J. Kirk, Regulatory Affairs & Quality Systems,Smithers, Wareham, MA, USA

Stanczyk, Frank, Department of Obstetrics and Gyne-cology, Department of Preventive Medicine, Universityof Southern California Keck School of Medicine, LosAngeles, CA, USA

Tan, Aimin, Bioanalytical, BioPharma Services Inc.,Toronto, ON, Canada

Tang, Daniel, ICON Development Solutions APAC, Pudong,Shanghai, China

Timmerman, Philip, Bioanalysis, Janssen Research &Development, Division of Janssen Pharmaceutica N.V.,Beerse, Belgium

Tse, Francis L.S., Drug Metabolism & Pharmacokinet-ics, Novartis Institutes for BioMedical Research, EastHanover, NJ, USA

Tweed, Joseph A., Regulated Bioanalytical, Pharmacoki-netics, Dynamics & Metabolism, Pfizer Inc., Groton, CT,USA

Unger, Steve, Bioanalytical Sciences, Worldwide ClinicalTrials, Austin, TX, USA

van Amsterdam, Peter, Abbott Healthcare Products BV,1380 DA Weesp C. J. van Houtenlaan 36, The Netherlands.

van Breemen, Richard B., Department of Medicinal Chem-istry & Pharmacognosy, University of Illinois College ofPharmacy, Chicago, IL, USA

van de Merbel, Nico, Bioanalytical Laboratories, PRA Inter-national, Assen, The Netherlands

Voelker, Troy, Tandem Labs, a Labcorp Company, Salt LakeCity, UT, USA

Wang, Laixin, Tandem Labs, a Labcorp Company, Salt LakeCity, UT, USA

Weng, Naidong, Bioanalysis, Janssen Research & Develop-ment, Global Development Operations, Raritan, NJ, USA

Wieling, Jaap, QPS Netherlands BV, Groningen, TheNetherlands

Williams, John, Vertex Pharmaceuticals Inc., Cambridge,MA, USA

Xia, Yuan-Qing, Product Applications, AB Sciex, Framing-ham, MA, USA

Yadav, Manish S., Clinical Research & Bioanalysis, AlkemLaboratories Ltd, Navi Mumbai, Maharashtra, India

Yang, Yi (Eric), PTS-DMPK Bioanalytical Science &Toxicokinetics, GlaxoSmithKline, King of Prussia, PA,USA

Yang, Ziping, Drug Metabolism & Pharmacokinetics,Novartis Institutes for BioMedical Research, EastHanover, NJ, USA

Yau, Martin, Lachman Consultant Services, Inc., Westbury,NY, USA

Yuan, Weiwei, QPS LLC, Newark, DE, USA

Zeng, Jianing, Bioanalytical Sciences, Bristol-MyersSquibb Co., Princeton, NJ, USA

Zhang, Duxi, Department of Bioanalysis, WuXi AppTec(Suzhou) Co., Ltd., Suzhou, Jiangsu, China

Zhang, Fagen, Toxicology & Environmental Research &Consulting, The Dow Chemical Company, Midland, MI,USA

Zhang, Jie, Drug Metabolism & Pharmacokinetics, NovartisInstitutes for BioMedical Research, East Hanover, NJ,USA

Zhou, Jin, Drug Metabolism & Pharmacokinetics,Boehringer-Ingelheim Pharmaceuticals Inc., Ridgefield,CT, USA

ABBREVIATIONS

AAPS American association of pharmaceuticalscientists

AAS Atomic absorption spectroscopyAC Absolute carryoverAch AcetylcholineACUP Animal care and use protocolADC Antibody–drug conjugateADME Absorption, distribution, metabolism, and

excretionAFA Adaptive focused acousticsALQ/AULOQ Above the upper limit of quantificationANDA Abbreviated new drug applicationANVISA National health surveillance agency (in

Portuguese, Agencia Nacional deVigilancia Sanitaria)

AP Analytical procedureAPCI Atmospheric pressure chemical ionizationAPI Atmospheric pressure ionizationAPPI Atmospheric pressure photoionizationASE Accelerated solvent extractionASEAN Association of southeast asian nationsAUC Area under the curveBA BioavailabilityBDMA ButyldimethylamineBDMAB Butyldimethylammonium bicarbonateBE BioequivalenceBIMO Bioresearch monitoringBLQ/BLLOQ Below the lower limit of quantificationBNPP Bis(4-nitrophenyl)-phosphateBSA Bovine serum albuminCAD Charged aerosol detectionCAD Collision-activated disassociationCAPA Corrective and preventive actionCD Compact disc

CDC Centers for disease control and preventionCDSCO Central drugs standard control organizationCE Collision energyCFR Code of federal regulationsCID Collision-induced dissociationCL ClearanceCNS Central nervous systemCoA Certificate of analysisCOV Compensation voltageCPGM Compliance program guidance manualCR Concentration ratioCRO Contract research organizationCs Calibration standardCSF Cerebrospinal fluidCSI Captive spray ionizationCV Coefficient of variationCXP Collision exit potentialCZE Capillary zone electrophoresisDBS Dried blood spotDDI Drug–drug interactionDDTC DiethyldithiocarbamateDDVP 2,2-Dichlorovinyl dimethyl phosphateDHEA DehydroepiandrosteroneDHT DihydrotestosteroneDIFP/DFP Diisopropyl fluorophosphateDM Drug metabolismDMS Differential mobility spectrometryDMS Dried matrix spotDNA Deoxyribonucleic acidDns-Cl Dansyl chlorideDns-Hz Dansyl hydrazineDP Declustering potentialDPS Dried plasma spotDPX Disposable pipette extraction

xvii

xviii ABBREVIATIONS

DQ Design qualificationDTNB 5, 5’-Dithiobis-(2-nitrobenzoic acid)DTT DithiothreitolEB Endogenous baselineEBF European bioanalysis forumEDMS Electronic data management systemEDTA Ethylenediaminetetraacetic acidEFPIA European federation of pharmaceutical

industries associationsEHNA Erythro-9-(2-hydroxy-3-nonyl) adenineELN Electronic laboratory notebookELSD Evaporative light scattering detectionEMA European medicines agencyEP Entrance potentialEPA Environmental protection agencyESI Electrospray ionizationFAIMS Field-asymmetric waveform ion mobility

spectrometryFDA Food and drug administrationFIH First-in-humanFOIA Freedom of information actFP Focusing potentialFTICR Fourier transform ion cyclotron resonanceFWHM Full width at half maximumGAMP Good automated manufacturing practiceGBC Global bioanalytical consortiumGC-MS Gas chromatography-mass spectrometryGCP Good clinical practiceGLP Good laboratory practiceGMP Good manufacturing practiceGPhA Generic pharmaceutical associationHAA Hexylammonium acetateHCT HematocritHETP Height equivalent of a theoretical plateHFIP HexafluoroisopropanolHILIC Hydrophilic interaction liquid

chromatographyHMP 2-Hydrazino-1-methyl-pyridineHP 2-HydrazinopyridineHPFB Health products and food branchHPLC High pressure liquid chromatography or

high performance liquidchromatography

HRMS High resolution mass spectrometryHSA Human serum albuminHTLC High-turbulence liquid chromatographyIA ImmunoaffinityIACUC Institutional animal care and use

committeeICH International conference on harmonizationICP-MS Inductively coupled plasma–mass

spectrometryID Inner diameterIDMS Isotope dilution mass spectrometry

IEC Ion-exchange chromatographyIMS Ion mobility spectrometryIND Investigational new drugIP Ion-pairingIQ Installation qualificationIS/ISTD Internal standardISA Incurred sample accuracyISR Incurred sample reanalysis or incurred

sample reproducibilityISS Incurred sample stabilityIV IntravenousKFDA Korea food and drug administrationLC-MS Liquid chromatography–mass

spectrometryLC-MS/MS Liquid chromatography-tandem mass

spectrometryLIMS Laboratory information management

systemLLE Liquid–liquid extractionLLOQ Lower limit of quantificationLOD Limit of detectionLUV Large unilamellar vesicleMAD Multiple ascending doseMAX Mixed mode anion exchangeMCD Maximum concentration differenceMCX Mixed mode cation exchangeMD Method developmentMDF Mass defect filterMEPS Microextraction by packed sorbentMF Matrix factorMFC Microfluidic flow controlMHFW Ministry of health and family welfareMHLW Ministry of health, labour and welfareMHRA Medicines and healthcare products

regulatory agencyMIP Molecularly imprinted polymersMIST Metabolites in safety testingMLV Multilamellar vesicleMRM Multiple reaction monitoringMS Mass spectrometryMTBE Methyl tert-butyl etherMV Method validationMVS Multichannel verification systemMWCO Molecular weight cutoffNCCLS National committee for clinical laboratory

standardsNCE New chemical entityNDA New drug applicationNHS National health serviceNIH National institutes of healthNL Neutral lossNME New molecular entityNMR Nuclear magnetic resonanceNPLC Normal phase liquid chromatography

ABBREVIATIONS xix

NRTI Nucleoside reverse transcriptaseinhibitor

NSB Nonspecific bindingNSI Nanospray ionizationOC Oral contraceptiveOECD Organization for economic cooperation

and developmentOEM Original equipment manufacturerOOS Out-of-specificationOQ Operational qualificationORA Office of regulatory affairsOSI Office of scientific investigationsPB Protein bindingPBMC Peripheral blood mononuclear cellPBS Phosphate buffered salinePCT Pressure cycling technologyPCV Packed cell volumePD PharmacodynamicsPDA Photodiode arrayPDF Portable document formatPEEK Polyether ether ketonePEG Polyethylene glycolPGC Porous graphitic carbonPI Principal investigatorPK PharmacokineticsPM Preventive maintenancePMP Pressure monitoring pipettingPMSF Phenylmethylsulfonyl fluoridePNPA p-Nitrophenyl acetatePoC Proof of conceptPPE Protein precipitation extractionPPT Protein precipitationPQ Performance qualificationPTM Posttranslational modificationQA Quality assuranceQAS Quality assurance statementQAU Quality assurance unitQC Quality controlQqQ Triple quadrupoleQqQLIT Hybrid triple quadrupole-linear ion trapQqTOF Hybrid quadrupole time-of-flightQ-TOF Quadrupole time-of-flightRAD Radioactivity detectionRAM Restricted access materialRBC Red blood cellRC Relative carryoverRE RecoveryRED Rapid equilibrium dialysisRF Response factorRFID Radiofrequency identifierRIA Radioimmunoassay

RNA Ribonucleic acidRT Retention timeRT Room temperatureRT-qPCR Real-time reverse transcription polymerase

chain reactionSAD Single ascending doseSALLE Salting-out assisted LLESAX Strong anion ion exchangeSBSE Stir bar sorptive extractionSCX Strong cation ion exchangeSD Standard deviationSDMS Scientific data management systemSE Standard errorSFC Supercritical fluid chromatographySFDA State food and drug administrationSIL-IS Stable isotope labeled internal standardSIM Selected ion monitoringsiRNA Small interfering RNASLE Supported liquid extractionS/N Signal-to-noiseSOP Standard operating procedureSPE Solid phase extractionSPME Solid phase microextractionSRM Selected reaction monitoringSSBG Sex steroid binding globulinSTD StandardSUV Small unilamellar vesicleSV Separation voltageTADM Total aspirate and dispense monitoringTDM Therapeutic drug monitoringTEA TriethylamineTEAA Triethylammonium acetateTEAB Triethylammonium bicarbonateTFA Trifluoroacetic acidTGA Therapeutic goods administrationTHU TetrahydrouridineTIC Total ion chromatogramTK ToxicokineticsTMA Trimethyl ammoniumTPD Therapeutic products directorateTSCA Toxic substance control actTTFA ThenoyltrifluoroacetoneUHPLC Ultra high performance liquid

chromatographyULOQ Upper limit of quantificationUPLC Ultra Performance liquid chromatographyURS User requirement specificationUV UltravioletWAX Weak anion exchangeWBC White blood cellWHO World health organization

PART I

OVERVIEW OF LC-MS BIOANALYSIS

1ROLES OF LC-MS BIOANALYSIS IN DRUGDISCOVERY, DEVELOPMENT, ANDTHERAPEUTIC DRUG MONITORING

Steve Unger, Wenkui Li, Jimmy Flarakos, and Francis L.S. Tse

1.1 INTRODUCTION

Bioanalysis is a subdiscipline of analytical chemistry forthe quantitative measurement of xenobiotics (chemicallysynthesized or naturally extracted drug candidates and genet-ically produced biological molecules and their metabo-lites or post-translationally modified products) and biotics(macromolecules, proteins, DNA, large molecule drugs,metabolites) in biological systems. Many scientific decisionsregarding drug development are dependent upon the accuratequantification of drugs and endogenous components in bio-logical samples. Unlike its sister subdisciplines of analyticalchemistry such as drug substance and drug product analy-sis, one very unique feature of contemporary bioanalysis isthat its measurement target is always at very low concentra-tion levels, typically at low ng/ml concentration range andeven at pg/ml for highly potent medicines. It is this verylow concentration, compounded by coexisting endogenousor exogenous compounds with similar chemical structures tothe target analytes at a much higher concentration (typicallyat μg/ml to mg/ml range), that challenges bioanalytical sci-entists to accurately and definitively measure the analytes ofinterest.

Since its commercial introduction in the 1980s, liq-uid chromatography–mass spectrometry (LC-MS), or muchmore predominantly, tandem mass spectrometry (LC-MS/MS) has rapidly become standard instrumentation inany well-equipped bioanalytical laboratory. LC-MS is a

combination of the physicochemical separation capabilitiesof liquid chromatography (LC) and the mass (MS or MS/MS)separation/detection capabilities of mass spectrometry. InLC-MS bioanalysis, assay selectivity can be readily achievedby three stages of separation of the analyte(s) of inter-est from unwanted components in the biological matrix:(1) sample extraction (protein precipitation, liquid–liquidextraction, solid-phase extraction, etc.), (2) column chro-matography, and (3) tandem mass spectrometric detectionin selected reaction monitoring (SRM) or multiple reac-tion monitoring (MRM) mode. Nevertheless, many fac-tors, including matrix effect, ion suppression, and in-source breakdown of labile metabolites, can compro-mise the reliability of a LC-MS bioanalytical assay.These factors should be carefully evaluated during methoddevelopment.

The focus of LC-MS bioanalysis in the pharmaceuti-cal industry is to provide a quantitative measurement ofthe active drug and/or its metabolite(s) for the accurateassessment of pharmacokinetics, toxicokinetics, bioequiva-lence (BE), and exposure–response (pharmacokinetics/ phar-macodynamics) relationships (Figure 1.1). The quality ofthese studies, which are often used to support regulatoryfilings and other evaluations, is directly related to the con-duct of the underlying bioanalysis. Therefore, the appli-cation of best practices in bioanalytical method develop-ment, validation, and associated sample analysis is key toan effective discovery and development program leading to

Handbook of LC-MS Bioanalysis: Best Practices, Experimental Protocols, and Regulations, First Edition. Edited by Wenkui Li, Jie Zhang, and Francis L.S. Tse.C© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

3

4 ROLES OF LC-MS BIOANALYSIS IN DRUG DISCOVERY, DEVELOPMENT, AND THERAPEUTIC DRUG MONITORING

DiscoveryPhase

IIPhase

III

~12 years

PhaseI

Screen compound libraries for leads Estimate safety margins for a

few candidates

Safetyassessment

Phase IV and

beyond

Therapeutic drug monitoring

andBioequivalence

Determine safety and exposure in humans (SAD, MAD)

Human ADME studiesMeasure parent and active metabolites in plasma and urine

Tissue distribution

Test compounds for physico-chemical and

in vitro DMPK properties

Bioequivalence andbioavailability studies

for generic drugsAnimal ADME studies, including bioavailability studies

Food effect assessment

Studies in pediatricMeasure metabolites for DDI and safety

(MIST)Metabolic stability

Animal pharmacology or PK studies Refine PK-PD relationships

Fit-for-purpose bioanalysis of tissue samples Population PK

Monitoring of drug levels with narrow therapeutic-safety margins to ensure that either under or over-dosage is avoided

Definitive metabolic stability

Reproductive TOX studies (rats and rabbits)

Studies in renal and/or hepatic impaired patients

Biomarker assays for proof-of-concept

Photo TOX studies (rat and mouse)

Define NOAEL and MTD in GLP TOX studies (2 weeks, 4 weeks)

Long term TOX studies (13 weeks, 26 weeks, 39 weeks, 2 years)

FIGURE 1.1 A flowchart of drug discovery and development, and postapproval studies of drugswhere LC-MS bioanalysis plays important roles.

the successful registration and commercialization of a drugproduct.

1.2 LC-MS BIOANALYSIS IN DRUG DISCOVERY

Before the introduction of combinatorial chemistry, manydrug candidates came from natural products where an activecompound was isolated and its chemical structure was char-acterized using NMR, MS, IR, and derivatization or selectivedegradation chemistry. Screening entailed an assessment ofbioactivity and physicochemical data compared to knowndatabases. High-resolution mass spectrometry played a crit-ical role allowing molecular formula searches from accuratemass data. Similarly, spectral databases allowed positive con-firmation or class assessments. This process helped to ensurethat novel compounds were selected. Since the introductionof combinatorial chemistry 20 years ago, the analyst’s role inearly drug discovery has shifted to the development of highlyefficient LC-MS analytical methods to support quantitativeanalysis. The drug discovery process begins with compoundlibrary development and ends with the selection of preclini-cal drug candidates for preclinical safety assessment. LC-MSbioanalysis plays an important role throughout this process.

1.2.1 Structure-Activity Relationships fromHigh-Throughput Screening

High-throughput LC-MS assays can be employed for thedetermination of solubility, membrane permeability or trans-port, protein binding, and chemical and metabolic stabilityfor a large number of compounds that have been identified as“hits” (Janiszewski et al., 2008). Thousands of compoundsper year go through some or all of these screening procedures.The in vitro studies validate in silico assessments performedprior to synthesis and select compounds for moving forwardin development.

1.2.2 Structure—PK-PD Relationships

Selected compounds from high-throughput screeningare subsequently evaluated in pharmacology models forefficacy. Provided the targeted biochemistry is applicableto LC-MS analysis, high-throughput screening of potentialbiomarkers can be performed in pharmacology studies viaeither a targeted pathway or a metabolomics approach. Ifsuccessful, discovery biomarkers may be useful in preclin-ical and clinical studies. Simple examples include steroidbiomarkers such as testosterone or dihydrotestosterone for

LC-MS BIOANALYSIS IN PRECLINICAL DEVELOPMENT OF DRUGS 5

5-α-reductase inhibitors or estrogen for selective estrogenreceptor modulators.

Integration of drug metabolism and pharmacokinetics(DMPK), pharmacology, and biology studies in drug dis-covery can greatly accelerate an understanding of thepharmacokinetic–pharmacodynamic (PK-PD) relationshipsof lead compounds. The minimum effective dose observed inthe pharmacology model is validated from knowledge of drugand active metabolite levels at the target site and comparedwith in vitro efficacy. Compounds known to have in vitropotency but are devoid of in vivo activity are suspected ofhaving poor bioavailability (BA) or other DMPK proper-ties (transport to target site, rapid clearance, etc.). Alterna-tively, compounds with an unanticipated high in vivo activitymay have superior access to the site of action or form activemetabolites.

LC-MS has a fundamental role in the success of manyof these discovery studies. An appropriately designed, earlyin vitro study can determine intrinsic clearance in multiplespecies. In vitro assessments have improved our ability to pre-dict systemic clearance using intrinsic clearance. However,predicting volume of distribution and tissue concentrations isfar more difficult. Combinatorial approaches such as cassettedosing or coadministration of many compounds is one meansof quickly assessing penetration into target sites. Typically,∼20 compounds are coadministered, but as many as 100 havebeen attempted (Berman et al., 1997). The specificity of MSdetection allows one to simultaneously measure numerouscompounds in biofluids and tissues and rapidly screen drugcandidates for their ability to penetrate into the site of action(Wu et al., 2000).

1.2.3 Candidate Selection

Within a therapeutic program, a limited number of com-pounds may be investigated in greater detail as possi-ble preclinical drug candidates. These include assessmentsat various doses in the rodent and nonrodent toxicologyspecies. Defining the systemic and local exposures, refiningPK-PD models and exploring dose-proportionality areamong the objectives of this phase. Studies with both singleand multiple ascending doses may be undertaken in an effortto assess accumulation, induction and toxicity. Whereas a“generic” LC-MS assay may suffice in supporting these non-GLP assessments of drug properties, one needs to be aware ofthe potential pitfalls, including stability of parent and metabo-lites and matrix effects from unknown metabolites, endoge-nous components, and dosing vehicles such as polyethyleneglycols, a frequently used formulation for IV dosage.

As a drug candidate progresses further, translationalmedicine often will define biomarkers from pharmacologyor metabolomics studies that can be used in clinical trials.Over the past 15 years, there has been considerable progressin the use of LC-MS to measure small biochemicals and

peptides. The ability to use biomarkers as a surrogate end-point and to ensure a reliable PK-PD relationship is a com-mon strategy for most drug development programs.

1.3 LC-MS BIOANALYSIS IN PRECLINICALDEVELOPMENT OF DRUGS

1.3.1 Toxicokinetics

Drug safety assessment studies regulated under good labo-ratory practice (GLP) are an important part of the preclin-ical development activities. In a typical toxicology study,toxicokinetic evaluation is performed in order to ascertainadequate drug exposure in the study animals. To sup-port bioanalysis of toxicokinetic samples from the GLPstudies, generic LC-MS methods used during drug dis-covery may no longer be suitable. Modification of thegeneric method or redevelopment of the respective methodis often needed, followed by full assay validation accord-ing to the current regulatory guidance and industrial prac-tices (EMA, 2011; FDA 2001; Viswanathan et al., 2007).These requirements are implemented to ensure adequate sen-sitivity, selectivity, accuracy, precision, reproducibility, anda number of other performance related criteria for a givenmethod.

Preclinical toxicity studies typically employ a broad doserange that could result in a wide range of circulating con-centrations of the test compound. Test samples containinganalyte levels exceeding the upper limit of quantification(ULOQ) need to be diluted, a step that can sometimes intro-duce errors. On the other hand, the lower limit of quantifica-tion (LLOQ) must be established so that the assay is sensitiveenough to measure trough levels from the lowest dose, yet nottoo sensitive that background noise (false positives) in spec-imens collected from control animals is detected. A usefulrule-of-thumb is to set the LLOQ at ca. 5% of the anticipatedpeak concentration following the low dose, which shouldallow accurate analyte measurement for approximately fourhalf-lives.

Different strains of rats such as Sprague Dawley,Wistar Hannover, and Fischer are used in toxicology stud-ies. The LC-MS assay method should be validated usingthe matrix from the same strain. The beagle dog is gen-erally the default nonrodent species. Nonhuman primates,such as cynomolgus, rhesus, or marmoset monkeys, areoccasionally used. The most common use of nonhuman pri-mates is when assessing immunogenicity of large moleculedrugs or when the metabolic profiles of dogs differ signif-icantly from human. Drug metabolizing enzymes, such asaldehyde oxidase, can have pronounced differences acrossspecies. Matching metabolic profiles to human assures goodsafety coverage for all metabolites. When metabolism differsacross species, metabolism-mediated toxicity can result in

6 ROLES OF LC-MS BIOANALYSIS IN DRUG DISCOVERY, DEVELOPMENT, AND THERAPEUTIC DRUG MONITORING

sensitivity within one species relative to others. For thisreason, there may be a need to measure metabolites inGLP preclinical studies. Although metabolite measurementin those toxicokinetic (TK) samples might be exempt fromfull GLP compliance due to various reasons, for example,absence of purity certification of reference metabolites andlack of full validation of the intended LC-MS assays, caremust be taken to ensure the integrity of the results gener-ated. Often, an assay separate from the parent measurementmay be set up for the occasional metabolite quantification.New guidance requires that steady-state exposures of sig-nificant metabolites in all species are obtained (Andersonet al., 2010). Non-GLP or tiered assays allow these decisionsto be made without extensive validation of multiple assays(Viswanathan et al., 2007).

In parallel with clinical drug development is the continuedtesting of the compound in animal toxicology studies. Thisincludes extending the safety in primary toxicology animalswith longer study durations. Dose range-finding studies areconducted in preparation for the 2-year carcinogenicity stud-ies in mouse and rat. Phototoxicity studies are performed inmice. Reproductive toxicology is performed in rats and rab-bits. Bioanalytical assays need to be validated in these addi-tional species. Again, metabolites unique to these speciesneed to be considered.

The bioanalyst should be prepared to support LC-MSbioanalysis of tissue samples for certain programs. Exten-sive validation and stability determinations might be needed,sometimes for both parent drug and metabolites. Having astable isotope labelled internal standard can help avoid prob-lems such as differences in extraction recovery and com-pensate for variability due to sample processing, transferand analysis of study tissue samples. Homogenization priorto freezing is also preferred. Nevertheless, one can neverfully ensure consistent analysis from tissue samples sincethe spiked quality control (QC) samples cannot fully mimicthe incurred tissues. The most definitive approach would beto compare tissue results obtained using LC-MS to thosefrom LC analysis in a radiolabeled study.

1.3.2 Preclinical ADME and Tissue DistributionStudies in Animals

Preclinical studies to elucidate the absorption, distribution,metabolism, and excretion (ADME) of drug candidates areusually conducted before and during the clinical phase.Radiolabeled drug is often needed for the animal ADMEor tissue distribution (quantitative whole body autoradiogra-phy) studies, although with today’s LC-MS instrumentation,much information can be gathered without the use of radio-labeled isotopes. Parent drug absorption and elimination canbe readily assessed using LC-MS assays. Metabolites can bedetermined using LC-MS under unit or high resolution condi-tions. Blood-to-plasma partitioning and protein binding, once

done exclusively using radiolabeled drug can now be per-formed using highly sensitive LC-MS assays. The questionof whether radiolabeled mass-balance studies in laboratoryanimals are still needed today has generated much discussion(Obach et al., 2012; White et al., 2013). The advance inLC-MS technology was the catalyst for this change.

1.4 LC-MS BIOANALYSIS IN CLINICALDEVELOPMENT OF DRUGS

1.4.1 First-in-Human Studies

Upon successful completion of the preclinical safety assess-ment of drug candidates, the investigational new drug(IND) submission is prepared. Traditionally, first-in-human(FIH) studies have included separate single and multipleascending dose (SAD and MAD) studies. Today, adaptivestudies can include a combination of SAD and MAD. Toensure safety, a sufficiently low starting dose is selected,and the supporting bioanalytical assay usually requires anLLOQ much lower than that used in toxicology studies. Fora drug candidate with a wide safety margin, a bioanalyticalmethod with a similar dynamic range will be needed. Whileit might be difficult to obtain a full PK profile on the earli-est doses of an ascending dose study, a full PK profile willbe required when an efficacious dose is reached. In additionto defining the maximum tolerable dose and possibly bio-logical effect, the DMPK objectives in FIH studies includedefining drug absorption, dose proportionality, and systemicclearance. Metabolite profiling and measurement will alsobe conducted to make sure unique human metabolites do notexist and major circulating metabolites at or above 10% oftotal drug-related exposure at steady state are also present atcomparable or greater exposure levels in at least one of themain preclinical toxicology species (FDA, 2008).

A bioanalytical LC-MS method should be developed andvalidated prior to completion of the study protocol. Impor-tant information such as conditions for blood sample col-lection, plasma harvest, sample storage, and transfer mustalso be verified. If samples need to be stabilized becauseof the presence of labile parent or metabolites, the informa-tion should be provided well in advance so that the clinicalstaff can be properly trained in the required sample handlingprocedures.

The SAD/MAD study may also include an arm to studythe food effect (fasted vs. fed) on the BA of the drug. Somedrugs bind to food resulting in decreased absorption. In con-trast, food can stimulate bile acid secretion that helps todissolve less soluble drugs, making them more bioavailable.A bioanalytical LC-MS method should, therefore, be evalu-ated in both normal and lipemic plasma. The assay shouldbe insensitive to changes in phospholipid concentration, a

LC-MS BIOANALYSIS IN CLINICAL DEVELOPMENT OF DRUGS 7

common issue in electrospray ionization that requires atten-tion during method development and validation.

Drug concentrations in urine are also typically measuredto assess renal clearance. Unlike plasma, blood or serum,urine does not normally contain significant amounts of pro-teins and lipids. The lack of proteins and lipids in urinesamples can be associated with the issue of nonspecificbinding or container surface adsorption of drug molecules,especially those lipophilic and highly protein bound, in quan-titative analysis of urine samples. The issue is often evidencedby the unusually low extraction recovery of the analytes ofinterest and/or nonlinearity of the calibration curves or highlyvariable QC sample results. Quick identification and effectiveprevention of analyte loss due to nonspecific binding or con-tainer surface adsorption must be conducted by bioanalyticalscientists prior to the study so that the correct collection andstorage condition can be provided (Li et al., 2010).

1.4.2 Human ADME Studies

Comprehensive information on the ADME of a drug inhumans can be obtained from mass-balance studies using aradiolabeled compound, and this should be an early objectivein clinical drug development (Pellegatti, 2012). Informationon drug tissue distribution in rodents (e.g., rat) and the antic-ipated therapeutic dose are needed for planning a humanADME study. Some knowledge of the drug metabolismin vitro and in animals can help to select the position anddesired specific activity of the radiolabel. Quantitative wholebody autoradiography is a common tool for tissue distribu-tion studies. Disposition of radioactivity into specific organsis quantified and scaled to human. Dosimetry calculations areperformed to ensure safe radioactivity exposure limits in dos-ing of humans. Typically the maximum exposure limit is 1mSv (ICRP 103, 2007). Traditional ADME studies generallyuse liquid scintillation counting and doses of ∼100 μCi of14C labeled drug mixed with unlabeled drug. LC-MS for mea-suring unlabeled drug is often used in human ADME studiesto differentiate the parent compound from its metabolite(s).For studies employing microdoses (<100 μg) or doses of lowradioactivity (<1 μCi), accelerator mass spectrometry maybe needed to measure the 14C labeled drug (Garner, 2005),whereas high sensitivity LC-MS methods have been usedto determine unlabeled drug concentrations (Balani et al.,2005).

ADME studies, though limited by their single dose nature,do illuminate what is important to measure in toxicologyand clinical studies to satisfy Metabolites in Safety Testingrequirements (Anderson et al., 2010). Obach et al. (2012)have advocated deferring the cost of this study until afterproof of concept (POC) and relying on pharmacokineticinformation derived from nonradiolabeled studies, namelySAD and MAD. The risk of delaying the human ADMEstudy is that unique human metabolites may be uncovered

after POC. The surprise of having significant metabolitesfound late in drug development can expose a lack of safetycoverage or protection of intellectual property if the metabo-lite is active. The advancement of more powerful high res-olution mass spectrometry for metabolite identification inLC-MS bioanalysis of early stage study samples helps tomitigate the risk.

1.4.3 Human Drug–Drug Interaction Studies

A drug–drug interaction (DDI) is a situation where a drugaffects the activity or toxicity of another drug when both arecoadministered. Interactions can be found where saturableor inducible enzymes or transporters are expressed and playa role in the absorption and disposition of the drug. DDIcan increase or decrease the activity of the drug or a neweffect can be produced that neither produces on its own. Thisinteraction can occur between the drug to be developed andother concomitantly administered drugs, foods, or medicinalplants or herbs. During clinical development, DDI studiesare normally conducted for the drug candidate in healthyvolunteers or patients to confirm any significant observationsseen during in vitro DDI studies.

From the perspective of LC-MS bioanalysis, assay speci-ficity against the coadministered medicines and their signif-icant metabolites needs to be demonstrated. In the case ofmetabolites that are difficult to obtain, interference could bediscounted based upon MS detection (e.g., differing MW orMRM). On the other hand, possible interference due to drugcandidates and/or their major metabolites on the accuracyof determination of DDI compounds and their significantmetabolites must be checked to ensure the quality of LC-MSbioanalytical results for the DDI assessment.

1.4.4 Renal Impaired and Hepatic Impaired Studiesin Human

Kidney (or renal) failure is a medical condition in which thekidneys fail to adequately filter toxins and waste productsfrom the blood. Similarly, liver (or hepatic) failure is theinability of the liver to perform its synthetic and metabolicfunction as part of normal physiology. Either can be acuteor chronic. Drug elimination may occur by filtration in thekidney or metabolism in the liver. When impacted by dis-ease, drug accumulation can result in toxicity. Depending onthe properties of metabolism and excretion of a drug candi-date, clinical studies in renal impaired or hepatic impairedpatients need to be conducted. In addition to conventionalplasma samples, urine samples may be collected and ana-lyzed. Some drugs may be metabolically activated, resultingin idiosyncratic liver toxicities. Therefore, it is important tounderstand both the impact of an impaired liver on the normalpharmacokinetic properties of a drug as well as the potentialof a drug to impact liver function.

8 ROLES OF LC-MS BIOANALYSIS IN DRUG DISCOVERY, DEVELOPMENT, AND THERAPEUTIC DRUG MONITORING

From the perspective of LC-MS bioanalysis, assaydynamic range must be suitable to measure exposures fromany given dose, or assay integrity of sample dilution mustbe checked to ensure data integrity for samples with unex-pected high analyte concentrations due to the impaired liveror kidney function.

1.4.5 Phase II and Phase III Studies

Moving beyond preliminary safety studies to POC studiesis a milestone goal for clinical drug development. A suc-cessful program will demonstrate POC before the end ofphase II studies. Therefore, moving from healthy subjectsto the intended patient population is an important transi-tion. However, patients might take more medications or areunder treatment with drug combinations. With this regard,the robustness of the intended LC-MS assay should be val-idated free from possible interference of combination drugsand their metabolites.

Phase II and III studies are larger and more expensive. Inorder to support the bioanalysis of a large number of samplesfrom these large multicentered trials, automation is an impor-tant consideration. For long-term, multicentered studies, theassay must be rugged enough to ensure storage stability. Awell-planned stability assessment of drug candidate and itsmetabolite(s) of interest is critical as stability must cover allreported results. Any significant assay bias must also be wellcharacterized. The entire bioanalytical work is represented inthe new drug application (NDA) submission. This includestabular and written summaries of assay validation perfor-mance of both nonclinical and clinical assays. Given that thedevelopment process of a drug may last more than a decade,it is important to maintain institutional knowledge to avoidgaps at filing.

1.4.6 “Fit-for-Purpose” Biomarker MeasurementUsing LC-MS in Clinical Samples

As drug candidates progress through POC studies, there isgreat need for LC-MS assays to measure biomarkers in clini-cal studies. There are numerous examples, including steroids,lipids, nucleotides, and peptides, which are directly amenableto LC-MS. Due to the endogenous nature of biomarkers, bio-analysis of those compounds usually encounters a series ofchallenges in maintaining analyte integrity from collectionto analysis, achieving specificity, and obtaining sufficientsensitivity, especially when endogenous concentrations aredownregulated. Those challenges entail special considerationand meticulous experimental design in method development,validation, and study conduct. Among the four commonapproaches to the preparation of standards, i.e. (1) authenticanalyte in authentic matrix, (2) authentic analyte in surro-gate matrix, (3) surrogate analyte in authentic matrix, and

(4) charcoal or chemical stripping and immunodepletion, thelast three are the ones most often applied.

Currently, there is no regulatory guidance specifically forbiomarker bioanalysis. Therefore, whatever needs to be donein LC-MS bioanalysis of biomarkers should fit for the pur-pose of the intended use of the data. This approach has gainedconsent within the bioanalysis community. The term “fit forpurpose” reflects flexible inclusion/exclusion of validationexperiments, experiment design, and acceptance criteria. Ingeneral, assessment of accuracy, precision, and stability isconsidered the essential part of assay validation, while oth-ers, for example, matrix effect and recovery, are consideredoptional, especially when a stable isotope labeled IS is used.

Another emerging trend in biomarker quantitation isthe LC-MS bioanalysis of peptide or protein biomarkersalthough ligand-binding assays, for example, enzyme-linkedimmuno sandwich assay (ELISA), still play an importantrole. Compared to ELISA, LC-MS assay development is rela-tively fast with no need to raise antibodies. More importantly,LC-MS assays can measure proteins as peptide surrogatewith similar sensitivity and specificity to many immunoas-says. However, introduction of stable label protein internalstandard can be very challenging and costly.

1.4.7 Other LC-MS Assays Needed for ClinicalDevelopment of Drugs

As clinical drug development progresses, there can be otherneeds for LC-MS assays. Metabolism-mediated toxicity oradverse events often trigger these requests. In toxicologystudies, this can include an assessment of parent and metabo-lite concentrations in various tissues from the most sensitivespecies. In man, penetration or distribution questions may bedifficult to answer. For blood–brain barrier penetration, onlycerebrospinal fluid surrogate sampling may be possible.

For antiinfective drugs, penetration studies are critical toensuring that trough concentrations greater than IC50 levelsare maintained where needed. When this is not achieved,resistance can develop. A similar objective to define cel-lular penetration can be achieved by analyzing peripheralblood mononuclear cells (PBMCs) after dosing with virol-ogy drug candidates. Plasma concentration is a poor indicatorof drug activities in the cell since the activation of the drug(nucleoside) to its triphosphate involves multiple enzymaticprocesses that may vary by individual (Rodman et al., 1996).The pharmacokinetics of the intracellular triphosphate is alsovery different from that of the nucleoside. For example, theintracellular triphosphate form of emtricitabine has a muchlonger half-life than the plasma half-life of emtracitabine(Wang et al., 2004). Analysis of drug concentrations at thetarget site is often fundamental to prove target engagementand can serve to build the clinical PK-PD model. For instance,both intracellular penetration and phosphorylation is needed