Handbook of LC-MS Bioanalysis E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Consolidates the information LC-MS bioanalytical scientists need to analyze small molecules and macromolecules The field of bioanalysis has advanced rapidly, propelled by new approaches for developing bioanalytical methods, new liquid chromatographic (LC) techniques, and new mass spectrometric (MS) instruments. Moreover, there are a host of guidelines and regulations designed to ensure the quality of bioanalytical results. Presenting the best practices, experimental protocols, and the latest understanding of regulations, this book offers a comprehensive review of LC-MS bioanalysis of small molecules and macromolecules. It not only addresses the needs of bioanalytical scientists working on routine projects, but also explores advanced and emerging technologies such as high-resolution mass spectrometry and dried blood spot microsampling. Handbook of LC-MS Bioanalysis features contributions from an international team of leading bioanalytical scientists. Their contributions reflect a review of the latest findings, practices, and regulations as well as their own firsthand analytical laboratory experience. The book thoroughly examines: * Fundamentals of LC-MS bioanalysis in drug discovery, drug development, and therapeutic drug monitoring * The current understanding of regulations governing LC-MS bioanalysis * Best practices and detailed technical instructions for LC-MS bioanalysis method development, validation, and stability assessment of analyte(s) of interest * Experimental guidelines and protocols for quantitative LC-MS bioanalysis of challenging molecules, including pro-drugs, acyl glucuronides, N-oxides, reactive compounds, and photosensitive and autooxidative compounds With its focus on current bioanalytical practice, Handbook of LC-MS Bioanalysis enables bioanalytical scientists to develop and validate robust LC-MS assay methods, all in compliance with current regulations and standards.
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Contents
Cover
Title Page
Copyright
Preface
Contributors
Abbreviations
Part I: Overview Of LC-MS Bioanalysis
Chapter 1: Roles of LC-MS Bioanalysis in Drug Discovery, Development, and Therapeutic Drug Monitoring
1.1 INTRODUCTION
1.2 LC-MS BIOANALYSIS IN DRUG DISCOVERY
1.3 LC-MS BIOANALYSIS IN PRECLINICAL DEVELOPMENT OF DRUGS
1.4 LC-MS BIOANALYSIS IN CLINICAL DEVELOPMENT OF DRUGS
1.5 LC-MS BIOANALYSIS OF LARGE MOLECULE DRUGS AND BIOPHARMACEUTICALS
1.6 GUIDANCE AND REGULATIONS FOR LC-MS BIOANALYSIS
1.7 GENERAL CONSIDERATIONS OF A ROBUST LC-MS BIOANALYTICAL METHOD
1.8 CONCLUSIONS
REFERENCES
Chapter 2: Overview: Fundamentals of a Bioanalytical Laboratory
2.1 INTRODUCTION
2.2 KEY ELEMENTS OF A BA LABORATORY
2.3 QUALITY ASSURANCE
2.4 SUPPORTING FUNCTIONS
2.5 CRO MONITORING
2.6 CONCLUSIONS
REFERENCES
Chapter 3: International Regulations and Quality Standards of Bioanalysis
3.1 INTRODUCTION
3.2 GLOBAL BIOANALYTICAL GUIDANCE
3.3 BIOANALYTICAL QUALITY
3.4 SCIENCE, QUALITY, AND REGULATION
ACKNOWLEDGMENTS
REFERENCES
Part II: Current Understanding of LC-MS Bioanalysis-Related Regulations
Chapter 4: Current Regulations for Bioanalytical Method Validations
4.1 INTRODUCTION
4.2 CONTEXT OF THE REGULATORY ENVIRONMENT
4.3 VALIDATIONS OF METHODS
4.4 CONCLUSION
REFERENCES
Chapter 5: Current Understanding of Bioanalytical Assay Reproducibility: Incurred Sample Reanalysis, Incurred Sample Stability, and Incurred Sample Accuracy
5.1 INTRODUCTION
5.2 INCURRED SAMPLE REANALYSIS
5.3 INCURRED SAMPLE STABILITY
5.4 INCURRED SAMPLE ACCURACY
5.5 SUMMARY
REFERENCES
Chapter 6: Lc-Ms Bioanalytical Method Transfer
6.1 INTRODUCTION
6.2 PREPARATION FOR METHOD TRANSFER
6.3 CURRENT UNDERSTANDING ON REGULATORY REQUIREMENTS FOR BIOANALYTICAL METHOD TRANSFER
6.4 METHOD TRANSFER
6.5 COMMON CAUSES OF BIOANALYTICAL METHOD TRANSFER FAILURE
6.6 INVESTIGATION OF A METHOD TRANSFER FAILURE
6.7 SUMMARY
REFERENCES
Chapter 7: Metabolites in Safety Testing
7.1 INTRODUCTION
7.2 TIMING OF ADME STUDIES WITH RADIOLABELED MATERIALS
7.3 FIH STUDIES
7.4 STANDARD FREE QUANTIFICATION AND ITS LIMITATIONS
7.5 TIERED OPTIONS FOR DETERMINATION OF HUMAN METABOLITE EXPOSURES AND RESPECTIVE LIMITATIONS
7.6 SUMMARY
REFERENCES
Chapter 8: A Comparison of FDA, EMA, ANVISA, and Others on Bioanalysis in Support of Bioequivalence/ Bioavailability Studies
8.1 INTRODUCTION TO BIOAVAILABILITY/BIOEQUIVALENCY STUDIES
8.2 REGULATIONS FROM THE US FDA
8.3 REGULATIONS FROM THE EUROPEAN MEDICINES AGENCY
8.4 REGULATIONS FROM THE BRAZILIAN SANITARY SURVEILLANCE AGENCY (ANVISA)
8.5 OTHER INTERNATIONAL GUIDELINES
8.6 CONCLUSION
REFERENCES
Chapter 9: A Comparison of the Guidance of FDA, OECD, EPA, and Others on Good Laboratory Practice
9.1 INTRODUCTION
9.2 FDA VERSUS EPA ON GLP
9.3 FDA GLP VERSUS OECD GLP PRINCIPLES
9.4 SOME COUNTRY SPECIFIC REQUIREMENTS ON GLP
9.5 GLP INSPECTION
9.6 SUMMARY
REFERENCES
Chapter 10: Current Understanding of Bioanalysis Data Management and Trend of Regulations on Data Management
10.1 INTRODUCTION
10.2 BIOANALYTICAL WORKFLOW AND DATA MANAGEMENT
10.3 COMPUTER SYSTEMS VALIDATION
10.4 CHALLENGES FOR BIOANALYTICAL DATA INTEGRITY AND SECURITY
10.5 FUTURE PERSPECTIVES
10.6 CONCLUSIONS
REFERENCES
Chapter 11: Regulatory Inspection Trends and Findings of Bioanalytical Laboratories
11.1 INTRODUCTION
11.2 CURRENT REGULATORY INSPECTION TRENDS
11.3 INADEQUATE INVESTIGATION—THE MDS CASE
11.4 DATA INTEGRITY CONCERN—THE CETERO CASE
11.5 DISCUSSION AND ANALYSIS OF SPECIFIC REGULATORY INSPECTION FINDINGS
11.6 RECOMMENDATIONS TO SUPPORT AN EFFECTIVE FDA INSPECTION—READINESS PREPARATION
REFERENCES
Part III: Best Practice in LC-MS Bioanalysis
Chapter 12: Assessment of Whole Blood Stability and Blood/Plasma Distribution of Drugs
12.1 ASSESSMENT OF WHOLE BLOOD STABILITY OF DRUGS
12.2 BLOOD/PLASMA DISTRIBUTION OF DRUGS
12.3 SUMMARY
ACKNOWLEDGMENT
REFERENCES
Chapter 13: Best Practice in Biological Sample Collection, Processing, and Storage for LC-MS in Bioanalysis of Drugs
13.1 INTRODUCTION
13.2 SAMPLE COLLECTION
13.3 DOCUMENTATION
13.4 REDUCING ADSORPTION OF ANALYTE TO CONTAINERS
13.5 SAMPLE COLLECTION FOR PEPTIDES AND PROTEINS
13.6 STABILIZING SAMPLES
13.7 BIOLOGICALLY HAZARDOUS SAMPLES
13.8 SAMPLE STORAGE
13.9 TRANSPORT AND SHIPPING OF SAMPLES
13.10 COMPLIANCE CHECKLIST
13.11 SUMMARY
ACKNOWLEDGMENT
REFERENCES
Chapter 14: Best Practices in Biological Sample Preparation for LC-MS Bioanalysis
14.1 WHY DO SAMPLE PREPARATION?
14.2 WHAT SHOULD YOU KNOW ABOUT THE SAMPLE?
14.3 KNOW YOUR TOOLS
14.4 KNOW YOUR NEEDS
14.5 CONCLUSION AND PERSPECTIVES
REFERENCES
Chapter 15: Best Practice in Liquid Chromatography for LC-MS Bioanalysis
15.1 INTRODUCTION
15.2 THEORETICAL CONSIDERATIONS
15.3 PRACTICAL CONSIDERATIONS FOR METHOD DEVELOPMENT
15.4 SAMPLE ANALYSIS
15.5 MAKING RUGGED METHODS
15.6 LESS IS MORE
15.7 HIGH-RESOLUTION AND HIGH-SPEED ANALYSIS
15.8 SPECIAL CHALLENGES AND OPPORTUNITIES
15.9 CONCLUSIONS
REFERENCES
Chapter 16: Best Practice in Mass Spectrometry for LC-MS
16.1 INTRODUCTION
16.2 ANALYZERS MOST OFTEN USED FOR LC-MS AND LC-MS/MS BIOANALYSIS
16.3 IONIZATION TECHNIQUES FOR UHPLC-MS
16.4 LC-MS IN QUANTITATIVE ANALYSIS
16.5 PERSPECTIVES
REFERENCES
Chapter 17: Use of Internal Standards in LC-MS Bioanalysis
17.1 INTRODUCTION
17.2 SELECTION AND USE OF IS
17.3 PERFORMANCE OF IS
17.4 CONCLUSION
ACKNOWLEDGMENT
REFERENCES
Chapter 18: System Suitability in LC-MS Bioanalysis
18.1 OVERVIEW
18.2 REGULATORY REQUIREMENTS FOR SYSTEM SUITABILITY
18.3 MONITORING INSTRUMENT PERFORMANCE
18.4 CONCLUSIONS
REFERENCES
Chapter 19: Derivatization in LC-MS Bioanalysis
19.1 INTRODUCTION
19.2 DERIVATIZATION REAGENTS ENHANCING IONIZATION EFFICIENCY IN LC/ESI-MS
19.3 DERIVATIZATION REAGENTS IN LC/ESI-MS/MS
19.4 CONCLUSION
REFERENCES
Chapter 20: Evaluation and Elimination of Matrix Effects in LC-MS Bioanalysis
20.1 INTRODUCTION
20.2 POTENTIAL IMPACT OF MATRIX EFFECTS
20.3 COMMON CAUSES OF MATRIX EFFECTS
20.4 MECHANISM OF MATRIX EFFECTS
20.5 METHODS TO IDENTIFY AND EVALUATE MATRIX EFFECTS
20.6 REGULATORY GUIDANCE ON EVALUATION AND AVOIDANCE OF MATRIX EFFECTS
20.7 METHODS TO AVOID OR ELIMINATE MATRIX EFFECTS
20.8 FUTURE PROSPECTS
ACKNOWLEDGMENTS
REFERENCES
Chapter 21: Evaluation and Elimination of Carryover and/or Contamination in LC-MS Bioanalysis
21.1 OVERVIEW
21.2 CURRENT UNDERSTANDING OF REGULATORY PERSPECTIVES ON CARRYOVER AND CONTAMINATION
21.3 CARRYOVER—WHAT IS IT?
21.4 CONTAMINATION—WHAT IS IT?
21.5 WHAT DO THE MANUFACTURERS SAY?
21.6 MANAGING CARRYOVER AND CONTAMINATION IN LC-MS BIOANALYSIS
21.7 SUMMARY
ACKNOWLEDGMENTS
REFERENCES
Chapter 22: Automation in LC-MS Bioanalysis
22.1 INTRODUCTION
22.2 AN OVERVIEW OF AUTOMATED SAMPLE PREPARATION IN LC-MS BIOANALYSIS
22.3 ROBOTIC LIQUID HANDLING PIPETTING MODES AND ASSOCIATED TECHNOLOGY
22.4 OPTIMIZING ROBOTIC LIQUID HANDLING PERFORMANCE
22.5 SOLID PHASE EXTRACTION
22.6 PROTEIN PRECIPITATION
22.7 LIQUID–LIQUID EXTRACTION
22.8 PRACTICAL CONSIDERATIONS: STRATEGY, QUALITY, AND COMPLIANCE
22.9 CONCLUDING REMARKS
REFERENCES
Chapter 23: LC-MS Bioanalysis of Drugs in Tissue Samples
23.1 INTRODUCTION
23.2 CLASSIFICATION OF TISSUES
23.3 WORKFLOW
23.4 TISSUE SAMPLE COLLECTION
23.5 TISSUE SAMPLE PREPARATION
23.6 CALIBRATION STANDARD AND QC SAMPLE PREPARATION
23.7 ANALYTE EXTRACTION
23.8 LC-MS/MS ANALYSIS
23.9 FIT-FOR-PURPOSE LC-MS/MS METHOD QUALIFICATION
23.10 DATA ANALYSIS AND REPORTING
23.11 SUMMARY
ACKNOWLEDGMENT
REFERENCES
Chapter 24: LC-MS Bioanalysis of Drugs in Urine
24.1 INTRODUCTION
24.2 BEST PRACTICE IN DEVELOPING A ROBUST URINE LC-MS QUANTITATION METHOD
24.3 SUMMARY
REFERENCES
Chapter 25: LC-MS Bioanalysis of Unbound Drugs in Plasma and Serum
25.1 INTRODUCTION
25.2 PROTEIN BINDING
25.3 REGULATORY REQUIREMENTS REGARDING UNBOUND DRUG CONCENTRATIONS IN SPECIAL POPULATIONS
25.4 TECHNIQUES FOR THE BIOANALYSIS OF UNBOUND DRUGS IN PLASMA AND SERUM
25.5 EXAMPLES
REFERENCES
Chapter 26: LC-MS Bioanalysis of Drugs in Bile
26.1 BILE AND BILIARY EXCRETION
26.2 BILE SAMPLE COLLECTION
26.3 BILE SAMPLE PREPARATION
26.4 BILE SAMPLE LC-MS BIOANALYSIS
26.5 SUMMARY
REFERENCES
Chapter 27: LC-MS Bioanalysis of Intracellular Drugs
27.1 INTRODUCTION
27.2 SAMPLE PREPARATION
27.3 LC-MS INTRACELLULAR DRUG BIOANALYSIS
27.4 APPLICATION
27.5 CONCLUSIONS
REFERENCES
Chapter 28: LC-MS Bioanalysis of Endogenous Compounds as Biomarkers
28.1 INTRODUCTION
28.2 APPROACHES FOR BIOMARKER QUANTITATION
28.3 BIOMARKER ASSAY VALIDATION AND STUDY CONDUCT
28.4 SUMMARY AND PROSPECTIVE
REFERENCES
Chapter 29: LC-MS Bioanalysis of Drugs in Hemolyzed and Lipemic Samples
29.1 INTRODUCTION
29.2 HEMOLYSIS
29.3 LIPEMIA
29.4 CONCLUSION
ACKNOWLEDGMENT
REFERENCES
Chapter 30: Best Practices in LC-MS Method Development and Validation for Dried Blood Spots
30.1 INTRODUCTION
30.2 METHOD DEVELOPMENT
30.3 METHOD VALIDATION
30.4 CONCLUSIONS
REFERENCES
Chapter 31: LC-MS Method Development Strategies for Enhancing Mass Spectrometric Detection
31.1 INTRODUCTION
31.2 DIFFERENTIAL MOBILITY SPECTROMETRY
31.3 MULTIPLE REACTION MONITORING CUBED
31.4 ATMOSPHERIC PRESSURE PHOTOIONIZATION
31.5 MS SIGNAL ENHANCEMENT VIA MOBILE PHASE ADDITIVES AND ANIONIC AND CATIONIC ADDUCTS PRECURSOR IONS
31.6 CONCLUSIONS
REFERENCES
Chapter 32: LC-MS Bioanalysis-Related Statistics
32.1 INTRODUCTION
32.2 BASIC STATISTICS
32.3 CALIBRATION
32.4 BIAS AND PRECISION
32.5 STABILITY
32.6 INCURRED SAMPLE REPRODUCIBILITY
REFERENCES
Chapter 33: Simultaneous LC-MS Quantitation and Metabolite Identification in Drug Metabolism and Pharmacokinetics
33.1 INTRODUCTION
33.2 UNIT RESOLUTION MASS SPECTROMETERS IN DRUG METABOLISM AND PHARMACOKINETICS
33.3 HIGH-RESOLUTION MASS SPECTROMETRY IN DRUG METABOLISM AND PHARMACOKINETICS
33.4 MASS DEFECT FILTER IN LC-MS
33.5 SIMULTANEOUS LC-MS QUANTITATION/ METABOLITE IDENTIFICATION
LC-MS WORKFLOW: QqQLIT
33.7 LC-MS WORKFLOW: HIGH-RESOLUTION MASS SPECTROMETRY
33.8 CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
Part IV: Representative Guidelines and/or Experimental Protocols of LC-MS Bioanalysis
Chapter 34: LC-MS Bioanalysis of Ester Prodrugs and Other Esterase Labile Molecules
34.1 INTRODUCTION
34.2 COMMON ESTERASES THAT CATALYZE HYDROLYSIS OF ESTER PRODRUGS AND OTHER MOLECULES
34.3 METHODOLOGY AND APPROACHES
34.4 SUMMARY
34.5 A REPRESENTATIVE PROTOCOL-LC-MS BIOANALYSIS OF BMS-068645 AND ITS ACID METABOLITE IN HUMAN PLASMA
REFERENCES
Chapter 35: LC-MS Bioanalysis of ACYL Glucuronides
35.1 INTRODUCTION
35.2 CHEMICAL REACTIVITY AND BIOANALYTICAL IMPLICATIONS
35.3 SAMPLE COLLECTION AND STORAGE
35.4 SAMPLE PREPARATION
35.5 LC-MS/MS QUANTIFICATION
35.6 INCURRED SAMPLE REANALYSIS OF AGs
35.7 SUMMARY
35.8 REPRESENTATIVE PROTOCOLS
REFERENCES
Chapter 36: Regulated Bioassay Of N-Oxide Metabolites Using LC-MS: Dealing with Potential Instability Issues
36.1 INTRODUCTION
36.2 FORMATION OF N-OXIDE METABOLITES
36.3 DISTRIBUTION AND EXCRETION OF N-OXIDES
36.4 METABOLISM OF N-OXIDES
36.5 BIOLOGICAL ACTIVITIES OF N-OXIDES
36.6 EXPERIMENTAL PROTOCOL
36.7 REGULATORY CONSIDERATIONS
36.8 EXAMPLES
36.9 CONCLUSIONS
REFERENCES
Chapter 37: Hydrolysis of Phase II Conjugates for LC-MS Bioanalysis of Total Parent Drugs
37.1 INTRODUCTION
37.2 METHODS AND APPROACHES
37.5 EXAMPLE PROTOCOL
REFERENCES
Chapter 38: LC-MS Bioanalysis of Reactive Compounds
38.1 INTRODUCTION
38.2 DETERMINATION OF REACTIVE COMPOUND EXPOSURE IN VIVO
38.3 MEASUREMENT OF SULFHYDRYL- CONTAINING COMPOUNDS
38.4 CONCLUSION
38.5 REPRESENTATIVE PROTOCOLS
REFERENCES
Chapter 39: LC-MS Bioanalysis of Photosensitive and Oxidatively Labile Compounds
39.1 INTRODUCTION
39.2 PHOTOSENSITIVE COMPOUNDS
39.3 OXIDATION OF COMPOUNDS
39.4 SUMMARY
39.5 PROTOCOL FOR DEVELOPING LC-MS/MS METHOD FOR BIOANALYSIS OF PHOTOSENSITIVE AND OXIDATIVELY LABILE COMPOUNDS
REFERENCES
Chapter 40: LC-MS Bioanalysis of Interconvertible Compounds
40.1 INTRODUCTION
40.2 INTERCONVERSION OF LACTONES AND HYDROXY ACIDS
40.3 STEREOISOMERIC INTERCONVERSION
40.4 CONSIDERATIONS FOR DEVELOPING BIOANALYTICAL METHODS FOR INTERCONVERTIBLE COMPOUNDS
40.5 VALIDATION AND QUALITY CONTROL OF METHODS FOR INTERCONVERTIBLE COMPOUNDS
40.6 SUMMARY
40.7 REPRESENTATIVE PROTOCOL FOR LC-MS/MS METHOD DEVELOPMENT FOR BIOANALYSIS OF INTERCONVERTIBLE COMPOUNDS
REFERENCES
Chapter 41: LC-MS Bioanalysis of Chiral Compounds
41.1 INTRODUCTION
41.2 APPLICATIONS
41.3 CURRENT CONSIDERATIONS AND PROTOCOLS IN DEVELOPING A ROBUST CHIRAL LC-MS/MS BIOANALYTICAL METHOD
41.4 CURRENT METHOD VALIDATION CONSIDERATIONS AND PROTOCOLS FOR CHIRAL BIOANALYTICAL LC-MS/MS
41.5 CONCLUSION
REFERENCES
Chapter 42: LC-MS Bioanalysis of Peptides and Polypeptides
42.1 INTRODUCTION
42.2 METHODS AND APPROACHES
42.3 EXAMPLE EXPERIMENTAL PROTOCOLS
42.4 CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
Chapter 43: LC-MS Bioanalysis of Nucleosides
43.1 INTRODUCTION
43.2 METHODS AND APPROACHES
REFERENCES
Chapter 44: LC-MS Bioanalysis of Nucleotides
44.1 INTRODUCTION
44.2 PREANALYTICAL METHODS AND APPROACHES
44.3 TROUBLESHOOTING
REFERENCES
Chapter 45: LC-MS Bioanalysis of Steroids
45.1 INTRODUCTION
45.2 METHODS AND APPROACHES
45.3 REPRESENTATIVE PROTOCOLS OF LC-MS BIOANALYSIS OF STEROID HORMONES
ACKNOWLEDGMENTS
REFERENCES
Chapter 46: LC-MS Bioanalysis of Liposomal Drugs and Lipids
46.1 INTRODUCTION
46.2 METHODS AND APPROACHES
46.3 PROTOCOLS
REFERENCES
Chapter 47: LC-MS Bioanalysis of Proteins
47.1 INTRODUCTION
47.2 METHODS
47.3 SUMMARY
REFERENCES
Chapter 48: LC-MS Bioanalysis of Oligonucleotides
48.1 INTRODUCTION
48.2 PHYSIOCHEMICAL PROPERTIES AND MODIFICATIONS OF OLIGONUCLEOTIDES
48.3 METHODS AND APPROACHES
48.4 PROTOCOLS
48.5 TROUBLESHOOTING OLIGONUCLEOTIDE LC-MS BIOANALYSIS
48.6 SUMMARY
REFERENCES
Chapter 49: LC-MS Bioanalysis of Platinum Drugs
49.1 INTRODUCTION
49.2 METHODS AND APPROACHES
49.3 TROUBLESHOOTING
REFERENCES
Chapter 50: Microflow LC-MS for Quantitative Analysis of Drugs in Support of Microsampling
50.1 INTRODUCTION
50.2 METHODS AND APPROACHES
50.3 PROTOCOLS
50.4 TROUBLESHOOTING
ACKNOWLEDGMENTS
REFERENCES
Chapter 51: Quantification of Endogenous Analytes in Biofluids by a Combination of LC-MS and Construction of Calibration Curves Using Stable-Isotopes as Surrogate Analytes with True Biological Control Matrices
51.1 INTRODUCTION
51.2 METHODS AND APPROACHES
REFERENCES
Appendix 1: Body and Organ Weights and Physiological Parameters in Laboratory Animals and Humans
Appendix 2: Anticoagulants Commonly Used in Blood Sample Collection
Appendix 3: Solvents and Reagents Commonly Used in LC-MS Bioanalysis
Appendix 4: Glossary of terms used in LC-MS Bioanalysis
Index
Copyright © 2013 by John Wiley & Sons, Inc. All rights reserved.
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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 for BioMedical Research, Jie Zhang, Ph.D., Novartis Institutes for BioMedical Research, Francis L.S. Tse, Ph.D., Novartis Institutes for BioMedical Research.
pages cm Includes 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 2013 615.1′901--dc23 2013004581
ISBN: 9781118159248
PREFACE
Bioanalysis is the most heavily regulated area within the discipline of drug metabolism and pharmacokinetics, which supports a large sector of drug development. The health authorities have very specific requirements regarding quality and integrity of bioanalytical results, and different customers usually have additional expectations on the performance of bioanalytical 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 US Food and Drug Administration (FDA) have renewed or are renewing their guidances on bioanalytical method validation (e.g., 21July2011/EMEA/CHMP/EWP/192217/2009) with the ultimate goal of improving the quality of bioanalytical results. Novel approaches to bioanalytical method development as well as advents of new liquid chromatographic (LC) techniques and mass spectrometric (MS) instruments have been reported. Various automatic laboratory procedures, electronic laboratory notebooks, and data management systems are now available. All of these culminate in a remarkable improvement in the quality, speed, and cost-effectiveness of bioanalytical work and contribute to the value we deliver to the patient.
Given the rapid changes within the field of bioanalysis and also in the larger area of drug development in which we operate, it is timely to conduct a broad overview of our discipline. This book is the first comprehensive handbook for LC-MS bioanalysis and provides an update on all important aspects of LC-MS bioanalysis of both small molecules and macromolecules. It not only addresses the needs of the bioanalytical scientists on the pivotal projects but also features perspectives on some advanced and emerging technologies including high-resolution mass spectrometry and dried blood spot (DBS) microsampling.
The 51 chapters of the book are divided into four parts. Part I provides a comprehensive overview on the role of LC-MS bioanalysis in drug discovery and development and therapeutic drug monitoring (Chapter 1), the key elements of a regulated bioanalytical laboratory (Chapter 2), and the current international regulations and quality standards of bioanalysis (Chapter 3).
In Part II, the global regulations and quality standards related to LC-MS bioanalysis are reviewed and compared. Chapter 4 highlights the current regulations governing bioanalytical method validations from a number of countries and regions including Brazil (ANVISA), Canada, China, the European Union (EMA), India, Japan, and the United States (FDA). This is followed by two in-depth reviews on the topics of assay reproducibility (Chapter 5) and method transfer (Chapter 6). Chapter 7 presents the current practices and regulatory requirements on Metabolites in Safety Testing (MIST). The guidances of regulatory bodies worldwide on bioanalysis for bioequivalence (BE)/bioavailability (BA) studies are compared in Chapter 8, whereas Chapter 9 concerns 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 evolvement of regulations on bioanalytical data management, which is discussed in Chapter 10. Chapter 11 concludes Part II by giving a detailed analysis of regulatory inspections including health authority expectation, reported inspectional trends, citations, and regulatory followup letters. Recent FDA 483 observations as well as other “hot topics” in bioanalysis compliance that have raised concerns about data integrity are reviewed. Applicable best practices in LC-MS bioanalysis are portrayed in Part III. From this section of the book, the reader will find sound scientific rationale and helpful practical instructions on the assessment of whole blood stability and blood/plasma distribution (Chapter 12), and on biological sample collection, processing and storage (Chapter 13). Chapter 14 introduces various sample preparation techniques for LC-MS bioanalysis, while the best practices in LC separation and MS detection are discussed in Chapters 15 and 16, respectively. A good bioanalytical method must be sensitive, specific, selective, reproducible, high-throughput, and fundamentally robust. Many factors that can contribute to the success of an assay are reviewed including the choice of internal standard (Chapter 17), evaluation of system suitability (Chapter 18), sensitivity enhancement via derivatization of analyte(s) of interest (Chapter 19), evaluation and elimination of matrix effect (Chapter 20), evaluation and elimination of carryover and/or contamination (Chapter 21), and robotic automation (Chapter 22). Chapters 23–29 describe the bioanalysis of drugs, biomarkers, and other analytes of interest in various body fluids and tissues, and Chapter 30 is devoted to DBS sampling and related bioanalytical issues. Chapter 31 offers some useful strategies for enhancing MS detection, and Chapter 32 shows the proper use of statistics as a tool for ensuring adequate method performance in LC-MS bioanalysis. The simultaneous quantitative and qualitative LC-MS bioanalysis of drugs and metabolites are discussed in Chapter 33.
Part IV aims to provide detailed instructions with representative experimental protocols for the LC-MS bioanalysis of various types of drug molecules commonly encountered in the bioanalytical laboratory today (Chapters 34–49). Chapter 50 describes a typical procedure using microflow LC-MS for the quantitative analysis of drugs in support of microsampling. Finally, a protocol on the quantification of endogenous analytes in biofluids without a true blank matrix is given in Chapter 51.
Our purpose in committing to this project was to provide scientists in industry, academia, and regulatory agencies with not only all the “important points to consider” but also all “practical tricks to implement” in LC-MS bioanalysis of various molecules according to current health authority regulations and industry practices. In this book, we are confident that we have accomplished our goal. The book represents a major undertaking, which would not have been possible without the contributions of all the authors and the patience of their families. We also thank the terrific editorial staff at John Wiley & Sons and give a special acknowledgment to Michael Leventhal, Associate Editor, and Robert Esposito, Associate Publisher, at John Wiley & Sons for their premier support of this project.
WENKUI LI, PhD JIE ZHANG, PhD FRANCIS L.S. TSE, PhD
CONTRIBUTORS
ABBREVIATIONS
AAPSAmerican association of pharmaceutical scientistsAASAtomic absorption spectroscopyACAbsolute carryoverAchAcetylcholineACUPAnimal care and use protocolADCAntibody–drug conjugateADMEAbsorption, distribution, metabolism, and excretionAFAAdaptive focused acousticsALQ/AULOQAbove the upper limit of quantificationANDAAbbreviated new drug applicationANVISANational health surveillance agency (in Portuguese, Agência Nacional de Vigilância Sanitária)APAnalytical procedureAPCIAtmospheric pressure chemical ionizationAPIAtmospheric pressure ionizationAPPIAtmospheric pressure photoionizationASEAccelerated solvent extractionASEANAssociation of southeast asian nationsAUCArea under the curveBABioavailabilityBDMAButyldimethylamineBDMABButyldimethylammonium bicarbonateBEBioequivalenceBIMOBioresearch monitoringBLQ/BLLOQBelow the lower limit of quantificationBNPPBis(4-nitrophenyl)-phosphateBSABovine serum albuminCADCharged aerosol detectionCADCollision-activated disassociationCAPACorrective and preventive actionCDCompact discCDCCenters for disease control and preventionCDSCOCentral drugs standard control organizationCECollision energyCFRCode of federal regulationsCIDCollision-induced dissociationCLClearanceCNSCentral nervous systemCoACertificate of analysisCOVCompensation voltageCPGMCompliance program guidance manualCRConcentration ratioCROContract research organizationCsCalibration standardCSFCerebrospinal fluidCSICaptive spray ionizationCVCoefficient of variationCXPCollision exit potentialCZECapillary zone electrophoresisDBSDried blood spotDDIDrug–drug interactionDDTCDiethyldithiocarbamateDDVP2,2-Dichlorovinyl dimethyl phosphateDHEADehydroepiandrosteroneDHTDihydrotestosteroneDIFP/DFPDiisopropyl fluorophosphateDMDrug metabolismDMSDifferential mobility spectrometryDMSDried matrix spotDNADeoxyribonucleic acidDns-ClDansyl chlorideDns-HzDansyl hydrazineDPDeclustering potentialDPSDried plasma spotDPXDisposable pipette extractionDQDesign qualificationDTNB5, 5'-Dithiobis-(2-nitrobenzoic acid)DTTDithiothreitolEBEndogenous baselineEBFEuropean bioanalysis forumEDMSElectronic data management systemEDTAEthylenediaminetetraacetic acidEFPIAEuropean federation of pharmaceutical industries associationsEHNAErythro-9-(2-hydroxy-3-nonyl) adenineELNElectronic laboratory notebookELSDEvaporative light scattering detectionEMAEuropean medicines agencyEPEntrance potentialEPAEnvironmental protection agencyESIElectrospray ionizationFAIMSField-asymmetric waveform ion mobility spectrometryFDAFood and drug administrationFIHFirst-in-humanFOIAFreedom of information actFPFocusing potentialFTICRFourier transform ion cyclotron resonanceFWHMFull width at half maximumGAMPGood automated manufacturing practiceGBCGlobal bioanalytical consortiumGC-MSGas chromatography-mass spectrometryGCPGood clinical practiceGLPGood laboratory practiceGMPGood manufacturing practiceGPhAGeneric pharmaceutical associationHAAHexylammonium acetateHCTHematocritHETPHeight equivalent of a theoretical plateHFIPHexafluoroisopropanolHILICHydrophilic interaction liquid chromatographyHMP2-Hydrazino-1-methyl-pyridineHP2-HydrazinopyridineHPFBHealth products and food branchHPLCHigh pressure liquid chromatography or high performance liquid chromatographyHRMSHigh resolution mass spectrometryHSAHuman serum albuminHTLCHigh-turbulence liquid chromatographyIAImmunoaffinityIACUCInstitutional animal care and use committeeICHInternational conference on harmonizationICP-MSInductively coupled plasma–mass spectrometryIDInner diameterIDMSIsotope dilution mass spectrometryIECIon-exchange chromatographyIMSIon mobility spectrometryINDInvestigational new drugIPIon-pairingIQInstallation qualificationIS/ISTDInternal standardISAIncurred sample accuracyISRIncurred sample reanalysis or incurred sample reproducibilityISSIncurred sample stabilityIVIntravenousKFDAKorea food and drug administrationLC-MSLiquid chromatography–mass spectrometryLC-MS/MSLiquid chromatography-tandem mass spectrometryLIMSLaboratory information management systemLLELiquid–liquid extractionLLOQLower limit of quantificationLODLimit of detectionLUVLarge unilamellar vesicleMADMultiple ascending doseMAXMixed mode anion exchangeMCDMaximum concentration differenceMCXMixed mode cation exchangeMDMethod developmentMDFMass defect filterMEPSMicroextraction by packed sorbentMFMatrix factorMFCMicrofluidic flow controlMHFWMinistry of health and family welfareMHLWMinistry of health, labour and welfareMHRAMedicines and healthcare products regulatory agencyMIPMolecularly imprinted polymersMISTMetabolites in safety testingMLVMultilamellar vesicleMRMMultiple reaction monitoringMSMass spectrometryMTBEMethyl tert-butyl etherMVMethod validationMVSMultichannel verification systemMWCOMolecular weight cutoffNCCLSNational committee for clinical laboratory standardsNCENew chemical entityNDANew drug applicationNHSNational health serviceNIHNational institutes of healthNLNeutral lossNMENew molecular entityNMRNuclear magnetic resonanceNPLCNormal phase liquid chromatographyNRTINucleoside reverse transcriptase inhibitorNSBNonspecific bindingNSINanospray ionizationOCOral contraceptiveOECDOrganization for economic cooperation and developmentOEMOriginal equipment manufacturerOOSOut-of-specificationOQOperational qualificationORAOffice of regulatory affairsOSIOffice of scientific investigationsPBProtein bindingPBMCPeripheral blood mononuclear cellPBSPhosphate buffered salinePCTPressure cycling technologyPCVPacked cell volumePDPharmacodynamicsPDAPhotodiode arrayPDFPortable document formatPEEKPolyether ether ketonePEGPolyethylene glycolPGCPorous graphitic carbonPIPrincipal investigatorPKPharmacokineticsPMPreventive maintenancePMPPressure monitoring pipettingPMSFPhenylmethylsulfonyl fluoridePNPAp-Nitrophenyl acetatePoCProof of conceptPPEProtein precipitation extractionPPTProtein precipitationPQPerformance qualificationPTMPosttranslational modificationQAQuality assuranceQASQuality assurance statementQAUQuality assurance unitQCQuality controlQqQTriple quadrupoleQqQLITHybrid triple quadrupole-linear ion trapQqTOFHybrid quadrupole time-of-flightQ-TOFQuadrupole time-of-flightRADRadioactivity detectionRAMRestricted access materialRBCRed blood cellRCRelative carryoverRERecoveryREDRapid equilibrium dialysisRFResponse factorRFIDRadiofrequency identifierRIARadioimmunoassayRNARibonucleic acidRTRetention timeRTRoom temperatureRT-qPCRReal-time reverse transcription polymerase chain reactionSADSingle ascending doseSALLESalting-out assisted LLESAXStrong anion ion exchangeSBSEStir bar sorptive extractionSCXStrong cation ion exchangeSDStandard deviationSDMSScientific data management systemSEStandard errorSFCSupercritical fluid chromatographySFDAState food and drug administrationSIL-ISStable isotope labeled internal standardSIMSelected ion monitoringsiRNASmall interfering RNASLESupported liquid extractionS/NSignal-to-noiseSOPStandard operating procedureSPESolid phase extractionSPMESolid phase microextractionSRMSelected reaction monitoringSSBGSex steroid binding globulinSTDStandardSUVSmall unilamellar vesicleSVSeparation voltageTADMTotal aspirate and dispense monitoringTDMTherapeutic drug monitoringTEATriethylamineTEAATriethylammonium acetateTEABTriethylammonium bicarbonateTFATrifluoroacetic acidTGATherapeutic goods administrationTHUTetrahydrouridineTICTotal ion chromatogramTKToxicokineticsTMATrimethyl ammoniumTPDTherapeutic products directorateTSCAToxic substance control actTTFAThenoyltrifluoroacetoneUHPLCUltra high performance liquid chromatographyULOQUpper limit of quantificationUPLCUltra Performance liquid chromatographyURSUser requirement specificationUVUltravioletWAXWeak anion exchangeWBCWhite blood cellWHOWorld health organizationPART I
OVERVIEW OF LC-MS BIOANALYSIS
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ROLES OF LC-MS BIOANALYSIS IN DRUG DISCOVERY, DEVELOPMENT, AND THERAPEUTIC DRUG MONITORING
STEVE UNGER, WENKUI LI, JIMMY FLARAKOS, AND FRANCIS L.S. TSE
1.1 INTRODUCTION
Bioanalysis is a subdiscipline of analytical chemistry for the quantitative measurement of xenobiotics (chemically synthesized or naturally extracted drug candidates and genetically produced biological molecules and their metabolites or post-translationally modified products) and biotics (macromolecules, proteins, DNA, large molecule drugs, metabolites) in biological systems. Many scientific decisions regarding drug development are dependent upon the accurate quantification of drugs and endogenous components in biological samples. Unlike its sister subdisciplines of analytical chemistry such as drug substance and drug product analysis, one very unique feature of contemporary bioanalysis is that its measurement target is always at very low concentration levels, typically at low ng/ml concentration range and even at pg/ml for highly potent medicines. It is this very low concentration, compounded by coexisting endogenous or exogenous compounds with similar chemical structures to the target analytes at a much higher concentration (typically at μg/ml to mg/ml range), that challenges bioanalytical scientists to accurately and definitively measure the analytes of interest.
Since its commercial introduction in the 1980s, liquid chromatography–mass spectrometry (LC-MS), or much more predominantly, tandem mass spectrometry (LC-MS/MS) has rapidly become standard instrumentation in any well-equipped bioanalytical laboratory. LC-MS is a combination of the physicochemical separation capabilities of liquid chromatography (LC) and the mass (MS or MS/MS) separation/detection capabilities of mass spectrometry. In LC-MS bioanalysis, assay selectivity can be readily achieved by three stages of separation of the analyte(s) of interest from unwanted components in the biological matrix: (1) sample extraction (protein precipitation, liquid–liquid extraction, solid-phase extraction, etc.), (2) column chromatography, and (3) tandem mass spectrometric detection in selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) mode. Nevertheless, many factors, including matrix effect, ion suppression, and in-source breakdown of labile metabolites, can compromise the reliability of a LC-MS bioanalytical assay. These factors should be carefully evaluated during method development.
The focus of LC-MS bioanalysis in the pharmaceutical industry is to provide a quantitative measurement of the active drug and/or its metabolite(s) for the accurate assessment of pharmacokinetics, toxicokinetics, bioequivalence (BE), and exposure–response (pharmacokinetics/ pharmacodynamics) relationships (Figure 1.1). The quality of these studies, which are often used to support regulatory filings and other evaluations, is directly related to the conduct of the underlying bioanalysis. Therefore, the application of best practices in bioanalytical method development, validation, and associated sample analysis is key to an effective discovery and development program leading to the successful registration and commercialization of a drug product.
FIGURE 1.1 A flowchart of drug discovery and development, and postapproval studies of drugs where LC-MS bioanalysis plays important roles.
1.2 LC-MS BIOANALYSIS IN DRUG DISCOVERY
Before the introduction of combinatorial chemistry, many drug candidates came from natural products where an active compound was isolated and its chemical structure was characterized using NMR, MS, IR, and derivatization or selective degradation chemistry. Screening entailed an assessment of bioactivity and physicochemical data compared to known databases. High-resolution mass spectrometry played a critical role allowing molecular formula searches from accurate mass data. Similarly, spectral databases allowed positive confirmation or class assessments. This process helped to ensure that novel compounds were selected. Since the introduction of combinatorial chemistry 20 years ago, the analyst's role in early drug discovery has shifted to the development of highly efficient LC-MS analytical methods to support quantitative analysis. The drug discovery process begins with compound library development and ends with the selection of preclinical drug candidates for preclinical safety assessment. LC-MS bioanalysis plays an important role throughout this process.
1.2.1 Structure-Activity Relationships from High-Throughput Screening
High-throughput LC-MS assays can be employed for the determination of solubility, membrane permeability or transport, protein binding, and chemical and metabolic stability for a large number of compounds that have been identified as “hits” (Janiszewski et al., 2008). Thousands of compounds per year go through some or all of these screening procedures. The in vitro studies validate in silico assessments performed prior to synthesis and select compounds for moving forward in development.
1.2.2 Structure—PK-PD Relationships
Selected compounds from high-throughput screening are subsequently evaluated in pharmacology models for efficacy. Provided the targeted biochemistry is applicable to LC-MS analysis, high-throughput screening of potential biomarkers can be performed in pharmacology studies via either a targeted pathway or a metabolomics approach. If successful, discovery biomarkers may be useful in preclinical and clinical studies. Simple examples include steroid biomarkers such as testosterone or dihydrotestosterone for 5-α-reductase inhibitors or estrogen for selective estrogen receptor modulators.
Integration of drug metabolism and pharmacokinetics (DMPK), pharmacology, and biology studies in drug discovery can greatly accelerate an understanding of the pharmacokinetic–pharmacodynamic (PK-PD) relationships of lead compounds. The minimum effective dose observed in the pharmacology model is validated from knowledge of drug and active metabolite levels at the target site and compared with in vitro efficacy. Compounds known to have in vitro potency but are devoid of in vivo activity are suspected of having poor bioavailability (BA) or other DMPK properties (transport to target site, rapid clearance, etc.). Alternatively, compounds with an unanticipated high in vivo activity may have superior access to the site of action or form active metabolites.
LC-MS has a fundamental role in the success of many of these discovery studies. An appropriately designed, early in vitro study can determine intrinsic clearance in multiple species. In vitro assessments have improved our ability to predict systemic clearance using intrinsic clearance. However, predicting volume of distribution and tissue concentrations is far more difficult. Combinatorial approaches such as cassette dosing or coadministration of many compounds is one means of quickly assessing penetration into target sites. Typically, ∼20 compounds are coadministered, but as many as 100 have been attempted (Berman et al., 1997). The specificity of MS detection allows one to simultaneously measure numerous compounds in biofluids and tissues and rapidly screen drug candidates 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 compounds may be investigated in greater detail as possible preclinical drug candidates. These include assessments at various doses in the rodent and nonrodent toxicology species. Defining the systemic and local exposures, refining PK-PD models and exploring dose-proportionality are among the objectives of this phase. Studies with both single and multiple ascending doses may be undertaken in an effort to 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 of the potential pitfalls, including stability of parent and metabolites and matrix effects from unknown metabolites, endogenous components, and dosing vehicles such as polyethylene glycols, a frequently used formulation for IV dosage.
As a drug candidate progresses further, translational medicine often will define biomarkers from pharmacology or metabolomics studies that can be used in clinical trials. Over the past 15 years, there has been considerable progress in the use of LC-MS to measure small biochemicals and peptides. The ability to use biomarkers as a surrogate endpoint and to ensure a reliable PK-PD relationship is a common strategy for most drug development programs.
1.3 LC-MS BIOANALYSIS IN PRECLINICAL DEVELOPMENT OF DRUGS
1.3.1 Toxicokinetics
Drug safety assessment studies regulated under good laboratory practice (GLP) are an important part of the preclinical development activities. In a typical toxicology study, toxicokinetic evaluation is performed in order to ascertain adequate drug exposure in the study animals. To support bioanalysis of toxicokinetic samples from the GLP studies, generic LC-MS methods used during drug discovery may no longer be suitable. Modification of the generic method or redevelopment of the respective method is often needed, followed by full assay validation according to the current regulatory guidance and industrial practices (EMA, 2011; FDA 2001; Viswanathan et al., 2007). These requirements are implemented to ensure adequate sensitivity, selectivity, accuracy, precision, reproducibility, and a number of other performance related criteria for a given method.
Preclinical toxicity studies typically employ a broad dose range that could result in a wide range of circulating concentrations of the test compound. Test samples containing analyte levels exceeding the upper limit of quantification (ULOQ) need to be diluted, a step that can sometimes introduce errors. On the other hand, the lower limit of quantification (LLOQ) must be established so that the assay is sensitive enough to measure trough levels from the lowest dose, yet not too sensitive that background noise (false positives) in specimens collected from control animals is detected. A useful rule-of-thumb is to set the LLOQ at ca. 5% of the anticipated peak concentration following the low dose, which should allow accurate analyte measurement for approximately four half-lives.
Different strains of rats such as Sprague Dawley, Wistar Hannover, and Fischer are used in toxicology studies. The LC-MS assay method should be validated using the matrix from the same strain. The beagle dog is generally the default nonrodent species. Nonhuman primates, such as cynomolgus, rhesus, or marmoset monkeys, are occasionally used. The most common use of nonhuman primates is when assessing immunogenicity of large molecule drugs or when the metabolic profiles of dogs differ significantly from human. Drug metabolizing enzymes, such as aldehyde oxidase, can have pronounced differences across species. Matching metabolic profiles to human assures good safety coverage for all metabolites. When metabolism differs across species, metabolism-mediated toxicity can result in sensitivity within one species relative to others. For this reason, there may be a need to measure metabolites in GLP preclinical studies. Although metabolite measurement in those toxicokinetic (TK) samples might be exempt from full GLP compliance due to various reasons, for example, absence of purity certification of reference metabolites and lack of full validation of the intended LC-MS assays, care must be taken to ensure the integrity of the results generated. Often, an assay separate from the parent measurement may be set up for the occasional metabolite quantification. New guidance requires that steady-state exposures of significant metabolites in all species are obtained (Anderson et al., 2010). Non-GLP or tiered assays allow these decisions to be made without extensive validation of multiple assays (Viswanathan et al., 2007).
In parallel with clinical drug development is the continued testing of the compound in animal toxicology studies. This includes extending the safety in primary toxicology animals with longer study durations. Dose range-finding studies are conducted in preparation for the 2-year carcinogenicity studies in mouse and rat. Phototoxicity studies are performed in mice. Reproductive toxicology is performed in rats and rabbits. Bioanalytical assays need to be validated in these additional species. Again, metabolites unique to these species need to be considered.
The bioanalyst should be prepared to support LC-MS bioanalysis of tissue samples for certain programs. Extensive validation and stability determinations might be needed, sometimes for both parent drug and metabolites. Having a stable isotope labelled internal standard can help avoid problems such as differences in extraction recovery and compensate for variability due to sample processing, transfer and analysis of study tissue samples. Homogenization prior to freezing is also preferred. Nevertheless, one can never fully ensure consistent analysis from tissue samples since the spiked quality control (QC) samples cannot fully mimic the incurred tissues. The most definitive approach would be to compare tissue results obtained using LC-MS to those from LC analysis in a radiolabeled study.
1.3.2 Preclinical ADME and Tissue Distribution Studies in Animals
Preclinical studies to elucidate the absorption, distribution, metabolism, and excretion (ADME) of drug candidates are usually conducted before and during the clinical phase. Radiolabeled drug is often needed for the animal ADME or tissue distribution (quantitative whole body autoradiography) studies, although with today's LC-MS instrumentation, much information can be gathered without the use of radiolabeled isotopes. Parent drug absorption and elimination can be readily assessed using LC-MS assays. Metabolites can be determined using LC-MS under unit or high resolution conditions. Blood-to-plasma partitioning and protein binding, once done exclusively using radiolabeled drug can now be performed using highly sensitive LC-MS assays. The question of whether radiolabeled mass-balance studies in laboratory animals are still needed today has generated much discussion (Obach et al., 2012; White et al., 2013). The advance in LC-MS technology was the catalyst for this change.
1.4 LC-MS BIOANALYSIS IN CLINICAL DEVELOPMENT OF DRUGS
1.4.1 First-in-Human Studies
Upon successful completion of the preclinical safety assessment of drug candidates, the investigational new drug (IND) submission is prepared. Traditionally, first-in-human (FIH) studies have included separate single and multiple ascending dose (SAD and MAD) studies. Today, adaptive studies can include a combination of SAD and MAD. To ensure safety, a sufficiently low starting dose is selected, and the supporting bioanalytical assay usually requires an LLOQ much lower than that used in toxicology studies. For a drug candidate with a wide safety margin, a bioanalytical method with a similar dynamic range will be needed. While it might be difficult to obtain a full PK profile on the earliest doses of an ascending dose study, a full PK profile will be required when an efficacious dose is reached. In addition to defining the maximum tolerable dose and possibly biological effect, the DMPK objectives in FIH studies include defining drug absorption, dose proportionality, and systemic clearance. Metabolite profiling and measurement will also be conducted to make sure unique human metabolites do not exist and major circulating metabolites at or above 10% of total drug-related exposure at steady state are also present at comparable or greater exposure levels in at least one of the main preclinical toxicology species (FDA, 2008).
A bioanalytical LC-MS method should be developed and validated prior to completion of the study protocol. Important information such as conditions for blood sample collection, plasma harvest, sample storage, and transfer must also be verified. If samples need to be stabilized because of the presence of labile parent or metabolites, the information should be provided well in advance so that the clinical staff can be properly trained in the required sample handling procedures.
The SAD/MAD study may also include an arm to study the food effect (fasted vs. fed) on the BA of the drug. Some drugs bind to food resulting in decreased absorption. In contrast, food can stimulate bile acid secretion that helps to dissolve less soluble drugs, making them more bioavailable. A bioanalytical LC-MS method should, therefore, be evaluated in both normal and lipemic plasma. The assay should be insensitive to changes in phospholipid concentration, a common issue in electrospray ionization that requires attention during method development and validation.
Drug concentrations in urine are also typically measured to assess renal clearance. Unlike plasma, blood or serum, urine does not normally contain significant amounts of proteins and lipids. The lack of proteins and lipids in urine samples can be associated with the issue of nonspecific binding or container surface adsorption of drug molecules, especially those lipophilic and highly protein bound, in quantitative analysis of urine samples. The issue is often evidenced by the unusually low extraction recovery of the analytes of interest and/or nonlinearity of the calibration curves or highly variable QC sample results. Quick identification and effective prevention of analyte loss due to nonspecific binding or container surface adsorption must be conducted by bioanalytical scientists prior to the study so that the correct collection and storage condition can be provided (Li et al., 2010).
1.4.2 Human ADME Studies
Comprehensive information on the ADME of a drug in humans can be obtained from mass-balance studies using a radiolabeled compound, and this should be an early objective in clinical drug development (Pellegatti, 2012). Information on drug tissue distribution in rodents (e.g., rat) and the anticipated therapeutic dose are needed for planning a human ADME study. Some knowledge of the drug metabolism in vitro and in animals can help to select the position and desired specific activity of the radiolabel. Quantitative whole body autoradiography is a common tool for tissue distribution studies. Disposition of radioactivity into specific organs is quantified and scaled to human. Dosimetry calculations are performed to ensure safe radioactivity exposure limits in dosing of humans. Typically the maximum exposure limit is 1 mSv (ICRP 103, 2007). Traditional ADME studies generally use liquid scintillation counting and doses of ∼100 μCi of 14C labeled drug mixed with unlabeled drug. LC-MS for measuring unlabeled drug is often used in human ADME studies to differentiate the parent compound from its metabolite(s). For studies employing microdoses (<100 μg) or doses of low radioactivity (<1 μCi), accelerator mass spectrometry may be needed to measure the 14C labeled drug (Garner, 2005), whereas high sensitivity LC-MS methods have been used to 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 toxicology and clinical studies to satisfy Metabolites in Safety Testing requirements (Anderson et al., 2010). Obach et al. (2012) have advocated deferring the cost of this study until after proof of concept (POC) and relying on pharmacokinetic information derived from nonradiolabeled studies, namely SAD and MAD. The risk of delaying the human ADME study is that unique human metabolites may be uncovered after POC. The surprise of having significant metabolites found late in drug development can expose a lack of safety coverage or protection of intellectual property if the metabolite is active. The advancement of more powerful high resolution mass spectrometry for metabolite identification in LC-MS bioanalysis of early stage study samples helps to mitigate the risk.
1.4.3 Human Drug–Drug Interaction Studies
A drug–drug interaction (DDI) is a situation where a drug affects the activity or toxicity of another drug when both are coadministered. Interactions can be found where saturable or inducible enzymes or transporters are expressed and play a role in the absorption and disposition of the drug. DDI can increase or decrease the activity of the drug or a new effect can be produced that neither produces on its own. This interaction can occur between the drug to be developed and other concomitantly administered drugs, foods, or medicinal plants or herbs. During clinical development, DDI studies are normally conducted for the drug candidate in healthy volunteers or patients to confirm any significant observations seen during in vitro DDI studies.
From the perspective of LC-MS bioanalysis, assay specificity against the coadministered medicines and their significant metabolites needs to be demonstrated. In the case of metabolites that are difficult to obtain, interference could be discounted based upon MS detection (e.g., differing MW or MRM). On the other hand, possible interference due to drug candidates and/or their major metabolites on the accuracy of determination of DDI compounds and their significant metabolites must be checked to ensure the quality of LC-MS bioanalytical results for the DDI assessment.
1.4.4 Renal Impaired and Hepatic Impaired Studies in Human
Kidney (or renal) failure is a medical condition in which the kidneys fail to adequately filter toxins and waste products from the blood. Similarly, liver (or hepatic) failure is the inability of the liver to perform its synthetic and metabolic function as part of normal physiology. Either can be acute or chronic. Drug elimination may occur by filtration in the kidney or metabolism in the liver. When impacted by disease, drug accumulation can result in toxicity. Depending on the properties of metabolism and excretion of a drug candidate, clinical studies in renal impaired or hepatic impaired patients need to be conducted. In addition to conventional plasma samples, urine samples may be collected and analyzed. Some drugs may be metabolically activated, resulting in idiosyncratic liver toxicities. Therefore, it is important to understand both the impact of an impaired liver on the normal pharmacokinetic properties of a drug as well as the potential of a drug to impact liver function.
From the perspective of LC-MS bioanalysis, assay dynamic range must be suitable to measure exposures from any given dose, or assay integrity of sample dilution must be checked to ensure data integrity for samples with unexpected high analyte concentrations due to the impaired liver or kidney function.
1.4.5 Phase II and Phase III Studies
Moving beyond preliminary safety studies to POC studies is a milestone goal for clinical drug development. A successful program will demonstrate POC before the end of phase II studies. Therefore, moving from healthy subjects to the intended patient population is an important transition. However, patients might take more medications or are under treatment with drug combinations. With this regard, the robustness of the intended LC-MS assay should be validated free from possible interference of combination drugs and their metabolites.
Phase II and III studies are larger and more expensive. In order to support the bioanalysis of a large number of samples from these large multicentered trials, automation is an important consideration. For long-term, multicentered studies, the assay must be rugged enough to ensure storage stability. A well-planned stability assessment of drug candidate and its metabolite(s) of interest is critical as stability must cover all reported results. Any significant assay bias must also be well characterized. The entire bioanalytical work is represented in the new drug application (NDA) submission. This includes tabular and written summaries of assay validation performance of both nonclinical and clinical assays. Given that the development process of a drug may last more than a decade, it is important to maintain institutional knowledge to avoid gaps at filing.
1.4.6 “Fit-for-Purpose” Biomarker Measurement Using LC-MS in Clinical Samples
As drug candidates progress through POC studies, there is great need for LC-MS assays to measure biomarkers in clinical studies. There are numerous examples, including steroids, lipids, nucleotides, and peptides, which are directly amenable to LC-MS. Due to the endogenous nature of biomarkers, bioanalysis of those compounds usually encounters a series of challenges in maintaining analyte integrity from collection to analysis, achieving specificity, and obtaining sufficient sensitivity, especially when endogenous concentrations are downregulated. Those challenges entail special consideration and meticulous experimental design in method development, validation, and study conduct. Among the four common approaches to the preparation of standards, i.e. (1) authentic analyte in authentic matrix, (2) authentic analyte in surrogate matrix, (3) surrogate analyte in authentic matrix, and (4) charcoal or chemical stripping and immunodepletion, the last three are the ones most often applied.
2
OVERVIEW: FUNDAMENTALS OF A BIOANALYTICAL LABORATORY
SHEFALI PATEL, QIANGTAO (MIKE) HUANG, WENYING JIAN, RICHARD EDOM, AND NAIDONG WENG
2.1 INTRODUCTION
Progression from a new drug candidate to a marketed pharmaceutical takes significant scientific resources and financial investment. While in the last decades the pharmaceutical industry has steadily increased its investment in drug discovery and development, fewer drugs have been approved by the Food and Drug Administration (FDA). The risk in drug development is still tremendously high, maybe higher than ever, due to the raised barrier of entry. “Me-too” drugs are less likely to be approved and only the best of the best with superior efficacy and safety will be able to enter the market. To select the optimal drug candidates and eliminate weak ones for efficient utilization of resources, questions concerning toxicity and efficacy need to be addressed during preclinical and clinical studies. Figure 2.1 summarizes some of the major types of studies to answer these questions.
FIGURE 2.1 Typical studies supported in a BA laboratory.
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PART II
CURRENT UNDERSTANDING OF LC-MS BIOANALYSIS-RELATED REGULATIONS
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CURRENT REGULATIONS FOR BIOANALYTICAL METHOD VALIDATIONS
MARK E. ARNOLD, RAFAEL E. BARRIENTOS-ASTIGARRAGA, FABIO GAROFOLO, SHINOBU KUDOH, SHRINIVAS S. SAVALE, DANIEL TANG, PHILIP TIMMERMAN AND PETER VAN AMSTERDAM
4.1 INTRODUCTION
Ensuring patient health and safety is a common objective for the pharmaceutical industry, their contract research partners and most certainly that of health authorities that regulate both. Over the past 30 years, drugs have become more and more potent and only advances in technology have allowed the measurement of drugs and their metabolites at the lower circulating concentrations. In parallel, the users of the drug concentration data, the pharmacokineticists, have asked for data with improved accuracy and precision to enable the development of better models that can support drug registrations. The response of health authorities to the increased significance of the pharmacokinetic data within a filing has been to increase their levels of scrutiny and implement new regulations and guidance designed to ensure sound science and data quality in bioanalytical validations and the analysis of nonclinical and clinical study samples for drugs and metabolites. Bioanalytical scientists operate in this milieu of sound science, data quality, regulatory compliance and advancing technology. Over the past decade, additional pressures have been felt as the pharmaceutical industry is facing pressures to reduce its operating costs while at the same time seeks to file its drugs across the world. Reducing costs for the bioanalysis has been achieved in many laboratories through implementing appropriate technology, but it is the globalization of the filings that has caused the bioanalyst to move from inwardly looking at their laboratory operations in relation to their own country's regulations, to outwardly looking at the regulations that govern bioanalysis from multiple countries and regions. While a core of commonality exists among the rules and recommendations promulgated by various health authorities, differences do exist and sometimes conflict. To aid the bioanalytical scientist in dealing with the variety of international regulations, this chapter is intended to provide insight into the current regulations applicable to the use of liquid chromatography–tandem mass spectrometry (LC-MS/MS) in bioanalysis from a number of countries and regions including Brazil, Canada, China, the European Union, India, Japan, and the United States.
4.2 CONTEXT OF THE REGULATORY ENVIRONMENT
