MASS SPECTROMETRY IN DRUG METABOLISM AND DISPOSITION docx

MASS SPECTROMETRY IN DRUG
METABOLISM AND DISPOSITION
ffirs 1 March 2011; 11:28:26
WILEY SERIES ON PHARMACEUTICAL SCIENCE
AND BIOTECHNOLOGY: PRACTICES, APPLICATIONS
AND METHODS
Series Editor:
Mike S. Lee
Milestone Development Services
Mike S. Lee  Integrated Strategies for Drug Discovery Using Mass Spectrometry
Birendra Pramanik, Mike S. Lee, and Guodong Chen  Characterization of Impurities
and Degradants Using Mass Spectrometry
Mike S. Lee and Mingshe Zhu  Mass Spectrometry in Drug Metabolism and Disposition:
Basic Principles and Applications
ffirs 1 March 2011; 11:28:26
MASS SPECTROMETRY
IN DRUG METABOLISM
AND DISPOSITION
Basic Principles and Applications
Edited by
Mike S. Lee
Milestone Development Services
Mingshe Zhu
Bristol-Myers Squibb
ffirs 1 March 2011; 11:28:26
Copyright r 2011 by John Wiley & Sons, Inc. All rights reserved.
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Library of Congress Cataloging-in-Publication Data:
Mass spectrometry in drug metabolism and disposition: basic principles and applications / edited by
Mike S. Lee, Mingshe Zhu.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-0-470-40196-5 (cloth) 1. Drugs—Metabolism—Analysis. 2. Metabolites—Spectra.
3. Mass spectrometry. 4. Mass spectrometry. I. Lee, Mike S., 1960- II. Zhu, Mingshe.
[DNLM: 1. Pharmaceutical Preparations—metabolism. 2. Biopharmaceutics—methods.
3. Drug Design. 4. Mass Spectrometry—methods. 5. Pharmacokinetics. QV 38]
RM301.55.M367 2011

615u.7—dc22
2010028341
Printed in Singapore
10987654321
ffirs 1 March 2011; 11:28:26
CONTENTS
FOREWORD ix
Tom Baillie
PREFACE xi
Mike Lee and Mingshe Zhu
CONTRIBUTORS xv
PART I BASIC CONCEPTS OF DRUG METABOLISM AND
DISPOSITION 1
1 Progression of Drug Metabolism 3
Ronald E. White
2 Common Biotransformation Reactions 13
Bo Wen and Sidney D. Nelson
3 Metabolic Activation of Organic Functional Groups Utilized
in Medicinal Chemistry 43
Amit S. Kalgutkar
4 Drug-Metabolizing Enzymes, Transporters, and
Drug–Drug Interactions 83
Steven W. Louie and Magang Shou
5 Experimental Models of Drug Metabolism and Disposition 151
Gang Luo, Chuang Lu, Xinxin Ding, and Donglu Zhang
6 Principles of Pharmacokinetics: Predicting Human
Pharmacokinetics in Drug Discovery 197
Takehito Yamamoto, Akihiro Hisaka, and Hiroshi Suzuki
7 Drug Metabolism Research as Integral Part of Drug Discovery
and Development Processes 229

W. Griffith Humphreys
v
TOC 1 March 2011; 11:35:40
PART II MASS SPECTROMETRY IN DRUG METABOLISM:
PRINCIPLES AND COMMON PRACTICE 255
8 Theory and Instrumentation of Mass Spectrometry 257
Ge
´
rard Hopfgartner
9 Common Liquid Chromatography–Mass Spectrometry
(LC–MS) Methodology for Metabolite Identification 291
Lin Xu, Lewis J. Klunk, and Chandra Prakash
10 Mass Spectral Interpretation 321
Li-Kang Zhang and Birendra N. Pramanik
11 Techniques to Facilitate the Performance of Mass
Spectrometry: Sample Preparation, Liquid Chromatography,
and Non-Mass-Spectrometric Detection 353
Mark Hayward, Maria D. Bacolod, Qing Ping Han, Manuel Cajina,
and Zack Zou
PART III APPLICATIONS OF NEW LCÀMS TECHNIQUES
IN DRUG METABOLISM AND DISPOSITION 383
12 Quantitative In Vitro ADME Assays Using LC–MS as a
Part of Early Drug Metabolism Screening 385
Walter Korfmacher
13 High-Resolution Mass Spectromet ry and Drug Metabolite
Identification 407
Russell J. Mortishire-Smith, Haiying Zhang, and Kevin P. Bateman
14 Distribution Studies of Drugs and Metabolites in Tissue
by Mass Spectrometric Imaging 449
Richard F. Reich, Daniel P. Magparangalan, Timothy J. Garrett,

and Richard A. Yost
15 Use of Triple Quadrupole–Linear Ion Trap Mass
Spectrometry as a Single LC–MS Platform in Drug
Metabolism and Pharmacokinetics Studies 483
Wenying Jian, Ming Yao, Bo Wen, Elliott B. Jones, and Mingshe Zhu
16 Quantitative Drug Metabolism with Accelerator Mass
Spectrometry 525
John S. Vogel, Peter Lohstroh, Brad Keck, and Stephen R. Dueker
17 Standard-Free Estimation of Metabolite Levels Using
Nanospray Mass Spectrometry: Current Statutes and
Future Directions 567
Jing-Tao Wu
vi
CONTENTS
TOC 1 March 2011; 11:35:40
18 Profiling and Characterization of Herbal Medicine and
Its Metabolites Using LC–MS 579
Zeper Abliz, Ruiping Zhang, Ping Geng, Dongmei Dai,
Jiuming He, and Jian Liu
19 Liquid Chromatography Mass Spectrometry Bioanalysis of
Protein Therapeutics and Biomarkers in Biological Matrices 613
Fumin Li and Qin C. Ji
20 Mass Spectrometry in the Analysis of DNA, Protein, Peptide,
and Lipid Biomarkers of Oxidative Stress 645
Stacy L. Gelhaus and Ian A. Blair
21 LC–MS in Endogenous Metabolite Profiling and Sm all-Molecule
Biomarker Discovery 685
Michael D. Reily, Petia Shipkova, and Serhiy Hnatyshyn
Appendix 723
Index 727

CONTENTS vii
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FOREWORD
Studies in the areas of drug metabolism and pharmacokinetics have assum ed
progressively greater importance in pharmaceutical research over the pa st two
decades, reflecting an increased awareness of the critical impact on successful
drug development of the absorption, metabolism, distribution, elimination, and
toxicity (ADMET) properties of candidate therapeutic agents. Indeed, the role
of drug metabolism studies in the pharmaceutical industry, formerly limited to
later phases of the development process, now spans the continuum from early
discovery efforts through lead optimization, preclinical development, clinical
trials, and postmarketing surveillance. Information on the identities and
exposure levels of drug metabolites, first in animals and subsequently in human
subjects, represents an essential component of preclinical and clinical safety
assessment programs and, in those cases where circulating metabolites are
pharmacologically active, provides the basis for assessing their pharmacoki-
netic/pharmacodynamic (PK/PD) relationships and contribution to the effects
of the parent drug. Chemically reactive drug metabolites, which can be detected
and characterized through specialized in vitro “trapping” techniques, generally
are viewed as risk facto rs in drug development in light of their association
with several forms of drug-induced toxicities, and early information on their
identities is key to the design of optimized new chemical entities that lack this
potential liability.
The detection, structural characterization, and quantitative analysis of drug
metabolites in complex biological matrices often is a challenging endeavor,
given the low levels that derive from highly potent parent compounds that are
administered at doses of a few milligrams per day or less. As a result, stringent
demands are placed on the analytical methodology employed for drug
metabolism studies conducted either in vitro or in vivo, in terms of sensitivity
and specificity of detection, and of wide dynamic range. In this regard, mass

spectrometry, which always has been an important technique in drug metabo-
lism studies, rapidly became the dominant technology in the field following the
introduction, in the early 1990s, of the first co mmercial LCÀMS/MS systems.
Over the past decade, remarkable technical advances have been made in ion
source design and ionization methods, rapid scanning and highly sensitive mass
analyzers, efficient methods for indu cing fragmentation of parent ions, rapid-
response detectors with wide dynamic range, and powerful data acquisition and
processing systems with sophisticated software packages and expert systems
designed specifically for investigations in drug metabolism. The evolution of
ix
fbetw 1 March 2011; 11:27:46
hybrid mass analyzers for MS/MS studies, and ancillary techniques such as ion
mobility spectrometry, have added new dimensions to the mass spectrometry
experiment, while the advent of high mass resolution capabilities on an LC time
scale (even with “fast” chromatography) is having a truly revolutionary impact
on the utility of LCÀMS/MS in this field.
Mass Spectrometry in Drug Metabolism and Disposition: Basic Principles and
Applications addresses each of these areas through a series of chapters authored
by eminent scientists well versed in the application of contemporary mass
spectrometry techniques to problems in drug metabolism and pharmacoki-
netics, with an emphasis on issues in drug discovery and development. The
reader cannot help but be impressed by the capabilities of the current
generation of LCÀMS/MS instruments, which provide a combination of
sensitivity, specificity, versatility, and speed of analysis that was difficult to
envisage only a few years ago, and which have transformed the way drug
metabolism studies are conducted. One can only wonder what lies in the years
ahead!
Thomas A. Baillie
Seattle, WA
August, 2010

x FOREWORD
fbetw 1 March 2011; 11:27:46
PREFACE
Two decades ago, drug metabolism research in the pharmaceutical industry
was limited to radiolabeled in vivo drug disposition studies conducted in late
stages of development. Drug metabolite identification was accomplished via a
long and tedious process: metabolite separation and isolation followed by mass
spectrometric and nuclear magnetic resonance analysis. Now, drug metabolism
plays a critical role in the drug discovery and development process from lead
optimization to clinical drug–drug interaction studies. Commercialized liquid
chromatography/mass spectrometry (LC/MS) platforms have become the
dominant analytical instrument employed in drug metabolism and pharmaco-
kinetics (DMPK) studies and revolutionized the productivity of drug metabo-
lism research. Certainly, the need for fast, sensitive and accurate measurements
of drugs and metabolites in complex biological matrices has driven the con-
tinued development of novel LC/MS technology. Drug metabolism science and
mass spectrometry technology have been integrated into an inseparable arena
in drug discovery and development as well in related academic research
activities. This book provides a unique thesis on mass spectrometry in drug
metabolism with specific emphasis on both principles and applications in
drug design and development. We believe that this integration will provide a
unique contribution to the field that details both drug metabolism and
analytical perspectives. Therefore, this book can be used as a teaching and/or
reference tool to delineate and understand the “why” and “how” with regard to
the many creative uses of mass spectrometry in drug metabolism and disposi-
tion studies. This work, authored by internationally renowned researchers,
represents a combination of complementary backgrounds to bring technical
and cultural awareness to this very important endeavor while serving to address
needs within academia and industry.
The book is organized into three parts. Part I provides the reader with the

basic concepts of drug metabolism and disposition. These concepts are
intended to build a unique foundation of knowledge for drug metabolism
and the subsequent studies performed during drug discovery and drug devel-
opment endeavors. The book begins with an elegant perspective on drug
metabolism. This review or perhaps “state of the union” provides unique
insight into where we are, how we got there, and where we appear to be headed.
Next we delve into the details of drug metabolism with a chapter on common
biotransformation reactions. Further detail is provided in Chapter 3 from a
medicinal chemistry perspective as the metabolic activation of organic
xi
fpref 1 March 2011; 11:35:10
functional groups is described along with considerations on how to address
the reactive metabolite issues in drug design. Chapter 4 provides an overview
on metabolizing enzymes, transporters, and their involvement with drug–drug
interactions. In vitro experimental approaches to assess and predict drug–
drug interactions in humans are elaborated. Chapter 5 starts with DMPK
strategies in a drug discovery setting followed by a comprehensive overview of
various experimental models applied in drug metabolism and disposition
studies. Selection and data interpretation of the appropriate model are also
discussed. Prediction of human pharmacokinetics is the focus of Chapter 6.
Basic concepts and principles are discus sed along with the use of mathematical
models to predict pharmacokinetics. The actual use of drug metabolism
information within the pharmaceutical industry is described in Chapter 7. In
this chapter, the reader will obtain insight into the strategies used to design
experiments for characterizing drug metabolism properties and addressing drug
metabolism related issues from drug discovery to regulatory registrations.
Part II of the book highlights the principles and common practices of
mass spectrometry in drug metabolism. The basic concepts and theory of
mass spectrometry are presented in Chapter 8. In this chapter the reader will be
able to obtain an updated thesis on the major components of this enabling

instrumentation as well as the various mass analyzer platforms in use today.
Some of the most common LC/MS-based methods used for metabolite
identification is described in Chapter 9. Strategies for identification are reviewed
and include a variety of mass spectrometry formats. Chapter 10 provides
a review of common fragmentation reactions in the gas phase that are the
foundation for mass spectral interpretation. Detailed examples provide
the reader with the necessary tools for metabolite structure eludication.
We dedicate an entire chapter to techniques that facilitate the performance of
mass spectrometry during metabolite studies. And so, Chapter 11 provides
concise background and industrial use of liquid chromatographic techniques as
well as other detection techniques that are used to enhance the analytical
performance of the mass spectrometer.
Part III of the book focuses on LC/MS techniques in drug metabolism and
disposition. The application of quantitative LC/MS in drug metabolism
and disposition is highlighted in Chapter 12. Critical studies that are routinely
performed in drug discovery that involve metabolic stability, enzyme kinetics,
metabolizing enzyme inhibition and induction, permeability and absorption,
and in vitro transporter experiments are described. Chapter 13 provides both
background and advantages of modern high-resolution mass spectrometry
along with the use of newly developed data-mining tools for in vitro and in
vivo drug metabolite identification. The understanding of the tissue distribution
of a drug and the corresponding metabolites is illustrated in Chapter 14. The
recent applications of imaging mass spectrometry for these studies are
described. Novel instrumentation and mass spectrometry scan functions of
hybrid triple quadrupole–linear ion trap mass spectrometry are discussed in
Chapter 15. The applications for both bioanalysis and metabolite identification
xii PREFACE
fpref 1 March 2011; 11:35:10
are highlighted. Quantitative drug metabolism studies using accelerator mass
spectrometry are introd uced and described in Chapter 16. The utility of

this powerful technique for microdosing and DMPK studies in early clinical
studies is described. Chapter 17 provides a provocative description of novel
approaches, typically used in the field of protein identification, to obtain
standard-free estimation of metabolite levels using nanospray mass spectro-
metry. A unique perspective on the profiling and characterization of Chinese
herbal medicine and their metabolites using LC/MS is provided in Chapter 18.
The approaches to determine the chemical composition of these medic ines and
their subsequent metabolites are discussed. The emerging area of bioanalysis of
protein therapeutics in biological matrices is discussed in Chapter 19. Unique
perspectives on digestion efficiency, internal standards, and biomarker valida-
tion are discussed in detail. Biomarker analysis is an exciting and emerging area
of interest. Chapter 20 provides the reader with real-world application of mass
spectrometry for the analysis of DNA, protein, peptide, and lipid biomarkers of
oxidative stress. Par t III of the book concludes with an updated perspective on
endogenous metabolite profiling and small-molecule biomarker discovery
(Chapter 21). In this chapter, the relationship between a perturbation and
effected biochemical pathways is described within the context of biomarker
discovery.
So, what criteria will emerge as the most desirable analytical figure of merit
for high-performance LC/MS analysis in drug metabolism? It is our sincere
hope that this book will provide an updated perspective on mass spectrometry
in drug metabolism and disposition with recent applications, novel technolo-
gies, and innovative workflows.
Finally, the authors wish to acknowledge the contributions of many who
transformed this book from idea to passion to reality. Specifically, the
contributions from the authors and their respective collaborators, the editorial
staff at John Wiley & Sons, and, of course, the families of the editors.
Mike S. Lee
Mingshe Zhu
PREFACE xiii

fpref 1 March 2011; 11:35:10
CONTRIBUTORS
ZEPER ABLIZ
Key Laboratory of Bioactive Substances and Resource Utilization of Chinese
Herbal Medicine, Ministry of Education
Institute of Materia Medica
Chinese Academy of Medical Sciences and Peking Union Medical College
Beijing, 100050 China
M
ARIA D. BACOLOD
Department of Chemistry
Lundbeck Research USA,
Paramus, New Jersey
T
OM BAILLIE
School of Pharmacy
University of Washington
Seattle, Washington
K
EVIN P. BATEMAN
Drug Metabolism and Pharmacokinetics
Merck Frosst Canada Ltd.
Quebec, H9H 3L1 Canada
I
AN A. BLAIR
Centers for Cance r Pharmacology and Excellence in Environmental Toxicology
University of Pennsylvania
Philadelphia, Pennsylvania
MANUEL CAJINA
Department of Chemistry

Lundbeck Research USA,
Paramus, New Jersey
DONGMEI DAI,
Key Laboratory of Bioactive Substances and Resource Utilization of Chinese
Herbal Medicine, Ministry of Education
Institute of Materia Medica
Chinese Academy of Medical Sciences and Peking Union Medical College
Beijing, 100050 China
xv
flast 1 March 2011; 11:34:37
XINXIN DING
Wadsworth Center,
New York State Department of Health
Albany, New York
S
TEPHEN R. DUEKER
Vitalea Science
Davis, California
T
IMOTHY J. GARRETT
Department of Medicine
University of Florida
Gainesville, Florida
S
TACY L. GELHAUS
Centers for Cancer Pharmacology and Excell ence in Environmental Toxicology
University of Pennsylvania
Philadelphia, Pennsylvania
P
ING GENG

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese
Herbal Medicine, Ministry of Education
Institute of Materia Medica
Chinese Academy of Medical Sciences and Peking Union Medical College
Beijing, 100050 China
Q
ING PING HAN
Department of Chemistry
Lundbeck Research USA,
Paramus, New Jersey
M
ARK HAYWARD
Analytical, Automation, and Formulation Laboratories
Department of Chemistry
Lundbeck Research USA
Paramus, New Jersey
J
IUMING HE
Key Laboratory of Bioactive Substances and Resource Utilization of Chinese
Herbal Medicine, Ministry of Education
Institute of Materia Medica
Chinese Academy of Medical Sciences and Peking Union Medical College
Beijing, 100050 China
xvi CONTRIBUTORS
flast 1 March 2011; 11:34:37
AKIHIRO HISAKA
Department of Pharmacy
The University of Tokyo Hospital
Faculty of Medicine
The University of Tokyo

Tokyo, 113-8655 Japan
S
ERHIY HNATYSHYN
Bioanalytical and Discovery Analytical Sciences
Applied and Investigational Metabonomics
Bristol-Myers Squibb
Princeton, New Jersey
G
E
´
RARD HOPFGARTNER
Life Sciences Mass Spectrometry
School of Pharmaceutical Sciences
University of Geneva
University of Lausanne
Geneva, Switzerland
W. G
RIFFITH HUMPHREYS
Department of Biotransformation
Bristol-Myers Squibb Research and Development
Princeton, New Jersey
Q
IN C. JI
Bioanalytical Sciences, Analytical Research & Development
Bristol-Myers Squibb
Princeton, New Jersey
W
ENYING JIAN
BA/DMPK
Pharmaceutical Research and Development

Johnson & Johnson
Raritan, New Jersey
E
LLIOTT B. JONES
Applied Biosystems Inc.
Foster City, California
A
MIT S. KALGUTKAR
Pharmacokinetics, Dynamics, and Metabolism Department,
Pfizer Global Research and Develo pment
Eastern Point Road, Groton, Conneticut
CONTRIBUTORS xvii
flast 1 March 2011; 11:34:37
BRAD KECK
Vitalea Science
Davis, California
L
EWIS J. KLUNK
Department of Drug Metabolism and Pharmacokinetics
Biogen Idec
Cambridge, Massachusetts
W
ALTER KORFMACHER
Exploratory Drug Metabolism
Merck Research Laboratories
Kenilworth, New Jersey
M
IKE LEE
Milestone Development Services
Newtown, PA

F
UMIN LI
Bioanalytical Department
Covance Laboratories
Madison, Wisconsin
J
IAN LIU
Key Laboratory of Bioactive Substances and Resource Utilization of Chinese
Herbal Medicine, Ministry of Education
Institute of Materia Medica
Chinese Academy of Medical Sciences and Peking Union Medical College
Beijing, 100050 China
P
ETER LOHSTROH
Vitalea Science
Davis, California
S
TEVEN W. LOUIE
Department of Pharmacokinetics and Drug Metabolism
Amgen, Inc.
Thousand Oaks, California
C
HUANG LU
Drug Metabolism and Pharmacokinetics
Millennium Pharmaceuticals, Inc
Cambridge, Massachusetts
xviii CONTRIBUTORS
flast 1 March 2011; 11:34:37
GANG LUO
Covance Laboratories

Madison, Wisconsin
D
ANIEL P. MAGPARANGALAN
Department of Chemistry
University of Florida
Gainesville, Florida
R
USSELL J. MORTISHIRE-SMITH
Janssen Pharmaceutical Companies of Johnson & Johnson
B-2340 Beerse, Belgium
S
IDNEY D. NELSON
Department of Medicinal Chemistry, School of Pharmacy
University of Washington
Seattle, Washington
C
HANDRA PRAKASH
Department of Drug Metabolism and Pharmacokinetics
Biogen Idec
Cambridge, Massachusetts
B
IRENDRA PRAMANIK
Chemical Research
Merck Research Laboratories
Kenilworth, New Jersey
R
ICHARD F. REICH
Department of Chemistry
University of Florida
Gainesville, Florida

M
ICHAEL D. REILY
Bioanalytical and Discovery Analytical Sciences
Applied and Investigational Metabonomics
Bristol-Myers Squibb
Princeton, New Jersey
P
ETIA SHIPKOVA
Bioanalytical and Discovery Analytical Sciences
Applied and Investigational Metabonomics
Bristol-Myers Squibb
Pennington, New Jersey
CONTRIBUTORS xix
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MAGANG SHOU
Department of Pharmacokinetics and Drug Metabolism
Amgen, Inc.
Thousand Oaks, California
H
IROSHI SUZUKI
Department of Pharmacy
The University of Tokyo Hospital
Faculty of Medicine
The University of Tokyo
Tokyo, 113-8655 Japan
J
OHN S. VOGEL
Vitalea Science
Davis, California
B

O WEN
Department of Drug Metabolism and Pharmacokinetics
Hoffmann-La Roche
Nutley, New Jersey
R
ONALD E. WHITE
White Global Pharma Consultants
Cranbury, New Jersey
J
ING-TAO WU
Drug Metabolism and Pharmacokinetics
Millennium Pharmaceuticals, Inc.
Cambridge, MA
L
IN XU
Department of Drug Metabolism and Pharmacokinetics
Biogen Idec
Cambridge, Massachusetts
M
ING YAO
Department of Biotransformation
Bristol-Myers Squibb Research and Development
Princeton, New Jersey
T
AKEHITO YAMAMOTO
Department of Pharmacy,
The University of Tokyo Hospital
Faculty of Medicine
The University of Tokyo
Tokyo, 113-8655 Japan

xx CONTRIBUTORS
flast 1 March 2011; 11:34:37
RICHARD A. YOST
Department of Chemistry
University of Florida
Gainesville, Florida
D
ONGLU ZHANG
Department of Biotransformation
Bristol-Myers Squibb
Princeton, New Jersey
H
AIYING ZHANG
Department of Biotransformation
Bristol-Myers Squibb Research & Development
Pennington, New Jersey
L
I-KANG ZHANG
Global Analytical Chemistry
Merck Research Laboratoriess
Kenilworth, New Jersey
R
UIPING ZHANG
Key Laboratory of Bioactive Substances and Resource Utilization of Chinese
Herbal Medicine, Ministry of Education
Institute of Materia Medica
Chinese Academy of Medical Sciences and Peking Union Medical College
Beijing, 100050 China
M
INGSHE ZHU

Department of Biotransformation
Bristol-Myers Squibb Research and Development
Princeton, New Jersey
Z
ACK ZOU
Department of Chemistry
Lundbeck Research USA,
Paramus, New Jersey
CONTRIBUTORS xxi
flast 1 March 2011; 11:34:37
PART I
Basic Concepts of Drug Metabolism
and Disposition
CH001 1 March 2011; 10:32:13
1 Progression of Drug Metabolism
RONALD E. WHITE
White Global Pharma Consultants, Cranbury, New Jersey
1.1 Introduction
1.2 Historical Phases of Drug Metabolism
1.2.1 The “Chemistry” Phase (1950À1980)
1.2.2 The “Biochemistry” Phase (1975ÀPresent)
1.2.3 The “Genetics” Phase (1990ÀPresent)
1.2.4 The “Biology” Phase (2010 and Beyond)
1.3 Next Step in the Progression of DM
1.3.1 New Regulatory Expectation
1.3.2 New Challenges for Technology
1.4 Perspective on the Magnitude of the Challenge
1.4.1 Ultimate Limits on Metabolite Quantitation
1.4.2 Practical Limits on Metabolite Quantitation
1.4.3 Natural Limit Due to Dose Size

1.5 Are There More Sensitive Alternatives to MS?
1.6 Summary
References
1.1 INTRODUCTION
In a certain sense, the field of drug metabolism (DM) is standing still. More
specifically, the basic experiment of drug metabolism (i.e., administering a new
drug to an animal or human and determining the structures, amounts, and
disposition of the metabolites) has changed very little over a period of decades.
Remarkably, the experimental design and resulting data set from a typical
absorption, distribution, metabolism, and excretion (ADME) study conducted
today would be instantly recognized and understood by DM scientists from 50
years ago. This is not the case with most other disciplines in the life sciences.
Mass Spectrometry in Drug Metabolism and Disposition: Basic Principles and Applications,
First Edition. Edited by Mike S. Lee and Mingshe Zhu.
r 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.
3
CH001 1 March 2011; 10:32:13
For instance, 20 years ago protein sequencing was based on peptide chemistry,
and the basic experiment was the assay of amino acids released from tryptic
peptides, a process that required months or years to complete. Now, protein
sequencing is based on nucleotide chemistry, and the basic experiment is the
automated assay of oligonucleotides from partial hydrolysis of complementary
deoxyribonucleic acid (cDNA) a process that takes about a day. The reason
that ADME experiments have not evolved much is that we have never devised a
surrogate for the whole human body for metabolism studies. All systems tried
up to this point (e.g., animals, transgenic animals, perfused organs, in vitro
incubations, three-dimensional (3D) microfluidic cell culture devices, in silico
calculations) fail to reliably predict the actual metabolic fate of NCEs. To be
sure, the technology we use for the ADME experiment has advanced greatly,
but even with the newest methods, DM is still essentially a chemical exercise at

its core, and the backbone technology remains mass spectrometry (MS).
However, if we consider the more complete picture, DM has expanded
enormously in scope and level of understanding in those 50 years. While the
chemistry-based core remains intact and actively growing, several other kinds
of DM studies have layered over the core, all coexisting and relevant in
contemporary DM science. Thus, in addition to the purely chemical description
of the structures of metabolites and probable chemical mechanisms of their
formation, we now have a very good biochemical understanding of the various
enzymes that catalyze these biotransformation processes, as well as a cellular
and genetic understanding of the expression and regulation of those enzymes.
We are even making progress toward reliable prediction of the fates of
xenobiotic substances in human beings (Anderson et al., 2009), although this
goal remains out of reach for the present. The current level of understanding of
DM is presented by several experts in Part I of this book.
1.2 HISTORICAL PHASES OF DRUG METABOLISM
Near the end of the twentieth century, I suggested that there had been four
overlapping phases of DM in industrial drug discovery and development
(White, 1998). These can be summarized as follows.
1.2.1 The “Chemistry” Phase (1950À1980)
During this period, only a descriptive account of the disposition of a new
chemical entity (NCE) was provided, largely consisting of chemical information.
Major urinary and fecal metabolites were isolated and identified by classic
chemical techniques including column and thin-layer chromatography, crystal-
lization, and derivatization. Eventually, spectroscopy was used for the structural
elucidation, including mass spectroscopy, infrared, and nuclear magnetic reso-
nance (NMR). These techniques required much smaller quantities to be isolated
and allowed high-performance liquid chromatography (HPLC) to replace
4 PROGRESSION OF DRUG METABOLISM
CH001 1 March 2011; 10:32:13
column chromatography. Interestingly, early in this period R.T. Williams pub-

lished his monograph Detoxication Mechanisms, which can be considered the
first identification of DM as a discrete field of study (Murphy, 2008). The
publication of that book also showed that academic researchers were beginning
to think about the biological basis and implications of DM, although this way of
thinking took some time to make an impact in the industrial world.
1.2.2 The “Biochemistry” Phase (1975ÀPresent)
Starting in the mid-1970s, we b egan to d etermine the un derlying biochemical
processes responsib le for the disposition of xenobiotics (e.g., which enzymes were
involved). Illustrating the indistinct separation of these phases, the pioneers in this
phase often came from a chemical background, and they sought to d escribe
the enzymes in chemical terms. The proteins were isolated so that they could
be treated as discreet chemical reagents, describable in classical chemical terms of
composition, reaction stoichiometry, thermodynamics, and reaction mechanism.
However, afte r about a decade or so, the chemical approach to studying
the enzymes transitioned to a biochemical and cell biology approach in which
enzyme kinetics and proteinÀprotein and membraneÀprotein interactions
became the “hottest” t opics. All of the important DM enzymes w ere ch aracter-
ized, named, and even made commercially available to industrial researchers. The
latest advance in the biochemistry phase was the realization that even the exposure
of drugs to t he drug-metabolizing enzymes was a biochemical event, mediated by
physical enzymes called transporters (Wu and Benet, 2005). Advances in our
understanding of the b iochemistry of DM in academic laboratories were reflected
in a greater expectation by regulatory agencies that a biochemical description be
provided in addition to the purely chemical d escription of DM of an NCE.
1.2.3 The “Genetics” Phase (1990ÀPresent)
In this phase, we began to account for individual variations in the pharmaco-
kinetic rates and molecular sites of metabolism by genotyping human test
subjects with respect to an ever-growing list of genetically polymorphic drug-
metabolizing enzymes. Equally important, regulation of DM enzymes was
recognized to occur mainly at the gene expression level, whether resulting from

heredity, disease process es, or environment. This pharmacogenetic character-
ization has become a routine expectation for the registration packages of NCEs
and continues to expand. As before, the new genetic information did not
replace any previous requirements for DM information but instead added an
additional dimension to that information package.
1.2.4 The “Biology” Phase (2010 and Beyond)
We are beginning to view drug metabolism in terms of systems biology. This
involves taking a holistic view of the simultaneous interaction of a xenobiotic
1.2 HISTORICAL PHASES OF DRUG METABOLISM 5
CH001 1 March 2011; 10:32:13
molecule with all the enzymes and receptors in the human body. Some of
these receptors are the pharmacological targets that lead to therapeutic benefits,
some are unintended targets that generate adverse events and toxicities, and some
are the enzymes and nuclear receptors of DM. In our overall description of the
disposition of the compound, interactions of the compound with these DM
targets are especially complex to relate to safety and efficacy. When designing a
practical clinical medicine, we need to establish a balance between too rapid
metabolism, leading to reduced efficacy, and too sluggish metabolism, leading to
accumulation and possible toxicity. In the clinic, we need to determine whether
metabolism decreases or increases the desired pharmacological effect (i.e., active
metabolites). And, finally, in this holistic biological view, we need to assess how
the metabolites interact with all the off-target human enzymes and receptors,
especially the phenomenon of reactive metabolites covalently binding to proteins
and nucleic acids, leading to toxic sequelae (Baillie, 2009).
These four phases are graphically depicted in Figure 1.1. They are layered in
the figure because we continue to do all the activities of each preceding phase as
we proceed through the evolution of industrial DM. Thus, the total amount of
DM characterization work for a new drug has increased dramatical ly over the
Cumulative Total DM Input
Needed For Registration

1950 1970 1990 2010
S
G
B
C
FIGURE 1.1 Progression of amounts of DM information required for regulatory
filing of a new chemical entity. The vertical axis is the total amount of DM information
in the registration application on a relative scale. The information classes are segregated
as discussed in the text. The chemistry component (C, black) continually increases with
time but abruptly increases about 2010 due to enhanced regulatory surveillance of
metabolites. Starting around 1975, biochemistry (B, dark gray) begins to be included in
the DM characterization and slowly increases with time. Genetics information (G, light
gray) continues to increase, mainly in clinical trials. The apparent jump in B and G work
around 2010 is due to the abrupt increase in C. Actual B and G work would not increase.
Systems biology (S, white) is nearly zero in 2010 but is expected to increase subsequently.
6
PROGRESSION OF DRUG METABOLISM
CH001 1 March 2011; 10:32:13
years. The meaning of the step increase in work at around 2010 in the figure will
be discussed in the next section.
1.3 NEXT STEP IN THE PROGRESSION OF DM
The first three of these historical phases of industrial DM serve to summarize
and rationalize the scientific questions of yesteryear and today. The questions
of tomorrow are described by the biology phase. However, now we can also
discern the beginning of an additional new trend that could be called the
“regulatory” phase. This phase is not primarily concerned with the physiolo-
gical process of DM, as are the other phases. Instead, the regulatory phase is
concerned with the human safety of the metabolites, once they are formed. But
even though the focus of this phase is safety, it may well pro duce the greatest
increment of additional DM work to be done in the future.

1.3.1 New Regulatory Expectation
Regulatory interest in metabolites has developed into a formal Guidance for
Industry (Safety Testing of Drug Metabolites) issued by the U.S. Food and Drug
Administration (FDA), which instructs sponsors on the qualitative a nd quantita-
tive characterization of metabolites in both clinical and preclinical toxicological
settings (U.S. FDA, 2008). A similar concern abo ut met abolite s afety is expressed
in a Guidance from the International Conference on Harmonisation (ICH),
currently in draft s tage (ICH, 2009). We may succinctly state the requirement as
follow s: Human circula tin g metabol ite s that ex ceed 10% of the total exposure of
all drug-related materials in circulation at pharmacokinetic steady state require
safety assessment b efore large-scale clinical trials can proceed. These Guidances
have implications for bioanalysis and safety assessment, but here we wish to focus
on the implications for b iotransformation studies. Development of these Gui-
dances, though initiated by an industry-sponsored group (Baillie et al., 2002), has
resulted in an in evita ble regulato ry emphasis on metabolite characterization much
earlier than traditionally carried out. Importantly, the relative levels of parent drug
and metab olites at pharmacokinetic stead y s tate ha ve be en little studied pre-
viously. Consequently, we can expect surprises, possibly even new phenomena, as
we watch the time evolution of drug metabolism during the approach to steady
state. Th e t raditional approach to d efinitive metabolite c haracterization ( i.e.,
single-dose
14
C-labeled c linical ADME studies performed during Phase II or III)
is in adequ ate for the new regulatory demands and eith er a new ap proach to
14
C
studies or new nonradiolabel-based method ology is r equired.
1.3.2 New Challenges for Technology
Distressingly, both the qualitative and quantitative requirements of the
new paradigm potentially push the existing technology past present limits.

1.3 NEXT STEP IN THE PROGRESSION OF DM 7
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