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IDENTIFICATION AND QUANTIFICATION
OF DRUGS, METABOLITES,
DRUG METABOLIZING ENZYMES,
AND TRANSPORTERS
Concepts, Methods, and Translational Sciences
IDENTIFICATION AND
QUANTIFICATION
OF DRUGS,
METABOLITES, DRUG
METABOLIZING
ENZYMES, AND
TRANSPORTERS
Concepts, Methods, and
Translational Sciences
Edited by
SHUGUANG MA
SWAPAN K. CHOWDHURY
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
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This book and the individual contributions contained in it are protected under copyright by the Publisher
(other than as may be noted herein).
Notices
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understanding, changes in research methods, professional practices, or medical treatment may become
necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using
any information, methods, compounds, or experiments described herein. In using such information or methods
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Contributors
Farah Al Qaraghuli Department of Pharma- Liam Evans Hypha Discovery Ltd., Oxford-
ceutical Sciences, School of Pharmacy and shire, United Kingdom
Pharmaceutical Sciences, The State University Raymond Evers Department of Pharmacokinet-
of New York at Buffalo, Buffalo, NY, United ics, Pharmacodynamics and Drug Metabolism,
States Merck & Co Inc., Kenilworth, NJ, United States
Ravindra Varma Alluri Clinical Pharmacology Robert S. Foti Pharmacokinetics, Pharmaco-
and Safety Sciences, R&D BioPharmaceuticals, dynamics and Drug Metabolism, Merck
AstraZeneca, Cambridge, United Kingdom Research Laboratories, Boston, MA, United
Sophie M.A. Argon Department of Pharma- States
ceutics, School of Pharmacy, University of Adrian J. Fretland DMPK, Research and Early
Washington, Seattle, WA, United States Development, Oncology R&D, AstraZeneca,
Piyush Bajaj Drug Safety Research and Evalua- Waltham, MA, United States
tion, Takeda Pharmaceutical International Co., Christopher Gemski Translational Research
Cambridge, MA, United States Bioassay and Immunogenicity Group, Drug
Tashinga E. Bapiro DMPK, Research and Early Metabolism and Pharmacokinetic Department,
Development, Oncology R&D, AstraZeneca, Takeda Pharmaceuticals International Co.,
Cambridge, United Kingdom Cambridge, MA, United States
Abdul Basit Department of Pharmaceutics, Anima Ghosal Independent Consultant,
University of Washington, Seattle; Depart- Piscataway, NJ, United States
ment of Pharmaceutical Sciences, Washington Jia Hao Drug Metabolism, Gilead Sciences
State University, Spokane, WA, United States Inc, Foster City, CA, United States
Andreas Brink Roche Pharma Research Satyajeet Haridas Translational Research Bio-
and Early Development, Roche Innovation assay and Immunogenicity Group, Drug
Center Basel, F. Hoffmann-La Roche Ltd, Basel, Metabolism and Pharmacokinetic Department,
Switzerland Takeda Pharmaceuticals International Co.,
Tingting Cai Laboratory Testing Division, Cambridge, MA, United States
WuXi AppTec, Nanjing, China Simon Hauri Roche Pharma Research and Early
Jose Castro-Perez Agios Pharmaceuticals, Inc., Development, Roche Innovation Center Basel,
Cambridge, MA, United States F. Hoffmann-La Roche Ltd, Basel, Switzerland
Jae H. Chang Preclinical Development, ORIC Nina Isoherranen Department of Pharma-
Pharmaceuticals, South San Francisco, CA, ceutics, University of Washington, Seattle,
United States WA, United States
Eugene Chia-Te Chen Department of Drug Wenying Jian DMPK, Janssen R&D, Spring
Metabolism and Pharmacokinetics, Genentech, House, PA, United States
South San Francisco, CA, United States Kevin Johnson Drug Metabolism and Pharma-
Marie Croft Pharmaron ABS, Germantown, cokinetics, Genentech, South San Francisco,
MD, United States CA, United States
xi
xii Contributors
Barry Jones DMPK, Research and Early Devel- Kaushik Mitra Department of Drug Metabo-
opment, Oncology R&D, AstraZeneca, Cam- lism and Pharmacokinetics, Janssen Research
bridge, United Kingdom and Development, Springhouse, PA, United
Robert S. Jones Drug Metabolism and Pharma- States
cokinetics, Genentech, South San Francisco, Diana Montgomery Department of Pharma-
CA, United States cokinetics, Pharmacodynamics and Drug
Jan Felix Joseph Freie Universitaet Berlin, Insti- Metabolism, Merck & Co Inc., Kenilworth, NJ,
tute of Pharmacy—Pharmaceutical Analysis; United States
Freie Universitaet Berlin, Department of Alexandra L. Orton DMPK, Research and Early
Biology, Chemistry, Pharmacy, CoreFacility Development, Oncology R&D, AstraZeneca,
BioSupraMol, Berlin, Germany Cambridge, United Kingdom
S. Cyrus Khojasteh Drug Metabolism and Katie H. Owens Department of Pharmaceutics,
Pharmacokinetics, Genentech, South San School of Pharmacy, University of Washing-
Francisco, CA, United States ton, Seattle, WA, United States
Yurong Lai Drug Metabolism, Gilead Sciences Axel P€ ahler Roche Pharma Research and
Inc, Foster City, CA, United States Early Development, Roche Innovation Center
Hoa Le Drug Metabolism, Gilead Sciences, Basel, F. Hoffmann-La Roche Ltd, Basel,
Foster City, CA, United States Switzerland
Xiaomin Liang Drug Metabolism, Gilead Sci- Maria Kristina Parr Freie Universitaet Berlin,
ences Inc, Foster City, CA, United States Institute of Pharmacy—Pharmaceutical Analy-
sis, Berlin, Germany
Liming Liu Product Development, Curon
Biopharmaceutical Ltd, Shanghai, People’s Shefali Patel DMPK, Janssen R&D, Spring
Republic of China House, PA, United States
Filipe Lopes Roche Pharma Research and Ichiko D. Petrie Department of Pharma-
Early Development, Roche Innovation Center ceutics, School of Pharmacy, University of
Basel, F. Hoffmann-La Roche Ltd, Basel, Washington, Seattle, WA, United States
Switzerland Richard Phipps Hypha Discovery Ltd., Oxford-
Justin Q. Ly Drug Metabolism and Pharmacok- shire, United Kingdom
inetics, Genentech, South San Francisco, CA, Chandra Prakash Agios Pharmaceuticals, Inc.,
United States Cambridge, MA, United States
Shuguang Ma Drug Metabolism and Pharma- Bhagwat Prasad Department of Pharmaceutics,
cokinetics, Genentech Inc., South San Francisco, University of Washington, Seattle; Department
CA, United States of Pharmaceutical Sciences, Washington State
Roshini Markandu DMPK, Research and Early University, Spokane, WA, United States
Development, Oncology R&D, AstraZeneca, Isabelle Ragueneau-Majlessi Department of
Cambridge, United Kingdom Pharmaceutics, School of Pharmacy, Univer-
Rosalinde Masereeuw Division of Pharmacol- sity of Washington, Seattle, WA, United States
ogy, Utrecht Institute for Pharmaceutical Venkatesh Pilla Reddy DMPK, Research and
Sciences, Utrecht, The Netherlands Early Development, Oncology R&D; Depart-
J. Eric McDuffie Investigative & Mechanistic ments of Modeling and Simulation, Early
Toxicology, Janssen Research & Development, Oncology Drug Metabolism and Pharma-
San Diego, CA, United States cokinetics, R&D Oncology, AstraZeneca,
Cambridge, United Kingdom
Contributors xiii
Ellen Riddle Drug Metabolism, Gilead Sciences Caisheng Wu School of Pharmaceutical Sci-
Inc, Foster City, CA, United States ences, Xiamen University, Xiamen, China
Qian Ruan Pharmaceutical Candidate Charac- Graeme C. Young GlaxoSmithKline Research
terization, Bristol-Myers Squibb, Princeton, and Development Ltd., David Jack Centre,
NJ, United States Ware, United Kingdom
Simone Schadt Roche Pharma Research and Jingjing Yu Department of Pharmaceutics,
Early Development, Roche Innovation Center School of Pharmacy, University of Washing-
Basel, F. Hoffmann-La Roche Ltd, Basel, ton, Seattle, WA, United States
Switzerland Lushan Yu Institute of Drug Metabolism and
Dhaval K. Shah Department of Pharmaceutical Pharmaceutical Analysis, Zhejiang University,
Sciences, School of Pharmacy and Pharma- Hangzhou, People’s Republic of China
ceutical Sciences, The State University of New Su Zeng Institute of Drug Metabolism and
York at Buffalo, Buffalo, NY, United States Pharmaceutical Analysis, Zhejiang University,
Julia Shanu-Wilson Hypha Discovery Ltd., Hangzhou, People’s Republic of China
Oxfordshire, United Kingdom Haeyoung Zhang Department of Pharma-
Kelly MacLennan Staiger Drug Metabolism, ceutics, University of Washington, Seattle,
Gilead Sciences Inc, Foster City, CA, United States WA, United States
Jonathan Steele Hypha Discovery Ltd., Oxford- Wanying Zhang Department of Pharma-
shire, United Kingdom ceutical Sciences, School of Pharmacy and
Manthena V.S. Varma Medicine Design, Pharmaceutical Sciences, The State Univer-
Worldwide R&D, Pfizer Inc., Groton, CT, sity of New York at Buffalo, Buffalo, NY,
United States United States
Matthew P. Wagoner Drug Safety Research Andy Z.X. Zhu Department of Drug Metabo-
and Evaluation, Takeda Pharmaceutical Inter- lism and Pharmacokinetics, Takeda Pharma-
national Co., Cambridge, MA, United States ceuticals International Co., Cambridge, MA,
United States
Naidong Weng DMPK, Janssen R&D, Spring
House, PA, United States Mingshe Zhu MassDefect Technologies, Prince-
ton, NJ, United States
Stephen Wrigley Hypha Discovery Ltd.,
Oxfordshire, United Kingdom
Foreword
It is my great pleasure to write the fore- identified many breakthroughs that have
word for this excellent book. The study of ultimately contributed to bringing drugs to
drug metabolism and disposition is a mature patients with an urgent need for better trea-
science, but still essential in drug discovery tments. It is very gratifying that the number
and development. Indeed, a quick search of of drugs approved by the FDA is increasing
PubMed, using “drug metabolism” as the steadily with 38 NMEs (new molecular en-
search term, came up with more than tity) and 10 BLAs (biological license applica-
18,000 hits. One of the earlier articles is by tion) approved by the US Food and Drug
Bernard Brodie, considered to be the founder Administration (FDA) in 2019 [2]. The level
of modern pharmacology and a major con- of innovation is best illustrated by about half
tributor to the study of drug metabolism. of the approved drugs in 2019 having a
His article—published in the Journal of Phar- breakthrough designation. Drug metabolism
macy and Pharmacology in 1956—is titled and pharmacokinetics (DMPK) scientists are
“Pathways of Drug Metabolism” and it is based fully embedded in project teams in drug dis-
on a lecture at the University of London [1]. covery and work hand in hand with medici-
It describes his work over the decade and nal chemists on the identification of drugs
some of it is based on collaborations with with superior properties. These scientists
other pioneers in the field such as Julius have become very good at dialing out the
Axelrod. One sentence still resonates very “known unknowns” such as metabolism by cy-
much and it actually covers much of the ma- tochrome P450 enzymes. However, this has
terial described in this book: actually resulted in having to deal with much
more complex drug disposition pathways in-
Finally, it is thought that a detailed knowl- volving less well-studied drug metabolizing
edge of enzymes involved in drug “detoxifica- enzymes and also more and more drug
tion” might help the medicinal chemist to transporters. This trend is also fueled by a
develop compounds of either high or low stabil- shift toward more and more drugs having
ity in the body, whichever would be more de- beyond the “rule of five” molecular proper-
sirable in gaining a desired therapeutic result. ties [3]. Indeed, the average molecular
weight of drugs approved by the FDA in
Drug metabolism sciences have advanced 2016 and 2017 was more than 500 Da and
tremendously since the publication of this ar- the median clogP of drugs approved from
ticle and this progress was to a large extent 2008 to 2017 is now 3.3, in part driven by try-
enabled by advances in the availability of ing, for example, to disrupt protein-protein
biochemical reagents and bioanalytical tech- interactions. An additional consequence of
niques, in particular mass spectrometry. this shift in properties is that in vitro to
Both academic and industrial scientists have in vivo extrapolation of ADME properties
xv
xvi Foreword
to predict the human pharmacokinetics has latter topic is covered in Chapter 5. The last
become more complex and the same can be chapter in the first section (Chapter 6)
said of predicting drug-drug interaction— focuses on accelerator mass spectrometry,
many of which now involve drug trans- a powerful tool to study ADME and the
porters. If anything, the involvement of absolute bioavailability in humans.
DMPK scientists is now more important than The second section addresses “Drug me-
ever because of the pursuit of novel molecular tabolizing enzymes, transporters and drug drug
modalities in academia and in the pharma- interactions.” Cytochrome P450-mediated
ceutical industry; a few that come to mind and non-cytochrome P450-mediated metab-
are antibody-drug conjugates, protein de- olism is discussed in Chapters 7 and 8.
graders, and macrocyclic peptides. DMPK In vitro to in vivo prediction of drug-drug in-
scientists can help make drugs out of these teraction is described in Chapter 9 while
hard-to-drug modalities. Finally, the integra- Chapter 10 specifically focuses on the role
tion of this diverse array of in vitro and in vivo of transporters in drug disposition and
data is enabled by computational modeling drug-drug interaction, and Chapter 11 on
and simulation. Physiologically based phar- the clinical relevance of these drug-drug
macokinetic (PBPK) modeling is a very pow- interactions. Making predictions using PBPK
erful tool to predict human pharmacokinetics modeling relies on accurate knowledge of
and study drug-drug interaction as well as physiologic constants and, most recently,
the pharmacokinetics in special populations mass spectrometry has been used to deter-
and infants. Pharmacokinetic/pharmaco- mine the abundance of drug metabolizing
dynamic modeling and its extension, quanti- enzymes and transporters in various tissues.
tative systems pharmacology, can help This is addressed in Chapter 12. Finally,
translate preclinical findings to the clinic. Chapter 13 focuses on disease-drug interac-
This book is comprised of many excellent tions mediated by therapeutic proteins such
chapters written by experts in the field that as interleukins.
address some of the challenges highlighted The third section focuses on “Strategy re-
in the previous paragraph. The first section lated to drug metabolism and safety.” Metabo-
focuses on “Techniques for identifying and lites in safety testing continues to be a
quantifying drugs and metabolites.” Chapters highly relevant area of research in drug dis-
1–3 introduce the readers to the latest ad- covery and development and it is addressed
vances in bioanalysis, in particular mass in Chapter 14. A close look at the (patent) lit-
spectrometry for the analysis of drugs, their erature indicates that finding drugs that bal-
metabolites, and endogenous biomarkers. In ance potency and metabolic stability can still
contrast to 20 years ago, high-resolution be problematic, and therefore many com-
mass spectrometry has now become routine panies incorporate deuterium to enhance
and it has had a great impact on the study metabolite stability and lower the dose—
of biotransformation. Of course, mass spec- see Chapter 15 for details. The focus on novel
trometry is frequently not sufficient to iden- chemical space and more molecular diversity
tify the definitive structure of a metabolite, has introduced more chirality in molecules
and hence Chapter 4 focuses on strategies and the impact of that on pharmacology, tox-
for generating metabolites and characteriza- icology, and drug metabolism is described in
tion via NMR. Supercritical fluid chromatog- Chapter 16. Drug-induced liver injury and
raphy has become easier to interface and it new predictive models for renal injury are de-
can greatly facilitate chiral separation; the scribed in Chapters 17 and 18, respectively.
Foreword xvii
Finally, Chapter 19 focuses on immuno- excellent set of chapters that describe the
genicity as a key component of antibody state of the art in ADME sciences as it
development. relates to drug discovery and development.
The fourth and last section is dedicated to The efforts by all authors, and in particular
“Translational sciences.” The use of geneti- the editors Shuguang Ma and Swapan
cally modified rodents is explored further Chowdhury, are greatly appreciated. This
in Chapter 20, while Chapter 21 speaks to book will be used as a resource for many
the use of CRISPR to advance in vitro ADME years to come.
models. In vitro to in vivo extrapolation of Cornelis E.C.A. Hop
hepatic and renal clearance is discussed in
Chapter 22. The breadth of our science is
nicely illustrated by Chapter 23 which de- References
scribes the role of mathematical modeling [1] B.B. Brodie, Pathways of drug metabolism, J. Pharm.
in translational sciences. Last, but by no Pharmacol. 8 (1) (1956) 1–17.
means least, Chapter 24 elegantly defines [2] A. Mullard, 2019 FDA drug approvals, Nat. Rev.
Drug Discov. 19 (2) (2020) 79–84.
ways to predict the human efficacious dose
[3] M.D. Shultz, Two decades under the influence of
using PK/PD modeling. the rule of five and the changing properties of ap-
As is the case with many compilations, a proved oral drugs, J. Med. Chem. 62 (4) (2019)
lot of effort has gone into assembling an 1701–1714.
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Preface
Some 15 years ago, one of us (SKC) com- and excretion (ADME), the potential for he-
piled the first edition of this book that patic and renal toxicity, immunogenicity of
covered the most up-to-date information biotherapeutics, and translational tools for
previously available on the strategies, predicting human dosage, safety, and effi-
methods, applications, and implications of cacy of small molecules and biologics.
scientific data on the role of enzymes and This book is organized into four sections:
transporters in the disposition of pharma- (1) techniques for identifying and quantify-
ceuticals. The book was a great success and ing drugs and metabolites, (2) drug metabo-
was widely used as a valuable resource by lism enzymes, transporters, and drug-drug
scientists in both industry and academia. interactions, (3) strategies related to drug
Since then, a lot has changed in the field of metabolism and safety, and (4) translatio-
drug metabolism, including how recent nal sciences. The book contains 24 chapters
scientific advances are being utilized to discovering the most recent, novel scientific
cover and develop safer medicines. There- breakthroughs and how they are utilized to
fore, it has become apparent that a new develop medicines in the modern era. It is
edition of the book is required to fully cap- our sincere hope that this material will serve
ture these profound changes, which involves as an important tool and desk reference for
the implementation of newer technologies in pharmacologists, toxicologists, clinical scien-
the discovery and development of medicines tists, and students interested in the fields
to treat a wide range of maladies. With much of pharmacology, biochemistry, and drug
enthusiasm from the publisher, we collabo- metabolism.
rated to assemble a comprehensive treatise Finally, we wish to acknowledge the con-
that would capture how the latest scientific tributions of the many scholars who partici-
findings are having a fundamental impact pated in and contributed to this book from
on the utilization of these novel advances conception and passion into print. We also
in drug research. This second edition is want to extend our gratitude to the contribu-
completely updated and provides an over- tions of the editorial staff and production
view of the last decade’s numerous improve- manager at Elsevier, and last but not least,
ments in analytical technologies for the the families of the editors for their encour-
detection and quantification of drugs, metab- agement, love, and support.
olites, and biomarkers. This new edition goes
beyond conventional LC-MS and features
all-new chapters, including: how to evaluate Shuguang Ma
drug absorption, distribution, metabolism, Swapan K. Chowdhury
xix
C H A P T E R
1
Bioanalysis of small and large
molecule drugs, metabolites, and
biomarkers by LC-MS
Naidong Weng, Shefali Patel, Wenying Jian
DMPK, Janssen R&D, Spring House, PA, United States
1 Introduction
Bioanalysis, often shortened to BA, is a subdiscipline within pharmaceutical research and

development (R&D). Contemporary bioanalysis quantitatively analyzes very low quantity
but highly variable levels (pg/mL-μg/mL) of drug candidates, their metabolites, endogenous
biomarkers, etc. in extremely complicated biological matrices such as plasma, blood, urine,
and tissues which are harvested from different types of animal species (rodents, dogs,
nonhuman primates, etc.) and humans [1]. Bioanalysis supports discovery, nonclinical
(tox), and clinical studies (Fig. 1). Fig. 1 shows typical studies an integrated bioanalytical func-
tion would support.
Bioanalytical data are used for calculating pharmacokinetic parameters such as bioavail-
ability, bioequivalence, drug and metabolites exposure, clearance, their distribution into
various body organs, correlation of pharmacokinetics (PK) effects and pharmacodynamics
(PD) changes, etc. Thus, bioanalysis plays a pivotal role in moving drug candidates from early
discovery all the way to regulatory filing and postmarket surveillance in the entire drug dis-
covery and development process. In today’s dynamic drug discovery and development
environment, bioanalytical scientists not only provide pivotal data but also actively engage
in project/program go/no-go discussions, along with colleagues from other functional areas.
While the most essential element of bioanalysis is to use analytical chemistry knowledge and
state-of-the-art instruments to provide reliable and accurate measurement, knowledge from
relevant disciplines such as biotransformation, pharmacokinetics, biology, pharmacology,
etc. is invaluable for ensuring appropriate conduct of bioanalysis.
Identification and Quantification of Drugs, Metabolites, Drug Metabolizing 3

Enzymes, and Transporters # 2020 Elsevier B.V. All rights reserved.
https://doi.org/10.1016/B978-0-12-820018-6.00001-6
4 1 Bioanalysis of small and large molecule drugs, metabolites, and biomarkers
Discovery Preclinical Clinical

In vitro Short term (2wk, 4 wk) TOX SAD, MAD
Plasma protein binding Long term (3mts +) TOX Metabolite assessment (MIST)
Transporter Reproductive TOX Food effect
Inhibition-induction Carcinoma studies Drug-drug interaction
Metabolic stability Micronucleus studies Comparator study
Plasma-blood distribution Animal ADME Human ADME
Bioavailability (IV/ORAL ) Population PK
In vivo Special population study (renal
Salt form selection impaired, pediatric, etc.)
Formulation Adaptive design clinical trial
Dose range finding Bioequivalence
Tissue distribution
Drug-like? Known liabilities? Superior efficacy?

FIG. 1 Exemplary bioanalytical support in drug discovery and development. TOX, toxicology; SAD, single ascend-
ing dose study; MAD, multiple ascending dose study; ADME, absorption, distribution, metabolism, and elimination;
IV, intravenous.
In this book chapter, we try to provide a brief overview of contemporary bioanalysis using
the LC-MS platform. It is an impossible task to provide a detailed and comprehensive review
of LC-MS bioanalysis in a book chapter. The case studies and literature are no doubt incom-
plete and biased toward our own experiences and publications. Interested readers are
referred to the excellent bioanalysis handbook by Li et al. for a more comprehensive overview
of this discipline [1]. Nevertheless, we hope the readers can appreciate the complexity and
dynamics of modern LC-MS-based bioanalysis for small and large molecule drugs, metabo-
lites, and biomarkers.
2 Complexity of contemporary bioanalysis
Bioanalytical support is required for important decision-making for all types of studies,
from discovery (non-GLP), to development (GLP), and to clinical (GCP) studies. Yet there
are many unique and complicated attributes of cost, quality, and timely delivery at each of
the abovementioned stages (or substage within each stage). For bioanalysis, the quality
and integrity of the bioanalytical data are ultimately the most important attributes. The right
scientific and compliance vigor must be applied to each study. The current regulatory land-
scape for regulated bioanalysis is highly complicated, with guidance from multiple regional
health authorities [2–6]. Since guidance from different regions are not totally harmonized, and
compounded by individual interpretation of different inspectors, it is still quite a challenge to
fully understand and execute the right level of compliance that can be acceptable in the global
filing. Efforts are currently being made to harmonize the guidelines into one single global
guideline ICH-M10 [7].
While timely delivery of bioanalytical data to support project decisions is a must-do item to
meet the ever-tightening drug discovery and development timelines, cost is another attribute
that should not be overlooked. The cost of bioanalysis activities should be carefully managed
to ensure that it stays within the predetermined budget. On the other side, the application of a
I. Techniques for identifying and quantifying drugs and metabolites

2 Complexity of contemporary bioanalysis 5
tiered approach, which typically consists of three tiers of assay qualification—screening as-
say, qualified assay, and validated assay with the increased levels of validation parameters—
can be used with a balance of scientific vigor and cost [8]. The European Bioanalytical Forum
(EBF) recommends exercising this approach for “nonregulated” nonclinical bioanalysis in
drug development and using “fit-for-purpose” elements in metabolite quantitation for
establishing safety coverage (MIST); urine bioanalysis; and tissue bioanalysis [9, 10]. Of
course, the actual implementation of which tier to use depends on each individual study.
For example, for urine bioanalysis, while a qualified assay could be used for most clinical
studies for understanding the urinary excretion of a drug candidate, validated assays should
be applied if renal clearance is the main route of elimination (PK end point) and/or the drug
target action is at the kidney.
There is an expansion of bioanalysis scope over the past decades. In the early days,
bioanalysis focused on supporting small molecule PK and bioequivalence (BE) from standard
formulations such as tablets and capsules. Analysis of metabolites and biomarkers rarely
occurred. Currently, PK and BE for both small and large molecules, as well as many hybrid
forms of large and small molecules such as antibody drug conjugate (ADC), are supported
by bioanalysis [11, 12]. Even for small molecules, advancement in formulation presents new
challenges for bioanalysis. For example, liposomes are widely applied in the pharmaceutical
industry due to their unique capabilities such as encapsulating and protecting the therapeutic
analytes from degradation, controlling the release rate, facilitating on-target delivery, and
reducing toxicity for drugs [13, 14]. For liposomal drug product development, validated
bioanalytical methods to determine the concentration of the encapsulated and nonencapsulated
forms (both the protein-free and protein-bound) of the active substance in biological samples
should be employed. However, establishing such bioanalytical assays can be quite challenging
due to the potential rapture of the liposome during sample collection, shipping, storage, and
analysis, which can artificially elevate the nonencapsulated concentrations [15, 16].
Peptide and protein drugs have evolved in recent years into mainstream therapeutics,
representing a significant portion of the pharmaceutical market [17]. Peptides and proteins
exhibit highly diverse structures and broad biological activities as hormones, neurotransmit-
ters, structural proteins, and metabolic modulators and therefore have a significant role as
both therapeutics and biomarkers. Unspecific proteolysis is a major elimination pathway
for peptides and proteins instead of the oxidative hepatic metabolism that is typical for most
small molecule drugs [18]. In addition to the typical PK support, bioanalysis has been actively
engaged in newly developed areas such as PKPD correlation, biomarker research in target
engagement/tissue distribution, simultaneous quantitation and metabolite identification
for small molecules and biologics, and antidrug antibody (ADA), etc., for many of which both
science and regulatory requirements are still evolving.
Historically, bioanalysis support can be categorized into two distinct areas—liquid chro-
matography for small molecules, usually chemically synthesized, and ligand binding assay
for large molecules, such as those which monoclonal antibodies typically produce by cell
culture. With the development of modern instrumentation, especially mass spectrometers,
measurement of large molecules by liquid chromatography in conjunction with a mass spec-
trometer (LC-MS) has become a reality. There is also a need to use LC-MS to investigate the
in vivo fate of some of the new modalities such as a half-life extended peptide constructed
by conjugating or fusing an otherwise short-lived peptide with an antibody or Fc to exten-
sively extend its in vivo half-life through decreased clearance and increased stability [19].

LC-MS has also been increasingly used for discovering and measuring endogenous protein or
peptide biomarkers [20, 21].
3 Bioanalytical requirements for supporting discovery, nonclinical, and clinical

studies
Let us take a further look at bioanalytical requirements for supporting discovery,

nonclinical development, and clinical studies using LC-MS methods. These three stages
should not be viewed as three separate and consecutive ones since each can extend well into
the next stage, in particular between nonclinical development and clinical phases. Many of the
longer-term toxicology studies are running in parallel with the clinical studies. Discovery
bioanalytical support is performed under non-GLP conditions. Generic LC-MS methods using
gradient mobile phase elution and protein precipitation sample extraction are typically used to
analyze the drug candidates in plasma and tissues. The results are used to rank the drug can-
didates as well as to obtain other important information such as tissue distribution and basic
pharmacokinetic parameters, for example, Cmax, Tmax, AUC, T1/2, and clearance. Usually an
internal bioanalysis/PK guideline is used for the conduct of this type of study. Some level of
method verification such as accuracy, precision, and critical stability is performed before the
sample analysis. Run acceptance criteria are usually a little bit wider than those used in GLP
studies. Even though discovery bioanalysis does not follow strict GLP rules, the data generated
in the discovery stage are still pivotal for compound selection, and therefore many companies
institute some levels of compliance to ensure data integrity. Nonclinical bioanalytical support
in development is under GLP regulations where the data generated should withstand rigorous
regulatory scrutiny [2, 3]. For each method, vigorous method development and validation is
performed as per regulatory guidance and internal Standard Operation Procedures (SOPs),
even though method validation itself is non-GLP. Any deviation during the sample analysis,
which follows the GLP principles, should be thoroughly investigated, documented, and jus-
tified. Once the candidate moves into the clinical stage, automation and other means of im-
proving the throughput of sample analysis become more relevant. The same clinical
bioanalytical method can be used by multiple bioanalytical chemists within or even among
different organizations to meet the demand for analyzing a large number of samples. At the
clinical stage, especially for studies in first in human (FIH), a bioanalytical method with better
sensitivity than those in nonclinical studies may be required. Extensive method optimization
for both sample preparation and LC-MS conditions to maximize recovery and minimize matrix
effects is often pursued.
4 Current regulatory landscape for bioanalysis
Both nonclinical development and clinical bioanalytical support are required to follow in-
ternal SOPs and regulatory recommendations. Dozens of SOPs are usually used to address
many aspects of bioanalytical activities ranging from instrument qualification, maintenance,
and calibration, to bioanalytical method validation, sample storage, analysis and destruction,
to data management and archiving, etc. The goal of these SOPs is to ensure the highest data

5 General considerations for bioanalysis for sample collection 7
TABLE 1 Bioanalytical full validation, partial validation and cross validation [2].
Full validation Partial validation Cross validation
Used for measuring analyte Modifications to a fully validated Where data are obtained from
concentrations in biological samples. analytical method may be evaluated different methods within or across
A full validation of a bioanalytical by partial validation. Partial studies, or when data are obtained
method should be performed when validation can range from as little as within a study from different
establishing a bioanalytical method one accuracy and precision laboratories applying the same
for the quantification of an analyte in determination to a nearly full method, comparison of those data is
clinical and in pivotal nonclinical validation. The items in a partial needed, and a cross validation of the
studies validation are determined according applied analytical methods should
to the extent and nature of the be carried out
Developing and validating an assay changes made to the method
for pivotal regulatory submission Data are obtained from different
(IND or NDA) Analytical site change using same fully validated methods within a
method (i.e., bioanalytical method study
Implementing an analytical method transfers between
that is reported in the literature laboratories) Data are obtained within a study
from different laboratories with the
A change in sample processing same bioanalytical method
procedures
integrity which can pass the regulatory scrutiny. These SOPs are usually drafted based on spe-
cific regulations and the regulatory guidance from the Food and Drug Administration (FDA)
and other international regulatory agencies, such as the European Medicines Agency (EMA),
and other federal/state requirements, as well as policies at each institute. One of the most im-
portant regulatory guidelines is the US FDA guidance on bioanalytical method validation [2],
which provides a general guideline on establishing important parameters, some of which are
specificity/selectivity, sensitivity, linearity ranges from a lower limit of quantitation (LLOQ) to
an upper limit of quantitation (ULOQ), accuracy, precision, stability, matrix effects, carryover,
recovery, dilution integrity, incurred sample reanalysis (ISR), etc. The method should also
demonstrate reproducibility and accuracy for incurred samples and its suitability for its
intended use. However, it is up to each institute to formulate its own SOPs based on this general
guidance. Typically, method validations can be categorized into full, partial, and cross valida-
tions (Table 1). Most of the validations are conducted in the form of full validation, whereas the
partial or cross validation can be used when a full validation is not required, as shown in the
examples in Table 1.
5 General considerations for bioanalysis for sample collection
During the project discussion, it is important to understand the objective of the study so
that important parameters such as matrix type, sample volume, ways of sampling (regular
sampling or microsampling), analytes to be measured (parent or parent plus metabolites),
anticoagulant selection, condition of centrifugation for harvesting plasma, sample storage
container, shipping condition and instruction, sample storage condition (20°C vs.
70°C), etc. can be provided to the project team. While during the early stage of drug

discovery/development it is not always possible to conduct the full spectrum of a stability

test, it is, however, always good practice to evaluate whether there is potential instability
of a metabolite that can impact the accurate quantitation of the parent analyte and/or the me-
tabolites. Detailed PK or TK (toxicokinetic) sample collection information, including labeling,
will be entered into the lab manual or study design. The following is just one typical example
in a lab manual supporting a Phase 1 clinical study. It includes three parts of the instruction
(material and labeling; preparation of PK samples; and shipment of PK samples). As one can
see from this example, an extremely detailed instruction is needed to ensure successful con-
duct of bioanalysis.
Materials and labeling:
• Blood must be collected in a glass or plastic K2EDTA containing blood collection tubes
(e.g., Vacutainer).
• No tubes with separation gel should be used. The resulting plasma samples must be stored
in polypropylene storage tubes with polypropylene or polyethylene caps.
• All tubes and containers will be labeled with preprinted labels. The preprinted information
will include the study number, participant identification number, treatment or treatment
period, scheduled sampling day and time as stipulated in the flow chart, and the analyte
name if applicable. No other information will be written on the labels. Labels should be
attached to the storage tubes at least 12 h before being frozen to ensure proper adherence.
• Labels should be applied to the sample tubes as follows:
• Apply labels to the sample tubes so that they do not overlap and obscure any
information. If possible, expose an area between the two ends of the label to allow
viewing of the contents of the tube.
• Do not alter the orientation of the label on the sample tube.
• Apply labels to all tubes in the same manner.
Preparation of pharmacokinetic samples:

• Collect 4 mL of blood into the appropriate K2EDTA-containing collection tube (e.g.,
Vacutainer) at each time point.
• Record the exact date and time of sampling.
• Before processing, gently invert the tubes 8–10 times to afford mixing; blood samples must
be kept on melting ice at all times during processing.
• Centrifuge blood samples within 1 h of collection in a centrifuge at 1300g for 10 min at 4°C,
to yield approximately 1.5–2 mL of plasma from each 4-mL whole blood sample. Plasma
samples must be kept on melting ice at all times during processing.
• Transfer plasma immediately to a prelabeled polypropylene tube. Plasma samples must be
kept on melting ice at all times until storage in a freezer.
• Store plasma samples in an upright position in a freezer, at a set temperature at 20°C until
transfer to the bioanalytical facility.
• The time between blood collection and freezing the plasma will not exceed 2 h.
• Ship samples according to the instructions provided. Ship specimens to the bioanalytical
facility, sorted by participant, sample collection date, and time.
• Questions regarding handling the plasma pharmacokinetic specimens should be
addressed to the contact person for the sponsor.

6 Diagnosis and mitigation of nonspecific adsorption loss for urine bioanalysis 9
Shipment of pharmacokinetic samples:
• All pharmacokinetic samples will be sent to the bioanalytical facility in a single shipment
at the end of the study or in multiple shipments as agreed upon with the bioanalytical
scientist.
• An inventory list must be included with each shipment. The sponsor-provided logs can be
used as an inventory list. The inventory list must note each specimen drawn for each
participant and note any missing specimens.
• For all international shipments, World Courier will be used. For domestic shipments, a
reliable domestic courier, such as Federal Express, will be used.
• As soon as the shipment day and air bill number(s) are available, the site will send an
e-mail to the principal investigator, sample management team, and site manager of the
bioanalytical facility. The e-mail must specify the study number, the number of
pharmacokinetic samples, the time of shipment pickup and the tracking number and
include an electronic sample inventory.
• Notify the bioanalytical scientist and the courier, at least 24 h in advance of the planned
shipment. Provide the courier with the appropriate account number to be used, if
applicable.
• Unless agreements were made with the principal investigator, samples will be shipped via
overnight delivery only on Monday through Wednesday, excluding holidays.
• Preferably the frozen samples will be shipped in boxes, sorted by participant and sampling
time. Boxes will be packed in bags that can withstand dry ice conditions (e.g., cryogenic
bags).
• Pack the frozen samples in a sufficient quantity of dry ice in appropriate containers, to
maintain a frozen state for at least 3 days.
• For all biological samples, follow the International Air Transport Association (IATA)
regulations for shipment.
• Ensure that the total package weight does not exceed 27.2 kg (60 pounds).
• Label the package with the sponsor name and study number.
• Include a return address (which includes the investigator’s name) on the outside of each
shipping container.
• Comply with all courier regulations for the shipment of biological specimens (include all
paperwork).
• Retain all documents indicating the date, time, and signature(s) of person(s) making the
shipment, in the study files.
6 Diagnosis and mitigation of nonspecific adsorption loss

for urine bioanalysis
For urine samples, it is always advisable to conduct a nonspecific adsorption loss exper-
iment before protocol finalization so that, if necessary, the means of preventing nonspecific
adsorption can be implemented. Nonspecific adsorption happens frequently in urine assays
because urine lacks proteins and lipids that can bind to the analytes or solubilize lipophilic
analytes [22]. A common approach to urine method development with a focus on overcoming

adsorption issues was published [23]. A simple and quick way to identify an adsorption prob-
lem is through a series of transfers and incubations. In this test, a drug solution in control
urine is prepared at a low quality control (QC) level in a test container. Dry and clean test
containers that are made from the same material and are of the same size as those for future
urine samples should be used. A portion of the urine solution is poured into a next container,
and then the transfer process is repeated for a few more containers. Between transfers, each
solution in the container should be left at room temperature for about 5–20 min to allow ad-
sorption to take place before continuing to the next transfer step. Finally, the urine sample
from each test container is assayed to determine the compound response. A sequential loss
of analyte response after each transfer indicates the existence of nonspecific adsorption loss.
To overcome the issue, antiadsorptive agents can be utilized, such as bovine serum albumin
(BSA), zwitterionic detergents such as 3-[3-cholamidopropyl)-dimethylammonio]-1-propane
sulfonate (CHAPS), sodium dodecyl benzene sulfonate (SDBS), beta-cyclodextrin, Tween 80,
and Tween 20.
7 Tissue bioanalysis
Knowledge of distribution of drugs, metabolites, biomarkers, etc. at specific locations in

the body of animal species and human subjects becomes more and more important for drug
discovery and development [24]. This knowledge is often obtained through the analysis of
tissue samples obtained from nonclinical and, to a lesser extent, clinical studies. Many factors
can affect the tissue distribution of drugs and metabolites. Passive diffusion across the cell
membrane is the primary pathway for the drug to distribute to the tissue. However, trans-
porter proteins can also assist or minimize the uptake of the drugs and metabolites into
the tissues. Other contributing factors such as drug metabolism, clearance rate, protein bind-
ing, permeability, molecular weight, log P, and pKa and other physicochemical properties can
also have a significant impact on tissue distribution. LC-MS has become the standard toolbox
for tissue sample analysis [25]. Different from plasma, which is in the liquid form, tissue sam-
ples are in a solid or semisolid format. Therefore, for typical LC-MS assays, they are homog-
enized prior to sample extraction. Soft tissues such as the brain, liver, lung, and kidney can be
easily homogenized. Tough tissues such as the muscle, heart, stomach, intestine, and colon
are more fibrous and need a more vigorous homogenization procedure. A higher shearing
force and longer duration of process may be required when a rotor blade homogenizer or
beads beater is used [26]. The heat generated during homogenization of tissues may cause
degradation of thermolabileanalyte(s), and therefore caution must be exercised. Hard tissues
such as skeletal bones, skin, and hair are mostly nonvascularized tissues and need special
treatment. In some extreme cases, enzymatic digestion or chemical treatment may be required
for some hard tissues [27]. Once the tissue sample is homogenized, the homogenate is ready
for extraction and the sample extraction approaches used for plasma samples should still ap-
ply. However, different from plasma samples, extraction recovery from solid tissue samples
cannot be assumed to be 100% and fortified QC samples, prepared in tissue homogenate, can-
not mimic the incurred samples [25]. Similarly, the internal standard, which was routinely
used to compensate for incomplete or variable extraction recovery for the plasma samples,

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9 General considerations for bioanalysis for extraction, chromatography, and MS detection 11
does not track the analytes in the tissues. Recovery evaluation using a radiolabeled incurred
sample [28] and recovery evaluation with orthogonal approaches [29] are just two examples
of addressing the issue of recovery evaluation.
8 Managing unstable metabolites such as acyl glucuronide
When moving into the development phase, information about biotransformation of the
drug candidate and its metabolites becomes more available. While the parent drug may
not be subjected to glucuronidation, its phase 1 metabolism, for example, oxidation of alcohol
to the carboxylic acid group, may generate metabolites that can form acyl glucuronide. If the
plasma samples were collected without acidification to stabilize the acyl glucuronide, the
phase 1 carboxylic acid metabolite may be overestimated due to the breakdown of acyl glu-
curonide during sample collection, storage, and analysis. It should also be noted that acidi-
fication of the plasma samples will affect the protein binding between the drug candidate and
the endogenous proteins such as albumin [30]. If the protein-binding measurement is needed
with the incurred samples, a separate pool of unbuffered plasma samples should be collected.
Acidification of the samples could also lead to the instability of other metabolites. In one ex-
ample of quantitation of diclofenac and metabolites in mouse plasma, acidification to stabilize
diclofenac acyl glucuronide resulted in increased oxidative degradation of 5-OH diclofenac
and ascorbic acid was added to the samples to prevent the degradation [31].
As we can tell from this simple acyl glucuronide example, a bioanalytical sample collection
strategy may evolve, depending on the stage of the drug discovery and development. For ex-
ample, at the early stage of drug development, information on parent exposure might be
enough for a go-no-go decision. There, a simple stabilization of acyl glucuronide by adjusting
the pH is used. With the project moving forward and gaining of information regarding acyl
glucuronide migration, which may raise toxicity concern [32], the measurement of both the
parent and acyl glucuronide metabolite is needed. Plasma protein binding for both the parent
and acyl glucuronide metabolite may be needed to better predict the initial human dose. One
would then be careful about the potential free fraction (Fu) change with pH adjustment
(nonphysiological pH).
9 General considerations for bioanalysis for extraction, chromatography,

and MS detection
Bioanalytical methods typically consist of analyte extraction from biological samples, liq-
uid chromatography to separate analytes of interest from endogenous components and me-
tabolites that may cause a matrix effect or selectivity issue, and MS detection, often in the
format of tandem mass spectrometers, to enhance assay selectivity and sensitivity. When de-
veloping a quantitative bioanalytical LC-MS method, one needs to holistically consider all
three parts as one integrated system and sometimes trade-offs need to be carefully balanced
[33]. One will always need to keep in mind the integrity of the analyte during the sample ex-
traction, postextraction, and chromatography. It should also be noted that some labile

metabolites, even though not being measured, can convert to the analyte during sample ex-
traction, chromatography, and MS detection and cause quantitation bias for the analytes of
interest.
Biological samples are seldom amenable to direct injection onto the LC-MS system.
Analytes of interest need to be extracted from the biological matrices prior to LC-MS analysis.
The purpose of extraction is to eliminate or reduce interferences which can co-elute with the
analytes and cause matrix effects or quantitation errors, and to concentrate the analytes and
improve their detection. When optimizing the extraction, analyte rather than the matrix is the
focus. This means that an extraction method with the highest recovery for the analyte may
also suffer from a severe matrix effect. The commonly used sample preparation methods
are direct injection, dilute and shoot, protein precipitation, liquid-liquid extraction (LLE)
(solid-phase supported liquid-liquid extraction), and solid-phase extraction (SPE), the last
three being the most frequently used. For example, protein precipitation is widely utilized
and is also frequently the preferred extraction method during drug discovery and preclinical
development. Analytes of interest are released from protein when a protein precipitation
reagent such as organic solvents (e.g., acetonitrile, methanol), acids, or salt (e.g., ammonium
sulfate) is added to the biological samples to denature the protein. The analyte stays in the
supernatant after the centrifugation and can be analyzed directly or can go through evapo-
ration/reconstitution steps to make the injection solution compatible with the chromato-
graphic condition. This procedure is of low cost, easy to perform, and can be performed in
the 96-well format. The extraction is also very mild and thus avoids potential analyte degra-
dation or conversion from metabolite to parent. With the advancement of the modern mass
spectrometer, assay sensitivity at low ng/mL, which in most cases is adequate for discovery
and nonclinical studies, is easily achievable. However, if a simple sample extraction proce-
dure such as protein precipitation is used, one may need to compensate for the potential
ion-suppression or in-source conversion such as that from the glucuronide metabolite to
the parent compound by using a more extensive chromatographic elution. The mechanism
for sample extraction and chromatography ideally should be orthogonal so that a better
method selectivity is provided. On the other side, SPE is very powerful for sample cleanup
as it provides some level of chromatographic separation between the analytes and other
matrix components. SPE is also easily automatable and provides high capacity for analyte
enrichment. However, some of the SPE conditions can be harsh, especially in the case of
strong cation or strong anion SPE where extreme pH conditions are used to elute the analytes.
This may lead to unwanted instability of analytes or the breakdown of conjugated metabolites
to the analyte of interest. Therefore, these SPE conditions should only be used when stability
and biotransformation of the analyte are well established.
For large molecule bioanalysis, two approaches, namely the bottom-up and top-down, are
typically used [34]. The bottom-up approach involves an enzymatic digestion and selection of
a surrogate peptide that can represent the large molecule, while the top-down LC-MS mea-
sures the intact large molecule directly. Both approaches still require a good sample clean to
remove the abundant endogenous proteins and other interferences. In addition to the classic
sample cleanup procedures such as SPE, other novel approaches are also developed for pro-
tein bioanalysis, notably protein precipitation/pellet digestion, abundant protein depletion,
and affinity enrichment. While the bottom-up approach, using a triple quadrupole mass spec-
trometer for the detection, typically has a superior sensitivity to the top-down approach by

9 General considerations for bioanalysis for extraction, chromatography, and MS detection 13
the high-resolution mass spectrometer such as TOF, the latter has several advantages too [35].
For the top-down intact protein LC-MS analysis, a generic MS method is typically used, and
little method development time is required. The method is independent of fragmentation ef-
ficiency, which is often not the case for surrogate peptides. The ability to acquire the complete
information of posttranslational modifications (PTM) and biotransformation allows
postacquisition data mining [19]. Some of the commonly observed challenges for sample
preparation of large molecules include loss of protein/peptide due to nonspecific adsorption;
analyte instability due to proteases; poor reproducibility of enzymatic digestion and high ma-
trix effects due to high concentration of endogenous proteins; incomplete recovery due to
binding of protein or peptides to high-abundance proteins and antibodies. Efforts have been
made to overcome these challenges. Possible solutions for nonspecific adsorption include use
of low-adsorption polypropylene and polyethylene containers; use of silanized glass con-
tainers; avoiding preparation of low concentration solutions in a matrix-free environment;
adding a carrier such as BSA to any matrix-free solutions; and spiking high concentration
stock solutions directly to plasma and preparing low concentration samples with serial dilu-
tion. Use of protease inhibitors such as diisopropylflurophosphate (DFP), sodium fluoride/
potassium oxalate/trichloroacetic acid, 4-(2-aminoethyl) benzene sulfonyl fluoride, hydro-
chloride (AEBSF)—Pefabloc, or phenylmethylsulfonyl fluoride (PMSF), storage of samples
in an ultralow freezer (<60°C), and handling the samples in an ice bath are recommended
as possible solutions for protein and peptide instability. There are several possible solutions
to remove abundant proteins [36]. The first step is to use urea, guanidine, or strong acid to
eliminate protein binding between target peptide/protein and endogenous abundant pro-
teins such as albumin. If the target protein/peptide is highly hydrophilic, TFA or TCA can
be used to precipitate abundant proteins while keeping the analytes in the aqueous supernatant
[37]. SPE (anion or cation exchange) can be used alone or in conjunction with protein
precipitation—for extracting hydrophilic proteins/peptides. Immuno-affinity extraction may
be used for achieving additional selectivity [38]. The largest challenge currently is still the rel-
atively poor sensitivity (compared to ELISA), but it has been significantly improved with nano-
LC-MS/MS and a better sample cleanup procedure such as immune-affinity extraction.
For LC-MS bioanalysis, chromatography plays a pivotal role to ensure assay selectivity and
robustness. Phase-II conjugated metabolites such as glucuronide and glutathione conjugates
need to be resolved from the analyte since they can break down back to the analyte in the ion
source and cause assay bias [39]. Isomers and endogenous interference need to be resolved
from the analyte. Compounds such as phospholipids and dosing vehicles like PEG need to be
resolved from analytes since they can cause ion suppression [40]. Reversed-phase LC has
been traditionally used for the quantitative LC-MS. With an increase of organic solvent con-
centration in the mobile phase, retention of the analyte decreases. However, one should be
aware of the potential bimodal retention on the reversed-phase column, leading to a
U-shaped retention profile where the initial retention decreases upon increasing the organic
content in the mobile phase but a further increase in the organic content results in increased
retention time for certain compounds, especially polar basic analytes, due to their interaction
with the residual silanol groups. This bi-model retention may cause a retention shift during
the run or irreproducibility of the method when a high organic content mobile phase is used
on a reversed-phase column [40]. This can also lead to the mismatch of injection solvent and
chromatography which may also lead to distorted chromatographic peaks [41]. In addition to

reversed-phase chromatography, separation based on other retention mechanisms can also be

used complementarily such as HILIC for polar analytes [42, 43] and normal phase chroma-
tography for chiral separations [44]. Large molecule LC-MS usually uses a shorter chain sta-
tionary phase such as C4, wider poor size (at least 300 Å), and elevated column temperature.
The principle of MS is the production of ions from analyzed compounds that are separated
or filtered based on their mass to charge ratio (m/z). Most of the applications for quantitative
bioanalysis use tandem mass spectrometers (MS/MS) that employ two mass analyzers—one
for the precursor ion in the first quadrupole and the other for the product ion in the third
quadrupole after the collision—activated dissociation of the precursor ion in a collision cell
(second quadrupole). Between the high-pressure LC and the MS operated under a high-
vacuum environment, interface connections that operate at atmospheric pressure, such as
electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and less fre-
quently atmospheric-pressure photo ionization (APPI), have matured into highly reliable
techniques necessary for quantitative LC-MS/MS bioanalysis. More recently, application
high-resolution mass spectrometer (HRMS) in bioanalysis has drawn a lot of attention
[45]. Due to its enhanced specificity using the high-resolution power and its capability of si-
multaneous quantitation and structural elucidation, HRMS could lead to a potentially rapid
and reliable method development for bioanalysis as well as sample analysis, thus generating
both cost and resource savings [46].
10 Selected applications for LC-MS bioanalysis
It is not the intention of this book chapter to cover every aspect of quantitative bioanalysis.
Rather, a few relatively newly developed areas are focused on to further illustrate the impact
of bioanalysis on drug discovery and development by using case studies from our own lab.
In the first example, we will discuss the status of using LC-MS, as a complementary tool to
the traditional ELISA methods, to analyze large molecules, particularly some of the novel
platform of biotherapeutics such as antibody-drug conjugate (ADC) and half-life extended
peptides. The value of multiple LC-MS methods such as the bottom-up and top-down as well
as simultaneous quantitation and catabolite identification is highlighted. The second example
is the development of microsampling technology which has been widely used to support both
preclinical and clinical studies. In comparison with the traditional plasma sampling technol-
ogy, microsampling presents some additional challenges for method establishment, valida-
tion, and sample analysis, from both the scientific and compliance points of view.
Biomarkers have become more and more important in drug discovery and development,
from confirmation of target engagement to patient stratification. As biomarkers are endoge-
nous molecules, their measurement is particularly challenging for bioanalytical scientists as
there is no “true” blank matrix to prepare standards and QC samples. It is also very difficult to
confirm the selectivity of a biomarker assay due its endogenous nature. Various strategies in
the third example have been proposed to mitigate these challenges. Lastly, the accurate read-
ing of important metabolites in the body has drawn a lot of attention recently since the pub-
lication of the FDA MIST guidance [47]. To establish safety coverage to ensure a safe clinical
trial is pivotal. It is not always straightforward to establish the right bioanalytical strategy for
metabolite measurement. In the last case study, the strategy for metabolite bioanalysis such as

10 Selected applications for LC-MS bioanalysis 15
measuring polar metabolites, assessment of chiral conversion, and selection of internal stan-
dard will be discussed.
10.1 LC-MS of large molecules

LBA is traditionally the primary platform for bioanalysis of large molecules and has the
advantages of providing superior sensitivity and high throughput. However, it may also suf-
fer from limitations such as cross-reactivity, requirement of highly specific reagents, and not
being able to directly elucidate structure information. In the past decade, LC-MS has increas-
ingly been applied for bioanalysis of large molecules as a complementary technique of LBA.
LC-MS has the unique advantages of being highly specific, more resistant to matrix interfer-
ence, and less stringent on reagent requirement. More importantly, LC-MS can provide valu-
able structure information which may be critical for understanding the ADME properties of
protein drug candidates.
As discussed earlier, there are generally two approaches of analyzing proteins using
LC-MS: bottom-up and top-down. In the bottom-up workflow, proteolytic (usually tryptic)
peptides generated by digestion are monitored as surrogates of the protein, typically on a tri-
ple quadruple mass spectrometer, which affords highly specific and sensitive detection.
However, there could be situations when the surrogate peptide does not adequately represent
the whole protein in terms of its functionality, stability, or coexistence of multiple isoforms.
High-level structural information such as integrity of a multiple subunit protein or proteolytic
catabolism is lost during digestion. This is when the top-down intact analysis of the protein
can play an important role. The top-down intact bioanalysis of large proteins (e.g., mAb) has
gained popularity in recent years thanks to applications of the advanced sample preparation
technique, such as immune-affinity capture, which enables effective sample cleanup [35].
Intact mass spectra obtained from HRMS may contain the peaks of not only the unchanged
intact molecule but also that of catabolites, making simultaneous quantitation and catabolite
identification possible [19].
In both the bottom-up and top-down approaches, the workflow involves an up-front sam-
ple preparation. Depending on the nature of the analyte, required sensitivity, and the biolog-
ical matrix, methodologies ranging from protein precipitation, solid phase extraction,
abundant protein depletion, and affinity capture can be chosen or combined, with increased
effectiveness in the sample cleanup. In the bottom-up approach, the surrogate (or signature)
peptide is detected and analyzed in the same way as small molecules, on a triple quadruple
instrument by multiple reaction monitoring (MRM) analysis, or in some labs, on HRMS by a
full scan or product ion analysis, a process also known as parallel reaction monitoring (PRM).
In comparison, intact protein analysis has more diversified choices when it comes to MS de-
tection [48]. Peptides or small proteins can be monitored in the same way as small molecules
or surrogate peptides on triple quadrupole or HRMS. Large proteins such as mAb are heavily
charged when analyzed under ESI, resulting in clusters of multiple charged ions which re-
quire resolution on HRMS. Peaks for a selected charged state (e.g., the three most abundant
peaks) can be extracted with a certain extraction window to reconstruct an extracted ion chro-
matogram (EIC) which can be used for quantitation [49]. This approach is more widely
adapted because it does not require additional software for data processing and thus can

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B O O K X V.
CHAPTER I.
S TAT E O F T H E WA R I N S PA I N .
Northern Provinces. The invasion of Gallicia, which had 1811.

been arrested by the arrival of the allies on the Coa, would
have been a most serious calamity. Abadia, a weak man, with
troops, distressed for provisions and clothing, was on bad terms with
the chief of his staff Moscosa, whom he feared, and on worse terms
with the junta. The great road to Coruña was open, Appendix, No. V.
and although general Walker, seeing the danger, Section 1.
advised that Ferrol, which was indefensible, should be dismantled,
and the guns, amounting to fifteen hundred, with the timber and
vessels of war in the harbour, transferred to Coruña, neither that nor
any other useful measure was executed.
Before this, overtures had been made to the Spanish government,
to take Spanish troops into British pay after the manner of the
Portuguese; but the regency remembering the prodigality of Canning
demanded three millions yearly, besides arms and clothing, without
which they said the Spaniards could make no efficient exertions! To
introduce British officers into the service on any other terms was not
possible, because the Spanish military were indignant at what they
termed the degradation of such a proposal. The Perceval faction
finding it thus, and wanting greatness of mind to support Wellington,
on a scale commensurate with his talents, then turned their attention
to the encouragement of the Partidas, as being less expensive, and
affording an example to the continental nations of popular and
protracted resistance to France.
Sir Howard Douglas, who succeeded general Walker as military
agent, (these officers must not be confounded with the military
agents originally sent out, and whose mischievous proceedings I
have had occasion to notice,) was directed to encourage those
bodies by increased supplies, and to combine their movements
better with each other and with the British squadron in the Bay of
Biscay. Lord Wellington at the desire of government, sent to the
guerilla chiefs, military presents, with a letter Sir H. Douglas’s
acknowledging the importance of their services, and Correspondence,
MSS.
this was not mere compliment, for he had indeed
derived great advantages from their exertions, and thought he had
derived more, because he only knew of their exploits by hearsay.
When he afterwards advanced into Spain and saw them closely, he
was forced to acknowledge that the guerillas, although active and
willing, and although their operations in general occasioned the
utmost annoyance to the enemy, were so little disciplined that they
could do nothing against the French troops unless the latter were
very inferior in numbers. If the French took post in a house or church
of which they only barricadoed the entrance, both regular troops and
guerillas were so ill equipped as military bodies, that their enemy
could remain in security until relieved. In like manner Napoleon
reprimanding his generals for suffering the Partidas to gain any
head, observed, that when cut off from communication with the
English ships they were a nullity!
Douglas arrived just as Dorsenne’s retreat enabled Sir H. Douglas’s
Abadia to resume his position on the frontier, but the Correspondence,
MSS.
army was in a miserable state; the wet season was
setting in upon men, destitute of even the necessaries of life,
although the province abounded in cattle and goods, which could be
easily procured, because money, although plentiful, was generally
hoarded, and commodities were therefore cheap, and could be
obtained in lieu of taxes at the market-price. An extraordinary
increase of the customs, arising from the trade of Santander and
Bilbao being transferred to Coruña by the war, also offered a
valuable resource; the harbour was filled with colonial goods, and as
the appetites of men generally stifle patriotism, and baffle power, a
licensed commerce was carried on with the enemy’s ports in Biscay;
yet without judgment as related to the war, for the return was iron, to
re-export to the colonies, whereas by an internal traffic of the same
kind, clothes and grain for the troops might have been had from
Castile and Leon. But confusion and corruption every where
prevailed, the exigences of the war were always the last things cared
for, and the starving soldiers committed a thousand excesses with
impunity, for where there is no food or pay, there can be no
discipline.
The people were oppressed with imposts, legal and illegal, and yet
the defalcation in the revenue was great, and the monopoly of
tobacco the principal financial resource, was injured by the
smuggling arising from the unsettled nature of the times. The annual
charge on the province was about £1,300,000, the actual receipts
were less than £500,000, and the junta endeavoured to supply the
deficiency by an extraordinary contribution from all property, save
that of day-labourers, which they expected would produce sixty
millions of reals (£750,000). But a corrupt and vexatious collection of
this tax tormented the people without filling the treasury; the clergy,
and the richer classes, were, as in Portugal, favoured, and it yielded,
in six months, less than a seventh part.
From this state of affairs two inferences may be safely drawn:—1º.
That England and not Gallicia had hitherto supported the war here,
as in other parts of the Peninsula. 2º. That as England had in 1808-9
paid to Gallicia three millions of hard dollars, and given other
succours sufficient for double the number of troops employed, the
deficiency of the revenue had been amply compensated, and the
causes of distress must be sought for in the proceedings of the
authorities, and in the anomalous nature of the war itself. The
successive juntas, apprehensive of offending the people, were
always inert in the civil administration, and either too corrupt, or too
incapable, to apply the succours from England, justly or wisely. The
junta of this period was, like its predecessors, factious and intriguing;
it was hostile to the junta of Leon, unfriendly to that of Asturias,
jealous and contemptuous of the military leaders; in return these last
abhorred the junta, and were tormented with factions of their own.
The regular officers hated the guerillas, and endeavoured to get the
controul of the succours granted, by England, to the latter; and as
they necessarily lived by plundering their own countrymen, they
strenuously opposed the arming of the peasants, partly from fear lest
the latter should resist this license, partly because the republican,
and anti-English spirit, which was growing up in the cortes had also
reached this quarter.
The clergy clung to the peasantry, with whom they had great
influence, but the army, which had imbibed liberal words, rather than
principles, was inimical to them. A press had been established at
head-quarters, from whence issued political papers either original, or
repeated from the libels at Cadiz, in which, the Portuguese were
called slaves, for submitting to British influence; and it was openly
avowed that the French yoke was preferable to that of England; the
guerilla system, and the arming of the people were also attacked,
and these writings were met by other political papers from the civil
press at Coruña and St. Jago. The frequent changes of commanders
rendered all the evils more prominent; for the local government had
legal power to meddle with the military arrangements, and every
change of commander produced a new difficulty. Thus the junta
refused to acknowledge Abadia as their president during the
absence of Castaños, he in return complained alike of their neglect
and of their interference; and when they proposed to establish a
general depôt at Lugo he marched a part of his army there to
prevent it.
But the occult source of most of these difficulties is to be found in
the inconsistent attempts of the British cabinet, to uphold, national
independence with internal slavery, against foreign aggression, with
an ameliorated government. The clergy who led the mass of the
people, clung to the English, because they supported aristocracy
and church domination; and they were also strongly for the Partidas,
because these were commanded by men who sprung directly from
the church itself, or from people who were attached to the church,
while the regular armies being officered by the friends of the cortes,
disliked the Partidas, both as interlopers and as political enemies.
The English ministers, hating Napoleon, not because he was the
enemy of England, but because he was the champion of equality,
cared not for Spain, unless her people were enslaved. They were
willing enough to use a liberal cortes to defeat Napoleon, but they
also desired to put down that cortes, by the aid of the clergy, and of
the bigoted part of the people: nevertheless as liberty will always
have more charms than slavery, they would have missed of both
objects, if the exigences of the continental system had not induced
the emperor to go to Moscow, where the snow destroyed him; and if
the very advocates of liberty in Spain had not in their madness,
resolved to oppress the Americans. The cortes, by discovering a
rabid love of power in practice, rendered their democratic doctrines
suspected, and lost partizans; but lord Wellington, in support of
aristocracy, used the greatest prudence in policy, and in his actions
was considerate and just.
In the first conference held at Coruña, after sir Howard 1811.
Douglas’s arrival, the junta, as the usual preliminary, Sept.
demanded more money from England; but he advised, instead, a
better management of their own resources, and pointed out the
military measures requisite to render the army efficient. He
recommended the adoption of the line of retreat upon Orense, rather
than upon Lugo and Coruña; and he endeavoured to establish a
permanent depôt in the island of Arosa, on the Vigo coast, as a
secure resource in the event of defeat; he also furnished the soldiers
with shoes and great-coats, the hospitals with blankets, and
completed the firelocks of the army to twenty-five thousand. There
were however abuses, which he could not remedy, and which would
seem rather to belong to the army of an Asiatic despot, than to an
European force fighting for independence. Innumerable baggage
animals devoured all the forage, and the personal servants and
cooks, who from custom never did duty, were above Appendix, No. I.
five thousand! a sixth part of the whole force! When Section 1.
the sick men were deducted, scarcely sixteen thousand infantry and
three squadrons of cavalry remained for service. Then there was so
little organization or arrangement that, although young, robust,
patient, and docile to the greatest degree, the troops could scarcely
be moved, even from one quarter to another, as a military body; and
the generals, unable to feed them on the frontier, more than once,
menaced, and in December did actually retire to Lugo, leaving the
province open to invasion.
Abadia at first exerted himself with activity, and appeared to enter
loyally into the ameliorations proposed. He gave the command of the
troops to Portasgo, repaired to Coruña himself, and organized the
province in seven military governments, under as many chiefs, one
for each division of the army. Every government was to raise a
reserve, and to supply and clothe the corresponding division on the
frontier. But in a little time this activity relaxed; he entered into
various intrigues, displayed jealousy, both of the peasantry and the
English, and no real improvement took place, save in that select part
of the army, which the Cadiz regency had destined for South
America, and had ordered him to equip from the English stores. This
was done at the very moment when a French army on the frontier
was again preparing to invade Gallicia, and sir Howard Douglas
vehemently opposed the disloyal proceeding; the junta also were
really averse to it, and Abadia pretended to be so; but he had a
personal interest in the colonies and secretly forwarded the
preparations. The regency, to evade Mr. Wellesley’s reproaches,
promised to suspend the embarkation of these troops, but the
expedition sailed from Vigo, and the organization of another, three
times its strength, including all the best artillery in the province, was
immediately commenced, and also sailed a few months later. This
then was the state of Gallicia in the latter end of 1811. She was
without magazines, hospitals, or system, whether civil or military, and
torn by faction, her people were oppressed, her governors foolish,
her generals bad: she had no cavalry, and the infantry were starving,
although the province easily supplied cattle for the allies in Portugal.
As a natural consequence, those famished soldiers were too
undisciplined to descend into the plains of Leon, and were
consequently of little weight in the general contest.
Under these circumstances, sir Howard Douglas had 1811.
nothing to work upon, save the Guerilla leaders, whose Nov.
activity he very considerably increased. His policy was to augment
the number of chiefs, but to keep the force of each low, lest, growing
proud of their command, they should consider themselves generals,
and become useless, as indeed had already happened to Campillo,
Longa, and Porlier, when they were made a part of the seventh
army. Nevertheless the advantage of this policy may be doubted, for
of all the numerous bands in the north, seven only were not
supported entirely by robbery. Mina, Pastor, Salazar, Pinto, Amor,
and the curate, whose united forces did not exceed Mr. Stuart’s Papers.
ten thousand men, were sustained by regular taxes, MSS.
customs, convent revenues, and donations; Longa supported his
from the produce of the salt-mines of Paza, but all the rest were
bandits, whose extinction was one of the advantages expected from
the formation of the seventh army.
It is now convenient to resume the narrative of military events.
In the Asturias, previous to Mendizabal’s arrival, and when Bonet
had marched to the Orbijo, Porlier surprised Santander, and
plundered some houses; but being followed by general Caucault, a
very active officer, he retired again to his strong-hold of Liebana. The
British cruizers, in concert with whom he acted, then destroyed
several coast-batteries, and the Iris frigate having arms on board,
came to the Bay of Biscay for the purpose of arranging an
intercourse with the Partidas of that province. But this was the period
when Reille and Caffarelli, were, as I have before noticed, chasing
Mina and Longa, whom they drove from the coast, into the
mountains of Leon, and thus marred the object of the Iris.
Nevertheless, when Mina was reinforced by the Valencians and
other fugitives from Catalonia, he returned to Navarre, and there
performed very considerable exploits, which, as belonging to other
combinations of the war, will be hereafter noticed.
While Caffarelli and Reille thus scoured the line of communication,
Dorsenne having the invasion of Gallicia in view, relieved Bonet on
the Esla, and sent him early in November, with eight thousand men
to re-occupy the Asturias as a preliminary measure. The Gallicians
foreseeing this, had detached Moscoso with three thousand five
hundred men to reinforce San Pol, who was at Pagares, below the
passes leading from Leon; and on the other hand Mendizabal uniting
the bands of Porlier and other chiefs, concentrated five thousand
men to the eastward on the Xalon. Eleven thousand men were
therefore ready to oppose the entrance of Bonet, but with the usual
improvidence of the Spaniards, the passes of Cubillas and Ventana,
to the westward of Pagares, were left unguarded. By these roads,
Bonet, an excellent officer, turned Moscoso, and drove him down the
Lena with loss and disgrace; then turning upon Mendizabal, he
chased him also in disorder from Lanes into the Liebana.
All the civil authorities immediately fled to Castropol, the Spanish
magazines fell into the hands of the French, and Bonet having
resumed his old positions at Oviedo, Gihon, and Grado, fortified
several posts in the passes leading to Leon, raised contributions,
and effectually ruined all the military resources of the Asturias. The
organization of the seventh army was thus for the time crushed, and
in Gallicia great mischief ensued. For the return of Moscoso’s
division and the want of provisions in the Bierzo, which had obliged
Abadia to retire to Lugo, while Dorsenne was menacing the frontier,
had thrown that kingdom into a ferment, which was increased by the
imposition of the new contributions. The people became exceedingly
exasperated and so unfavourably disposed, that it was common to
hear them say, “the exactions of a French army were a Sir H. Douglas’s
relief in comparison to the depredations of the Spanish Correspondence,
MSS.
troops.”
During these transactions in the north, Drouet had joined Girard at
Merida, and menaced the allies in the Alemtejo, hoping thus to draw
Wellington from the Coa; but the demonstration was too feeble, and
the English general thought it sufficient to reinforce Hill with his own
brigade from Castello Branco. These movements were undoubtedly
part of a grand plan for invading Portugal, if the emperor could have
arranged his affairs peaceably with Russia. For to move once more
against Lisbon, by Massena’s route, was not promising, unless the
northern provinces of Portugal were likewise invaded, which required
the preliminary occupation of Gallicia, at least of the interior. In the
south also, it was advisable to invade Alemtejo, simultaneously with
Beira; and the occupation of Valencia and Murcia was necessary to
protect Andalusia during the operation. The plan was vast,
dangerous, and ready for execution; for though the wet season had
set in, an attack on the northern parts of Portugal, and the invasion
of Gallicia, were openly talked of in Dorsenne’s army, Caffarelli was
to join in the expedition, and Monthion’s reserve, which was to
replace Caffarelli’s on the line of communication, was already six
thousand strong. Ney or Oudinot were spoken of to command the
whole, and a strong division was already in march to reinforce the
army of the south, arrangements which could have reference only to
Napoleon’s arrival; but the Russian war soon baulked the project,
and Wellington’s operations, to be hereafter noticed, obliged
Dorsenne to relinquish the invasion of Gallicia, and caused Bonet
once more to abandon the Asturias.
Thus, with various turns of fortune, the war was managed in the
northern provinces, and no great success attended the French arms,
because the English general was always at hand to remedy the
faults of the Spaniards. It was not so on the eastern line of invasion.
There Suchet, meeting with no opponent capable of resisting him,
had continued his career of victory, and the insufficiency of the
Spaniards to save their own country was made manifest; but these
things shall be clearly shewn in the next chapter, which will treat of
the conquest of Valencia.
CHAPTER II.
C O N Q U E S T O F VA L E N C I A .
In August, and the beginning of September, Suchet, while 1811.

preparing for this great enterprise, had dispersed the bands of August.
Villa Campa and the other chiefs, who during the siege of Taragona
vexed Aragon. He had sent his feeble soldiers to France, receiving
conscripts in their places, and although the harvest was very bad,
formed large magazines in Morella and Tortoza. Eight thousand men
had been left in Catalonia under general Frere, another eight
thousand were placed under general Musnier, to protect Aragon, and
twenty-four thousand of all arms remained for the invasion of
Valencia, but this force Suchet thought inadequate, and demanded a
reinforcement from the army of reserve, then in Navarre. Napoleon,
whose system of war, whatever has been said to the contrary, was
eminently methodical, refused. He loved better to try a bold push, at
a distant point, with a few men, than to make an overwhelming
attack, if he thereby weakened his communications; he judged
courage and enterprise fittest for the attack, prudence and force for
the support. And yet he designed to aid Suchet’s operations
vigorously when the decisive blow could be struck. Then not only the
divisions of the reserve were to march, but combined movements, of
detachments from nearly all the armies in the Peninsula, were
arranged; and we shall find, that if Wellington, by menacing Ciudad
Rodrigo, saved Gallicia, the French army of the north, in return, by
menacing Gallicia, fixed the allies on the Agueda, and so protected
Suchet’s invasion of Valencia.
Three roads led to the Guadalaviar, one from Tortoza by the 1811.
sea-coast, one by Teruel and Segorbe, and one by Morella Sept.
and San Mateo. That from Tortoza, and that by Teruel, were
carriage-roads, but the first only was fit for heavy artillery, and it was
blocked, partially by the fortress of Peniscola, and completed by the
fort of Oropesa. Wherefore, though the infantry and cavalry could
move on a bye-road to the right, the convoys and the guns, which
were at Tortoza, could not pass until Oropesa was reduced.
Nevertheless the French general, well knowing the value of boldness
in war, resolved to mask Peniscola, to avoid Oropesa, to send his
field artillery by Teruel, and uniting his troops near Saguntum, to offer
battle to Blake; and if the latter declined it, to reduce Oropesa and
Saguntum, trusting for subsistence to the “huerta” or garden of
Valencia, until the arrival of his convoys.
He had, however, organized his system of supply with care. From
Morella and Tortoza, brigades of mules, after the manner adopted in
the British army, were to carry provisions to the troops, and sheep
and cattle were delivered to each regiment for its subsistence in
advance. This last plan, which sir John Moore had also projected in
his campaign, Suchet found advantageous; and I am persuaded that
the principle should be extended, so that all things requisite for the
subsistence, and fighting of troops should be organized regimentally,
and the persons employed wear the uniform of their different corps.
Jealousies between the functionaries, of different branches of the
service, would then be unknown; and the character of all subordinate
persons, being under the guardianship of the battalions to which they
belonged, would be equally praiseworthy, which cannot now be said.
While Suchet was thus gathering his strength, Valencia was a prey
to disorder. About the period of the siege of Taragona, Palacios,
notwithstanding his high monarchical principles, which caused him to
be dismissed from the regency, had been appointed captain-general
of Valencia, Murcia, and Aragon; and he immediately raised a strong
party amongst the friars and other opponents of the cortes. When
after the dispersion of the Murcian army at Baza, Blake had rallied
the fugitives, and in virtue of his power as regent, assumed the chief
command at Valencia, Palacios’ faction opposed him, and
endeavoured to draw the soldiers and the populace to Capt. Codrington’s
their side, by proposing to inundate the plain of papers, MSS.
Murviedro, and to defend the strong country in advance. Blake,
however, resolved to act on the flanks of the French army by
detachments, and, in this view, sent C. O’Donnel, with the divisions
of Obispo and Villa Campa, to Albaracin, supporting them with four
thousand men at Segorbe and Liria. He charged Mahy, who
commanded five thousand infantry, and seven hundred cavalry of
the Murcian army, to surprise the French detachment of the army of
the centre, posted at Cuença. He detached Bassecour with two
thousand men to Requeña, and the same time, directed Duran and
the Empecinado, to unite, and invade Aragon; and it was to aid in
this expedition that Mina quitted the mountains of Leon.
Blake had, exclusive of Mahy’s and Bassecour’s divisions, about
twenty thousand infantry, and two thousand cavalry. Three thousand
five hundred men were placed in Saguntum, which was provisioned
for three months; two hundred were in Oropesa, and fifteen hundred
in Peniscola; and there were so many Partidas, that the whole
country seemed to be in arms, but the assembling of these people
being very uncertain, Blake could not depend upon Roche, MSS.
having a permanent partizan force, of more than eight
thousand. The Valencian army contained the Albuera Tupper, MSS.
divisions, St. Juan’s, Miranda’s, and Villa Campa’s Mr. Wellesley,
veterans; it was therefore, not only numerous, but the MSS.
best Spain had yet produced; and Valencia itself was exceedingly
rich in all things necessary for its supply: but there was Doyle, MSS.
no real power, the building, though fair enough
outside, had the dry rot within. The French had many Appendix, No. I.
secret friends, faction was as usual at work, the Section 3.
populace were not favourable to Blake, and that general had rather
collected than organized his forces, and was quite incapable of
leading them. He was unpopular, both at Cadiz and Valencia, and
the regency of which he formed a part was tottering. The Cortes had
quashed Mahy’s command of the Murcian army, and even recalled
Blake himself; but the order, which did not reach him until he was
engaged with Suchet, was not obeyed. Meanwhile that part of the
Murcian army, which should have formed a reserve, after Mahy’s
division had marched for Cuença, fell into the greatest disorder:
above eight thousand men deserted in a few weeks, and those who
remained were exceedingly dispirited. Thus all interest became
concentrated in the city of Valencia; which was in fact the key of all
the eastern coast because Carthagena required an army to defend
it, and could only be fed from Valencia, and Alicant was then quite
defenceless.
It was in this state of affairs, that Suchet commenced the invasion.
His army was divided into three columns, and, on the 15th of
September one moved by the coast-road, one by Morella and San
Mateo, and one by Teruel, where an intermediate magazine was
established; but this latter column instead of Suchet.
proceeding directly to Segorbe, turned off to its left,
and passed over the Sierra de Gudar to Castellon de la Plana,
where the whole three were united on the 20th. The Vacani.
main column, commanded by Suchet in person, had
masked Peniscola on the 15th, and invested Oropesa by a
detachment on the 19th; but as the road run directly under the fire of
the last place, the main body moved by the rugged route of Cabanes
to Villa Franca, leaving the battering-train still at Tortoza.
During these operations Blake appeared inclined to Roche, MSS.
fight, for he brought Zayas up in front of Murviedro,
and called in Obispo; Mahy, who had done nothing on the side of
Cuença, was also in march to join him; but all these divisions
marched slowly, and with confusion; and a slight skirmish at
Almansora, on the Mingares, where a few French dragoons put a
great body of Spanish infantry to flight, made Blake doubt the
firmness of his troops. He therefore left O’Donnel with four thousand
men on the side of Segorbe, and then retired himself with fifteen
thousand behind the Guadalaviar. Valencia was thus Tupper, MSS.
thrown into great confusion, but Bassecour’s division
was at hand, and Suchet fearing to attack so large an army in an
entrenched camp (which had cost two years to construct), while his
own communication with Tortoza was intercepted, merely dispersed
the armed peasants which had assembled on his flank, and then
turned against Murviedro.
SIEGE OF SAGUNTUM.
This celebrated place, situated about four leagues from Valencia,

was a rocky mountain, covered with the ruins of the ancient city, and
the remains of Moorish towers and walls, which being connected by
modern works, formed four distinct posts covering the whole summit
of the rock: but in consequence of the usual Spanish procrastination
the heavy guns prepared to arm it were not yet mounted, and only
seventeen pieces of inferior size were available for defence. The
modern town of Murviedro, situated at the foot of the rock, was
covered by the river Palancia, and by a canal, and occupied by some
Spanish picquets; but the 23d Habert, having passed the water,
invested the rock on the east, while Harispe invested it on the west
and south, and a third division drove the Spanish posts from
Murviedro and entrenched itself in the houses. The rest of the army
was disposed in villages, on the hills to the north-west, and patroles
were pushed towards Valencia. Thus the rock of Saguntum was
invested, but it was inaccessible to the engineer, save on the west,
where the ascent, although practicable, was very rough and difficult.
It would have been impregnable, if the Spaniards had mounted their
large guns; for the French were obliged to bring earth from a
distance, to form the batteries and parallels, and to set the miner to
level the approaches, and their parapets were too thin to withstand
heavy shot.
The first point of resistance was an ancient tower called San
Pedro, and immediately above it was the fort of San Fernando,
which could not be attacked until San Pedro fell, and, from its height,
then only by the miner. But near the eastern extremity of the rock,
there were two ancient breaches which the Spaniards were still
engaged repairing, and had only stopped with timber; a large tank
offered cover for the assembling of troops close to these breaches,
and Suchet resolved to try an escalade. To effect this, three columns
were assembled before daybreak on the 28th in the tank, a strong
reserve was held in support, and a false attack was directed against
the San Pedro to distract the attention of the besieged: but in the
previous part of the night, the Spaniards having sallied were
repulsed, and the action having excited both sides, a French soldier
fired from the tank before the appointed time, whereupon the
columns rushing forward, in disorder, planted their ladders, and the
garrison would have carried the place by noise, but the garrison
thrust the ladders from the walls, and drove the stormers back, with
the loss of three hundred men. After this check, as the artillery was
still at Tortoza, Suchet ordered a part of his army to attack Oropesa,
employed another part in making a road, for the guns, to reach the
battery raised against the tower of San Pedro, and then turned his
own attention to the movements of Blake.
That general following his first plan of action against the French
flanks, had during the investment of Saguntum, sent C. O’Donnel
with Villa Campa’s division and St. Juan’s cavalry, to Betera, and
Beneguazil, and Obispo’s division to Segorbe; thus forming a half
circle round the French army, and cutting its communication with
Teruel, near which place Mahy had by this time arrived. Suchet
however caused Palombini to attack Obispo, whose whole division
dispersed after a skirmish with the advanced guard, and the Italians
then returned to the siege. The next night Harispe marched against
O’Donnel, who was well posted at Beneguazil behind a canal, having
his centre protected by a chapel and some houses; nevertheless the
Spaniards were beaten with loss at the first shock, and fled in
disorder over the Guadalaviar. During these events Blake remained
an idle spectator of the defeat of his division, although he had a large
body of troops in hand, and was within a few miles of the field of
battle.
The French train now advanced from Tortoza, and four 1811.
pieces were placed in battery against Oropesa. On the 10th Octobe
r.
Suchet took the direction of the attack in person, and the fort
situated upon an isolated rock, was breached in a few hours; but the
garrison of the King’s Tower (a separate work placed on a small
promontory, and commanding the harbour) refused to surrender, and
was carried off, on the 11th, under the French fire, by the
Magnificent. The French general having thus with a loss of only thirty
men opened the road for his artillery, returned to Saguntum and
pushed the siege of that place; but the difficulties were very great,
the formation of the road to the batteries was itself a work of pain,
and although his indefatigable troops had formed a breaching battery
on the 12th, while seven small mortars and howitzers, placed on the
right and left, had nearly silenced the Spanish fire, the muskets of
the besiegers alone brought down from fifteen to twenty men.
On the 17th the breaching battery being armed, opened its fire
against the tower, and the new masonry crumbled away at once; yet
the ancient work resisted the guns like a rock. On the 18th the fire
recommenced, when the wall gave way to the stroke of the guns,
and the assault was ordered; but from the height of the tower, which
overlooked the works at a short distance, the preparations were
early discovered, the Spaniards collecting on the breach repaired it
with sand-bags, and regardless of the French fire, with loud cries
provoked the attack. At five o’clock, four hundred men rushed
forward as swiftly as the steepness of the ascent would permit.
Soon, however, the head of the column was checked, the rear began
to fire, the whole got into confusion, and when one-half had fallen
without making the slightest impression on the defenders, the
attempt was abandoned. After this signal failure the French erected a
second battery of six pieces, one hundred and forty yards from the
tower, and endeavoured to push the approach close to the foot of the
breach, yet the plunging fire of the besieged baffled them;
meanwhile Andriani the governor, having communication by signal
with the ships in the Grao, was encouraged to continue his gallant
defence, and was informed that he was already promoted for what
he had done. But to understand Suchet’s embarrassments, from the
protracted resistance of Saguntum, we must take a view of Lacy’s
contemporary operations in Catalonia, and the proceedings of the
Partidas against the French communications and posts in Aragon.
C ATA L O N I A .
It will be recollected that the blockade of Figueiras August.

produced sickness in M‘Donald’s army, and that the return of
Suchet to Aragon, and the parcelling of his troops on the lines, from
Lerida to Montserrat, Tortoza, and Taragona, had completely
extinguished the French power in the field; because the divisions of
the army of Aragon which still remained in Lower Catalonia, being
destined for the enterprize against Valencia, could not be employed
in harassing expeditions. Lacy was therefore enabled,
notwithstanding the troubles which followed the fall of Taragona, to
reorganize about eight thousand men in two divisions, the one under
Eroles, the other under Sarsfield; the junta also called out the tercios
of reserve, and arms and ammunition being supplied by the English
navy, Lacy was soon in a condition to act offensively. Thus the taking
of Montserrat was very injurious to the French, for it is generally
supposed that Friere’s division, if held together in the field, would
have prevented this reaction in the principality. Lacy at first
suggested to the British navy the recapture of the Medas Islands,
and it was effected in the latter end of August, by the Undaunted,
Lavinia, and Blossom, aided by a small party of Spaniards, the whole
under the command of captain Thomas. The enterprise itself was
one of more labour than danger, and the Spanish allies were of little
use, but the naval officers to whose exertions the success was
entirely due, were indignant at finding that colonel Green, who
served as a volunteer, endeavoured to raise his own Appendix, No. II.
reputation with the Catalans by injuring the character Section 2.
of those under whom he served.
Immediately after the fall of Montserrat, Lacy and the junta had
proposed the fortifying of Palamos or Blanes, to be held as a marine
depôt and strong-hold, in common with the British navy, but with a
strange folly expected that sir E. Pellew, who had no troops, would
defend them from the enemy while establishing this post. Finding
this scheme received coldly by the admiral, they turned their
attention inland, and blowing up the works of Berga, fixed upon the
position of Busa, as a place of strength and refuge. This remarkable
rock which is situated between the Cardener and Memoir upon Busa,
Bindasaes rivers and about twenty miles from by Capt.
Zeupfinning, MSS.
Cardona could be reached by one road only, and that
a very rugged one. The rock itself fourteen miles in circumference,
healthy and full of springs, is fertile, and produces abundance of
forage, and fuel. It is cut off from the rest of the world by frightful
precipices, and could neither be forced, nor starved into a surrender.
Busa, Cardona, Solsona, and Seu d’Urgel were therefore guarded
by the tercios of reserve and Lacy soon commenced offensive
excursions with the regular army, against the long lines of the French
communication.
In September while the Somatenes interrupted the passage 1811.
of the convoys to Montserrat, Eroles made an unsuccessful Sept.
attack on the fort of Moncada near Barcelona; Lacy who had
returned from an incursion in the French Cerdaña where he had
gathered some booty, then united Eroles and Sarsfield’s troops, and
surprised the town of Igualada, where he killed two hundred French,
but not daring to attack the castle retired to Calaf, and from thence
again detached Eroles to Jorbas, to attack a French convoy coming
to Igualada. Eroles beat the escort, and captured the convoy, and
then the French quitted the fortified convent of Igualada, and joined
the garrison of Montserrat, when the whole, fearful of being invested
and so starved, abandoned that important point, and marched
through Barcelona to Taragona; the Spaniards immediately occupied
Montserrat, and recovered a large store of clothing and cavalry
equipments, which had been hidden in a vault and were
undiscovered by the enemy. Eroles, pursuing his success, forced the
garrisons of Belpuig, and Cervera, about five hundred in all, to
surrender, and thus the whole line of communication, between Lerida
and Barcelona, fell into the power of the Catalonians. The
confidence of the people then revived; Sarsfield occupied Granollers,
and the passes leading into the valley of Vich; Manso and Rovira
menaced the Ampurdan; and Eroles suddenly passing by Seu
d’Urgel into the Cerdaña, defeated, at Puigcerda, some national
guards commanded by general Gareau, who had been sent there
after Lacy’s invasion. He afterwards raised large contributions on the
frontier, burnt a French town, and returning with his spoil by the way
of Ribas, and Ripol, took post in the pass of Garriga, while Milans
occupied Mataro, and both watched to intercept a convoy which
M‘Donald was preparing for Barcelona.
Sarsfield at the same time embarked his division and sailed to the
coast of the Ampurdan, but the weather would not permit him to
land. Nevertheless the attention of the French general was
distracted, and the convoy did not move. Lacy then recalled
Sarsfield, and projected the surprise of Barcelona itself, but after
putting his troops in march, feared the execution and relinquished
the attempt. Meanwhile one swarm of the smaller Partidas menaced
the French communication between Mequinenza and Tortoza, and
another swarm settled on the plains about Lerida.
The state of Aragon was equally alarming. Duran and the
Empecinado had received Blake’s orders to unite near Cuença, for
the purpose of invading Aragon; but the secret junta of the district
were averse to the plan, and the troops of the latter chief refused to
move, and even came to blows with the junta’s people. In this
confusion general d’Armanac, who had retired from Cuença,
returned, and dispersed the whole. The Empecinado however
collected them again, and having joined Duran, their united powers
being about six thousand infantry and two thousand five hundred
horse, moved against Calatayud; Mina also acting in concert with
them, quitted the mountains of Leon and entered Navarre with about
five thousand men, and some minor partisans were already acting
against different parts of Aragon. The whole were in want of clothing
and ammunition, but Mr. Tupper, the consul at Valencia, having safe
means of communication with the interior supplied them.
General Musnier’s force was so scattered that he could not fight
either of the large Partidas, without exposing some important point to
the other, and the 29th of September the Empecinado took
possession of the pass of Frasno, while Duran invested the fortified
convent of Calatayud. This place was garrisoned by some French
and Italian troops, who differed upon the defence, and when the
explosion of two mines had killed a number of them they
surrendered. Musnier collected some men to succour the place, but
unable to force the pass of Frasno, retired; yet being reinforced on
the 5th, he again advanced, and a column sent from Navarre by
general Reille also came up; whereupon the Spaniards disappeared
until the French retired, and then reoccupied Calatayud. They were
now in full communication with Mina and a general plan of invasion
was discussed, but as Duran and Mina could not accord each acted
separately.
Severoli’s division eight thousand strong and just arrived 1811.
from Italy, then reinforced Musnier, and on the 9th driving the Octobe
r.
Spaniards from Calatayud pursued them on the roads to
Molino, Daroca, and Medinaceli. On the other side of the Ebro
however Mina fell on the post of Exca in the Cinco Villas; the
garrison broke through his investment in the night, but he pursued
them almost to the gates of Zaragosa, and then turning off towards
Ayerbe, attacked that post and menaced the communication by
Jaca. The commandant of Zaragosa had sent an Italian battalion to
look after the flying garrison of Exca, which was found at Zuera, and
the united forces amounting to eleven hundred infantry and sixty
cavalry followed Mina and came up with him at Ayerbe; the guerilla
chief instantly turned with a part of his troops, and the Italians
retreated towards Huesca, but having to cross a plain were all killed
or taken.
Reille and Musnier hearing of this misfortune spread their columns
in all directions to intercept Mina, but he evaded their toils, and
although sharply chased and several times engaged, reached
Motrico on the Biscay coast with his prisoners. The Iris frigate which
was then harassing the enemy’s coast line took some of them off his
hands, and the remainder, three hundred in number, were sent to
Corunna by the Asturian mountains, but only thirty-six arrived, the
rest were shot by the escort, under pretence that they made a noise
near a French post!
While these events were passing on the left of the Ebro,
Mazzuchelli’s brigade followed the Empecinado, and having
defeated him in a sharp action, at Cubiliejos de la Sierra, brought off
the garrison of Molino and dismantled that fort; but the smaller
Partidas infested the road between Tortoza and Oropesa, and in this
disturbed state of affairs reports were rife that an English force was
to disembark at Peniscola. Blake also sent Obispo’s division against
Teruel, which was thus menaced on all sides, for Mahy was still in
those parts. Thus the partizan warfare seemed interminable, and
Suchet’s situation would really have been very dangerous, if he had
been opposed by a man of ability. He had an inferior force and was
cooped up between the enemy’s fortresses; his communications
were all interrupted; he had just met with two signal failures at
Saguntum, and he was menaced by a formidable army which was
entirely master of its operations. Blake however soon relieved him of
his difficulties.

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Bioanalysis, often shortened to BA, is a subdiscipline within pharmaceutical research and

Identification and Quantification of Drugs, Metabolites, Drug Metabolizing 3

Discovery Preclinical Clinical

Drug-like? Known liabilities? Superior efficacy?

2 Complexity of contemporary bioanalysis

I. Techniques for identifying and quantifying drugs and metabolites

I. Techniques for identifying and quantifying drugs and metabolites

3 Bioanalytical requirements for supporting discovery, nonclinical, and clinical

Let us take a further look at bioanalytical requirements for supporting discovery,

4 Current regulatory landscape for bioanalysis

I. Techniques for identifying and quantifying drugs and metabolites

5 General considerations for bioanalysis for sample collection

I. Techniques for identifying and quantifying drugs and metabolites

discovery/development it is not always possible to conduct the full spectrum of a stability

Preparation of pharmacokinetic samples:

I. Techniques for identifying and quantifying drugs and metabolites

6 Diagnosis and mitigation of nonspecific adsorption loss

I. Techniques for identifying and quantifying drugs and metabolites

Knowledge of distribution of drugs, metabolites, biomarkers, etc. at specific locations in

I. Techniques for identifying and quantifying drugs and metabolites

8 Managing unstable metabolites such as acyl glucuronide

9 General considerations for bioanalysis for extraction, chromatography,

I. Techniques for identifying and quantifying drugs and metabolites

I. Techniques for identifying and quantifying drugs and metabolites

I. Techniques for identifying and quantifying drugs and metabolites

reversed-phase chromatography, separation based on other retention mechanisms can also be

10 Selected applications for LC-MS bioanalysis

I. Techniques for identifying and quantifying drugs and metabolites

10.1 LC-MS of large molecules

I. Techniques for identifying and quantifying drugs and metabolites

Northern Provinces. The invasion of Gallicia, which had 1811.

In August, and the beginning of September, Suchet, while 1811.

This celebrated place, situated about four leagues from Valencia,

It will be recollected that the blockade of Figueiras August.

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