Identification and Quantification of Drugs Metabolites Drug Metabolizing Enzymes and Transporters Concepts Methods and Translational Sciences 2Nd Edition Shuguang Ma Editor Full Chapter

Identification and Quantification of
Drugs, Metabolites, Drug Metabolizing

Enzymes, and Transporters: Concepts,
Methods and Translational Sciences
2nd Edition Shuguang Ma (Editor)
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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
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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.
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,

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

be universally applied to data acquired on instruments from different vendors. Alternatively,

multiple charged ions are deconvoluted to generate non- or zero-charged peaks which can be
in turn quantified based on the intact molecule, a process requiring a specific software func-
tion. In the case when multiple components, such as those of catabolites or different glyco-
sylation isoforms, are present in the sample, deconvolution is a more direct and effective
way to visualize the data.
10.1.1 LC-MS bioanalysis of mAb

Monoclonal antibodies and related products represent one of the most promising and fast-
growing classes of therapeutics. Even though the bioanalytical platform for mAb has been
predominantly LBA, there have been increased numbers of applications on quantitation of
mAb using LC-MS. This is particularly true for discovery study support when a specific re-
agent may not be readily available at an early stage before the identification of the final can-
didate for development. The general workflow for nonclinical LC-MS bioanalysis of mAb
involved trypsin digestion and LC-MRM analysis of surrogate peptides, which are typically
the conserved sequences of the human Fc region on the mAb. The choice of sample prepara-
tion depends on the required sensitivity and type of biological matrix. Protein precipitation
followed by digestion of the pellet, a process known as pellet digestion, has been widely used
because it is easy to operate and does not require specific reagents [50]. For better sensitivity,
immune-affinity capture using antihuman Fc can provide an effective and selective cleanup
of human mAb from nonclinical samples. For clinical samples, more specific capturing re-
agents such as the antiidiotype antibody against epitope in CDR of the mAb can be used
to avoid pulling down endogenous human IgGs. Alternatively, the target protein of the
mAb can also be used as an affinity capture reagent. Either way, caution has to be exercised
to evaluate interference from endogenous targets on the capturing process and to understand
whether the measured concentrations are those of free (not occupied by target) or total mAb.
Due to the highly specific detection by LC-MS, it is possible to monitor certain modifications
on the molecules. One example is deamidation, a common and spontaneous biotransforma-
tion of mAb. It has been shown that deamidation at a certain region of mAb could impact its
binding affinity, potency, safety, and pharmacokinetics [51]. The surrogate peptide
containing Asn, the deamidation product Asp, and the succinimide intermediate can be sep-
arated on LC and monitored by MRM to evaluate the relative levels of each species, which
helps understand the dynamics of this in vivo biotransformation [52, 53].
The emerging technique of intact protein bioanalysis has also been exemplified on mAb in
a few publications. Our lab published a workflow of quantifying large proteins in biological
samples by using immune-affinity capture coupled to LC-HRMS analysis [35]. The
deconvoluted intact mass spectra were processed for quantitation using a research version
of the software. This deconvoluted approach was also compared with EIC and demonstrated
similar bioanalytical performance in terms of sensitivity, linearity, accuracy, and precision.
More importantly, the intact concentration data generated for an in vivo monkey PK study
showed results consistent with those by the more commonly used bottom-up approach,
confirming the suitability of quantifying mAb using the intact quantitation method [49].
For better sensitivity and detection resolution, mAb can be broken down into relatively
smaller “intact” pieces by using reducing reagents to dissociate the light chains and heavy
chains. Furthermore, mAb can be specifically cleaved around the hinge region by the IgG

protease such as IdeS to reduce the size of the heavy chain. This procedure generates three
types of fragments, light chain, Fd, and Fc/2, each 25 kDa. The application of this
“middle-down” approach has been demonstrated for monitoring of in vivo changes of critical
quality attributes (CQA) such as glycosylation of mAb, which may play important roles in
their function and PK properties [54].
10.1.2 LC-MS bioanalysis of ADC

ADCs have shown great promise as novel therapeutics for treatment of cancer due to their
capability to deliver potent cytotoxic agents to targeted cells, thereby reducing systemic ex-
posure, increasing drug concentrations at the disease site, and broadening the therapeutic
window. Due to their highly heterogeneous and complex structure and complicated biotrans-
formation, multiple analytes are to be measured for an ADC to fully describe its PK in vivo: (1)
total antibody for antibody related properties; (2) conjugate for activity related properties;
and (3) the released small molecule payload and/or its catabolites for toxicity [55]. LC-MS
has been applied in the bioanalysis of each of the analytes to different extents. The released
payload/catabolites, due to their small molecule nature, are commonly analyzed using
LC-MS. Total antibody is typically measured by LBA but can also be quantified by using
immuno-affinity capture followed by enzyme digestion and LC-MS/MS analysis of the sur-
rogate peptide. Conjugates are assessed in one of two forms, conjugated antibody or conju-
gated payload. A conjugated antibody is usually quantified by LBA but can also be measured
by hybrid LBA LC-MS using the antipayload capture, followed by digestion and detection of
the surrogate peptide from the antibody. Alternatively, for a payload conjugated by an enzy-
matic/chemical cleavable linker, the conjugated payload can be quantified by using the
antiantibody capture, followed by release of the payload, which in turn is measured by
the LC-MS method [56]. In addition to the bottom-up method, the top-down intact analysis
has been applied to in vivo samples to understand the dynamic change of DAR (drug anti-
body ratio), metabolism of payloads on the antibody, and the fate of the dissociated payload
which may bind to endogenous proteins such as albumin [57–59].
10.1.3 LC-MS bioanalysis of PDC

In recent years, novel nonantibody scaffold proteins have emerged as a platform to con-
struct protein-drug conjugates to overcome challenges associated with antibodies such as
complex structure and requirement for a mammalian expression system. These novel pro-
teins possess a similar targeting binding capability but are of a much smaller size and have
a simpler structure than antibodies. They are usually domain-size proteins absent of disulfide
bonds or glycosylation, can be readily generated in the bacterial expression system in large
quantities, and of superior thermostability making production, purification, and storage rel-
atively easy [60]. They can be mutated to incorporate a linkage site for cytotoxic payload to
construct protein-drug conjugates (PDC). Similar to ADC, it requires multiple analytes to un-
derstand the PK properties of a PDC. In our lab, an affinity-capture based LC-MS/MS method
was developed to simultaneously quantify total protein and conjugated protein in plasma
and tissue for a novel PDC constructed with Centyrin, an engineered scaffold protein based
on the consensus sequence of fibronectin type III domains (FN3 domains) from human
Tenascin C [61]. Fig. 2 depicts the workflow which utilizes immobilized ion affinity chroma-
tography (IMAC) to extract the analytes, followed by trypsin digestion and LC-MRM analysis

FIG. 2 (A) The workflow to quantify total and

Centyrin drug conjugate in
conjugated Centyrin in mouse plasma and tissue
plasma/tissue homogenate
samples. (B) The scheme showing the surrogate
peptides representing conjugate and total
Add stable isotope labeled
Centyrin, respectively. Overall structure of
centyrin IS
Centyrin drug conjugate is shown as two identi-
cal Centyrin motifs (light blue color), each
IMAC purification
containing one conjugation site and an albumin Trypsin digestion
binding domain (orange color). Reprint with permis-
sion from C. Shi, S. Goldberg, T. Lin, V. Dudkin, W. Digestion by trypsin
Widdison, L. Harris, et al., Bioanalytical workflow for
for 4 h
novel scaffold protein-drug conjugates: quantitation of Surrogate Surrogate
total Centyrin protein, conjugated Centyrin and free LC-MRM analysis of surrogate peptide for peptide for
payload for Centyrin-drug conjugate in plasma and peptides conjugated total
Centyrin Centyrin
tissue samples using liquid chromatography-tandem (A) (B)
mass spectrometry, Bioanalysis 10 (20) (2018)
1651–1665.
of surrogate peptides. The peptide containing the linker and payload was monitored to quan-
tify the conjugated Centyrin while peptide from a region that does not contain the linker was
analyzed as a surrogate of the total Centyrin protein [62, 63]. This approach can be considered
as a choice for PK support of other scaffold PDCs as well as next generation site-
specific ADCs.
10.1.4 LC-MS bioanalysis of protein biomarkers

Endogenous peptides or proteins such as cytokines, hormones, or enzymes often need to
be monitored as biomarkers to elucidate the status of diseases, engagement of target, and
effect of drug treatment. The challenges of analyzing biomarkers such as choice of matrix is
well discussed in other sections of this chapter. Peptide/protein biomarkers present the ad-
ditional challenges of being of large size, potentially unstable, heterogeneous, and less
abundant. Novel LC-MS methodologies have been developed to overcome some of the lim-
itations. The heterogeneity of a protein, such as those in posttranslation modifications,
could convey biological or pharmacological meaning such as stage of a disease. LC-MS,
thanks to its high specificity in detection of molecule structure, can be very effective in elu-
cidating those heterogeneous modifications. For example, we have developed an intact
LC-HRMS method to quantify the relative abundance of three different glycosylation forms
of the apolipoprotein ApoC3 in human plasma and reveal that the ratio of these forms is
correlated to the status of diabetes [64]. The highly abundant nature of the ApoC3 protein
made a simple SPE step sufficient for the sample extraction procedure. For protein bio-
markers that exist at extremely low concentrations, sequential protein and surrogate pep-
tide immune-affinity capture has been proven to be able to effectively achieve LLOQ at low
pg/mL level for protein biomarkers [65].
10.1.5 LC-MS bioanalysis of drug metabolizing enzymes and transporters

Quantitation of drug metabolizing enzymes and transporters in cell lines and tissues has
been considered crucial for understanding drug disposition using in vitro-in vivo extrapola-
tion (IVIVE) and PBPK modeling. Tremendous efforts have been made in recent years to

develop reliable methods for absolution quantification of these challenging analytes using
LC-MS methods [66]. However, large variability still exists among data obtained from differ-
ent laboratories. In general, variables involved in analyzing metabolizing enzymes and trans-
porters include but are not limited to incomplete and inconsistent recovery of protein from
membrane fractionation and extraction, incomplete and inconsistent digestion of the protein,
ion suppression, poor choice of surrogate peptides, and challenges in correlation of expres-
sion level and activity. Some of them can be overcome by using a stable isotope labeled
internal standard and thorough evaluation of choice of surrogate peptides [67]. Optimization
of solubilization and denaturation steps for digestion is also critical because membrane pro-
teins may have transmembrane domains that are not directly accessible for the protease en-
zyme. Ideally, it is recommended to perform quantitation using a purified protein standard.
However, most of the drug metabolizing enzymes and transporters are membrane proteins
that are difficult to express and purify in large quantitation. Therefore, the common
approaches for quantifying those proteins are based on peptide standards, not being able
to fully address the variability in extraction recovery and digestion efficiency. For example,
a comparison of different methods showed that subcellular membrane fractionation gave in-
complete enrichment of the proteins leading to underestimation of protein concentration in
comparison to methods using whole tissue lysates [68]. Immuno-affinity capture at the pep-
tide level by using antibodies against the surrogate peptide has been applied to improve sen-
sitivity of the assays. An innovative approach using peptide group-specific antibodies that
recognize the short common C-terminal tryptic peptide sequence shared by multiple drug
metabolizing enzymes and transporters has been shown to effectively enrich the surrogate
peptides for quantitation without the need for the membrane fractionation step [69].
10.1.6 LC-MS bioanalysis of half-life extended biotherapeutics

Many peptides or proteins exhibit excellent bioactivity but are limited in their potential as
biotherapeutics due to their short in vivo half-life caused by proteolytic degradation and/or
urinary clearance. Strategies to extend half-life, such as conjugation to polymers, fusion/con-
jugation to Fc, mAb, or serum albumin, have been extensively investigated in recent years
[70]. These novel modalities require innovative bioanalytical approaches to assess their sta-
bility and ADME properties. The bottom-up approach, by monitoring surrogate peptides,
originated from different parts of the protein, for example, peptide from functional region
versus that from the half-life extension scaffold, can reveal the dynamic change of the concen-
trations of each part of the molecule during circulation. The top-down approach, on the other
hand, elucidates high-level structural alteration such as proteolytic degradation and helps
pinpoint the catabolic soft spots which can be potentially modified to improve the stability.
In our lab, both approaches are utilized in an integrated way for novel half-life extended mol-
ecules. This workflow is exemplified by a recent publication using dulaglutide, a GLP1-Fc
fusion protein, as the model molecule [19]. In this work, dulaglutide dosed to mice, along with
its in vivo catabolites, was purified from plasma using an anti-Fc antibody, followed by tryp-
sin digestion and LC-MS/MS analysis on a triple quadrupole instrument, or by LC-HRMS
analysis on a triple TOF mass spectrometer without digestion. In Fig. 3, the total Fc concen-
tration (red line) was measured using a surrogate peptide on Fc by the bottom-up approach
while the top-down intact assay was able to simultaneously quantify the intact concentration
(green line) and reveal the proteolytic cleavage sites on the molecules. The data revealed that

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Chippewa.
At his request they seated him on a log, with his back leaning
against a tree. He then commenced painting his face and singing his
death-song. As his enemies approached he only sang a louder and a
livelier strain; and when several had gathered around him, flourishing
their scalping-knives, and screeching forth their demoniac yells of
exultation, not a look or gesture manifested that he was even aware
of their presence. At lengthy they seized him and tore his scalp from
his head. Still seated with his back against a large tree, they
commenced shooting their arrows into the trunk around his head,
grazing his ears, neck, etc., until they literally pinned him fast,
without having once touched a vital part. Yet our hero remained the
same imperturable stoic, continuing to chant his defiant strain, and
although one of the number flourished his reeking scalp before his
eyes, still not a single expression of his countenance could be
observed to change. At last one of the number approached him with
a tomahawk, which, after a few unheeded flourishes, he buried in the
captive’s skull, who sank in death, with the war song still upon his
lips. He had, indeed, succeeded well in teaching his enemies “how a
Chippewa could die.”
The reader has already made the acquaintance of that renowned
Mandan chief Mahtotopa; here is another episode in that hero’s
history:
A party of 150 Scheyenne warriors had invaded the territory of the
Mandans; Mahtotopa, the young but already famous warrior of whom
we have spoken, went in pursuit of them at the head of fifty of the
bravest of his tribe. At the end of two days he came up with them.
The Mandans, inferior in number, hesitated to engage in combat,
when by a sudden impulse, Mahtotopa planted his lance,
ornamented with a piece of red stuff, in the ground in token of
defiance. The Scheyennes who were approaching to attack the party
were arrested by the sight of this courageous act, and their chief
advancing alone to meet the young Mandan warrior enquired who he
was who defied alone the enemy?
“It is Mahtotopa, second chief in command of the brave and
valiant Mandans.”
“I have often heard him spoken of,” replied the Scheyenne; “he is
a great warrior. Would he dare to advance and fight against me
alone while our warriors look on?”
“Is it a chief who speaks to Mahtotopa?”
“See the scalp which hangs from the bit of my horse,” answered
the Scheyenne; “see my lance ornamented with the fur of the ermine
and the feather of the eagle of war.”
“You have spoken enough,” said the Mandan.
The Scheyenne chief set off at full gallop and planted his lance by
the side of that of Mahtotopa. The warriors of the two tribes drew
near and formed a great circle. The two champions advanced into
the middle of these lists formed by human warriors. They were on
horseback, decorated with feathers and wearing their finest
garments. They each fired a shot without effect; Mahtotopa then
showed his adversary his powder-flask, which had been pierced by a
ball, and threw it on the ground as well as his gun, which had thus
become useless. The Scheyenne chief in order to fight with equal
arms did the same, and for some moments they galloped one round
the other discharging arrows with incredible rapidity. The horse of the
Mandan rolled on the ground pierced by an arrow, and when
Mahtotopa arose to continue the fight his adversary sprang from his
horse and once more the combat became equal. Soon the warriors
were exhausted. Then the Scheyenne drew his knife and brandished
it in the air. “Yes,” answered Mahtotopa, who understood this
unspoken invitation. The two warriors disencumbered themselves of
their quivers and shields; but the Mandan had not his knife; he had
forgotten it in his cabin; this did not stop him; he parried the blows of
his adversary with the wood of his bow, which he wielded like a club.
He soon succeeded in forcing his enemy to relax his hold on his
weapon; the knife fell, the combatants threw themselves on each
other and tried to get possession of the weapon which lay at their
feet; it was taken and wrenched back again several times by both
adversaries, and each time it was dyed with the blood of one or the
other. At length Mahtotopa seized it a last time and plunged it to the
hilt in the heart of the Scheyenne chief, then drew it out, took off his
adversary’s scalp and showed the trophy of his victory to the
spectators. Such a scalp as this would be more precious in the eyes
of Mahtotopa than any dozen of such bloody trophies he might
previously have possessed. Few Indian warriors of the “old school”
but who could point in the same fashion to one poor scrap of skin
and hair with special exultation, while with pomp and pride they
describe to the curious listener the peculiar circumstances under
which the trophy was obtained. Take the following little anecdote
related by a somewhat celebrated Ojibbeway “brave” as an example:
“This scalp I nailed separately because I took it under curious
circumstances and like to recall it to my memory. I went on the war
trail just ten years ago against the Sioux band of the chief Wabasha.
There were eighty of us Ojibbeways, and we went down the
Chippeway River in canoes. When we found ourselves close to the
enemy we turned into an arm of water which we thought was the
main channel; but it was only a bayou which lost itself in swamp and
rushes, and on attempting to push through all our canoes stuck in
the mud. The Sioux fleet was coming up to cut us off in our hole, and
we left our canoes and went on foot. The Sioux fired on us from the
water and we replied from land; but the distance was too great, and
no one was wounded. One of the boldest and bravest of the Sioux,
however, pushed on far in advance in order to cut us off. He came
too near the bank and was shot by one of our men and he fell back
in his canoe which began drifting down the stream. His body hung
over the side of the boat into the water. I saw this, and feeling
desirous to have his scalp I leaped into the water and swam after the
canoe. There was plenty of risk, for the other Sioux were now
paddling up; besides, it was not at all certain the man was really
dead. I did not care though, but swam on, seized the canoe and the
man, and had his scalp with a couple of cuts. Ha, ha! I waved it once
to the Sioux, pushed the canoe with the half-dead quivering fellow
towards them, and soon joined my party again. We all escaped, and
only our enemies had cause to lament. He was their best warrior,
and so I nailed his scalp, the only one taken that time, here on my
hatchet which I carry about with me.”
The following tradition of a war exploit of the same tribe, recorded
by the Rev. P. Jones, will show the confidence they place in dreams:
“A canoe manned with warriors was once pursued by a number of
others, all filled with their enemies. They endeavoured to escape,
paddling with all their might, but the enemy still gained upon them;
then the old warriors began to call for the assistance of those things
they had dreamt of during their fast-days. One man’s munedoo was
a sturgeon, which being invoked, their speed was soon equal to that
of this fish, leaving the enemy far behind; but the sturgeon being
short-winded was soon tired, and the enemy again advanced rapidly
upon them. The rest of the warriors, with the exception of one young
man who, from his mean and ragged appearance, was considered a
fool, called the assistance of their gods, which for a time enabled
them to keep in advance. At length, having exhausted the strength of
all their munedoos, they were beginning to give themselves up for
lost, the other canoes being now so near as to turn to head them,
when just at this critical moment the foolish young man thought of his
medicine-bag, which in their flight he had taken off from his side and
laid in the canoe. He called out, ‘Where is my medicine-bag?’ The
warriors told him to be quiet; what did he want with his medicine-bag
at this perilous time? He still shouted, ‘Where is my medicine-bag?’
They again told him to paddle and not trouble them about his
medicine-bag. As he persisted in his cry, ‘Where is my medicine-
bag?’ one of the warriors seeing it by his side took it up and threw it
to him. He, putting his hand into it, pulled out an old pouch made of
the skin of a saw-bill, a species of duck. This he held by the neck to
the water. Immediately the canoe began to glide swiftly at the usual
speed of a saw-bill; and after being propelled for a short time by this
wonderful power, they looked back and found they were far beyond
the reach of the enemy, who had now given up the chase. Surely this
Indian deserved a patent for his wonderful propelling power, which
would have superseded the use of the jarring and thumping steam-
boats, now the wonder and admiration of the American Indian. The
young man then took up his pouch, wrung the water out of it, and
replaced it in his bag; telling the Indians that he had not worn his
medicine-bag about his person for nothing,—that in his fast he had
dreamt of this fowl, and was told that in all dangers it would deliver
him, and that he should possess the speed and untiring nature of the
saw-bill duck. The old warriors were astonished at the power of the
young man whom they had looked upon as almost an idiot, and were
taught by him a lesson, never to form a mean opinion of any persons
from their outward appearance.”
The canoe of the Indian has been several times mentioned in
these pages, and as it plays a very important part in the career of the
savage in question, in times of peace as well as of war, it may not be
amiss here to furnish some particulars as to its construction. Of its
antiquity there is very little doubt; for being of a simple construction,
and the materials for it at hand, we suppose that it would occur to the
simplest savage, that if it was necessary to go some distance on the
water, he must have something to float upon, and that wood or the
lightest part of it—bark—was just the thing that was required. So that
if it can exactly be computed how long it is since the North American
Indian first took up his abode in those vast regions which he so long
possessed undisturbed, and deduct a few years for him to look about
his new home, we shall have the exact age of the canoe; at any rate,
the discoverers of America found the canoe along with the Indians,
and the natives called them canoas, which were hollowed out of
trees. The way that the tribes belonging to the Algongian stock, who
are essentially fishermen and sailors, build their canoes is as follows:
—
The birch is the tree selected for the purpose, and the bark is that
part of it of which the skeleton of the canoe is built. The Indians
select the largest and smoothest trees; so that they can obtain large
pieces of bark and prevent too much sewing. The inner side of the
bark is scraped with knives, and it is then given over to the women to
sew. The men then get ready the framework of the boat, which is of
cedar. “They have usually a sort of model, or a frame of the figure
and size of a canoe, round which the branches or ribs are bent. In
the centre the arches are large, growing smaller towards either end.
These ribs are peeled wonderfully thin, because lightness and easy
carriage are the chief qualities of a canoe. Between the upper end of
the ribs or rarangues, as they are called, a thin cross-piece is
fastened, to keep them in a horizontal position. This is for the
purpose of giving strength to the sides.” These boats have no keel,
but the rarangues, and lanes, or cross-pieces, are tied to a piece of
wood at the top.
The Indians use neither nails nor screws in the manufacture of the
canoe; everything about it is either tied or sewn together. This,
however, does not seem to deteriorate its strength or utility. When
the framework is completed, the bark covering, previously alluded to,
and which is made by the women, is spread over it, and the edge
turned down over the “maître” and firmly bound to it. The interior of
the canoe is then lined with thin boards, laid across the ribs, which
they call les lisses. These protect the bottom from the feet of the
passenger, and injury from the sails. They are remarkably thin and
light, and not much stouter than the sides of a cigar-box. Of course
the canoes are not suited for the nailed boots of a European or the
transport of ironshod boxes, but only for the soft mocassined feet of
the Indians, and the still softer bundles of fur.
“All the wood-work in the canoe is derived from the cédre blanche,
for this wood is very elastic, does not split, has but light specific
gravity, and is easily cut with a knife. The material for the cords and
strings is also obtained from the same tree, though they also use the
bark taken from the root of the epinetee blanche, a species of
spruce. All this is prepared by the women, who are always busy in
twisting ‘watals,’ owing to the large quantities used. They can make
either twine or stout cords out of it, and for their fishing nets, the
ropes often reach a length of fifty yards. These cords last a long
time, and resist the influence of water, and they can be laid up for
two years without deteriorating. If damped, they become as supple
as leather.
“The canoe is sharp, front and back, and the ends stand up a
little: these ends are often gaily decorated in the large canoes. A
small piece of wood is inserted in either end to give it increased
strength. This, too, is often carved and painted into the shape of a
queer-looking manikin.
“After the canoe is completed, the material is left to dry. For this
purpose pieces of wood are inserted in every part to keep it well
extended, and it is then hung up in the air. Botching all the little
holes, seams, and stitches is the final process. For this purpose the
resin of the pine or fir is used, and is laid on in thick patches
wherever a hole would allow the water entrance. The weak parts of
the bark or the holes of branches are also covered with this resin.”
In the canoe building, as, indeed, in all labours, a great part of the
work falls to the women. They do all the sewing and tying, and often
are compelled to take part in the hammering and botching. When the
little craft is afloat, the squaws assist in the paddling; and very often
are more skilful in this respect than the men. Usually, however, when
a family is moving about, the man and wife paddle side by side. In
the primitive mode of sailing, one sits at the stern and one at the
bow, both paddling with short broad paddles. The one in the bow
looks out for shallow rocks and rapids, which might prove
dangerous; he then signals to the one in the stern, to whose care the
propelling of the boat is principally entrusted, who directs the boat
accordingly. The lightness of the canoe, and the extraordinary skill of
the Indians in guiding it, enables it to skim over the surface of the
water with marvellous rapidity. The most surprising part of the
business is the great load these canoes can carry. Mr. Kohl makes
mention of one he saw, which contained a family of twenty persons,
with their goods and chattels! They had come some hundred and
fifty miles in their little boat,—over cataracts and rapids; besides they
had a quantity of deer and bear skins with them, and several live
dogs. The whole weight must have exceeded a ton!
Throughout the whole of Polynesia, as in savage North America,
the native, wherever you find him, regards war as the first business
of his life, as the only means of earning fame and riches. Without
doubt this yearning for perpetual strife has now somewhat subsided,
but within the memory of the still young, the said yearning was at its
highest. Samoa furnishes an apt instance; and even within the last
few years, when Mr. Turner was there located as missionary, he
found that the murder of a chief, a disputed title, or a desire on the
part of one, two, or more of the districts to be considered stronger
and of more importance than the rest, were frequent causes of war.
Hostilities were often prevented by such acts as giving up the culprit,
paying a heavy fine, or bowing down in abject submission, not with
ropes round their necks, but carrying firewood and small stones used
in baking a pig, or perhaps a few bamboos. The firewood, stones,
and leaves were equivalent to their saying, “Here we are your pigs to
be cooked if you please; and here are the materials with which to do
it.” Taking bamboos in the hand was as if they said, “We have come,
and here are the knives to cut us up.” A piece of split bamboo was of
old the usual knife in Samoa.
If, however, the chiefs of the district were determined to resist,
they prepared accordingly. The boundary which separated one
district from another was the usual battle field, hence the villages
next to that spot on either side were occupied at once by the troops.
The women and children, the sick and the aged, were cleared off to
some fortified place in the bush, or removed to some other district
which was either neutral or could be depended upon as an ally.
Moveable property was either buried or taken off with the women
and children. The wives of the chiefs and principal men, generally
followed their husbands wherever they might be encamped, to be
ready to nurse them if sick or wounded. A heroine would even follow
close upon the heels of her husband in actual conflict, carrying his
club or some other part of his armour; it was common for chiefs to
take with them a present of fine mats when they went to another
district to solicit help in war, but there was no standing army or
regularly paid soldiers anywhere.
Polynesian Weapons.
When the chiefs decided on war, every man and boy under their
jurisdiction old enough to handle a club, had to take his place as a
soldier, or risk the loss of his lands and property and banishment
from the place. In each district there was a certain village or cluster
of villages known as the advance troops. It was their province to take
the lead and in battle their loss was double the number of that of any
other village. Still they boasted of their right to lead, and would on no
account give it up to others, and talked in the current strain of other
parts of the world, about the glory of dying in battle. In a time of
peace the people of these villages had special marks of respect
shown to them, such as the largest share of food at public feasts,
flattery, etc. While war was going on the chiefs and heads of families
united in some central spot, and whatever they decided on, either for
attack or defence, the young men endeavoured implicitly to carry
out. Their weapons were of old, clubs, spears, and slings;
subsequently, as iron was introduced, they got hatchets, and with
these they made their most deadly weapon, viz., a sharp tomahawk
with a handle the length of a walking stick. After that again, they had
the civilized additions of swords, pistols, guns, and bayonets. Around
the village where the war party assembled, they threw a rough
stockade, formed by any kind of sticks or trees cut into eight feet
lengths and put close to each other upright, with their ends buried
two feet in the ground. The hostile parties might be each fortified in
this way, not more than a mile from each other, and now and then
venture out to fight in the intervening space, or to take each other by
surprise at weak or unguarded points. In their war canoes they had
some distinguishing badge of their district hoisted on a pole, a bird it
might be, or a dog, or a bunch of leaves. And for the bush-ranging
land forces, they had certain marks on the body by which they knew
their own party, and which served as a temporary watchword. One
day the distinguishing mark might be blackened cheeks, the next two
strokes on the breast, the next a white shell suspended from a stripe
of white cloth round the neck, and so on; before any formal fight they
had a day of feasting, reviewing, and merriment. In action they never
stood in orderly ranks to shoot at each other. According to their
notions that would be the height of folly. Their favourite tactics were
rather of the surprise and bush skirmishing order. Prisoners, if men,
were generally killed; if women, distributed among the conquerors. In
the battle which was fought in 1830, to avenge the death of
Tamafainga, a fire was kindled, and prisoners to the extent of four
hundred, some say, were burned, but probably it did not reach the
half of that number.
Their heroes were the swift of foot, like Achilles or Asahel; men
who could dash forward towards a crowd, hurl a spear with deadly
precision; and stand for a while, tilting off with his club other spears
as they approached him within an inch of running him through. They
were ambitious also to signalize themselves by the number of heads
they could lay before the chief. No hero at the Grecian games
rejoiced more over his chaplet than did the Samoan glory in the
distinction of having cut off a man’s head. As he went along with it
through the villages on the way to the place where the chiefs were
assembled, waiting the hourly news of the battle, he danced and
capered and shouted, calling out every now and then the name of
the village, and adding, “I am so and so, I have got the head of such
a one.” When he reached the spot where the chiefs were met, he
went through a few more evolutions and then laid down the head
before them. This, together with the formal thanks of the chiefs
before the multitude for his bravery and successful fighting, was the
very height of a young man’s ambition. He made some giddy
frolicsome turns on his heels and was off again to try and get
another victim. These heads were piled up in a heap in the mapae or
public assembly. The head of the most important chief was put on
the top, and as the tale of the battle was told they would say, “There
were so many heads surmounted by the head of so-and-so,” giving
the number and the name. After remaining for some hours piled up
they were either claimed by their relatives or buried on the spot.
A rare illustration of this ambition to get heads occurred about ten
years ago. In an unexpected attack upon a village one morning, a
young man fell stunned by a blow. Presently he recovered
consciousness, felt the weight of some one sitting on his shoulders
and covering his neck, and the first sounds he heard was a dispute
going on between two as to which of them had the right to cut off his
head. He made a desperate effort, jostled the fellow off his back,
sprang to his feet, and with his head all safe in his own possession,
soon settled the matter by leaving them both far behind him.
The headless bodies of the slain scattered about in the bush after
a battle, if known, were buried, if unknown left to the dogs. In some
cases the whole body was pulled along in savage triumph, and laid
before the chiefs. One day when Mr. Turner was in a war-fort,
endeavouring to mediate for peace, a dead body of one of the
enemy was dragged in, preceded by a fellow making all sorts of
fiendish gestures, with one of the legs in his teeth, cut off by the
knee.
If the war became general, and involved several districts, they
formed themselves into a threefold division of highway, bush, and
sea fighters. The fleet might consist of three hundred men in thirty or
forty canoes. The bushrangers and the fleet were principally
dreaded, as there was no calculating where they were or when they
might pounce unawares upon some unguarded settlement. The fleet
met apart from the land forces and concocted their own schemes.
They would have it all arranged, for instance, and a dead secret, to
be off after dark to attack a particular village belonging to the enemy.
At midnight they land at an uninhabited place some miles from the
settlement they intend to attack. They take a circuitous course in the
bush, surround the village from behind, having previously arranged
to let the canoes slip on quietly and take up their position in the
water in front of the village. By break of day they rush into the
houses of the unsuspecting people before they have well waked up,
chop off as many heads as they can, rush with them to their canoes,
and decamp before the young men of the place have had time to
muster or arm. Often they are scared by the people who during the
war keep a watch night and day at all the principal openings in the
reef; but now and then the plot succeeds and there is fearful
slaughter. In one of these early morning attacks from the fleet the
heads of thirteen were carried off. One of them was that of a poor old
man who was on his knees at his morning devotions, when off went
his head at a blow. In another house that same morning there was a
noble instance of maternal heroism in a woman who allowed herself
to be hacked from head to foot bending over her son to save his life.
It is considered cowardly to kill a woman, or they would have
dispatched her at once. It was the head of her little boy they wanted,
but they did not get it. The poor woman was in a dreadful state, but,
to the surprise of all, recovered.
To the king of Samoa was reserved the power of sparing life.
When led to the king’s presence the captive warriors usually
prostrated themselves before him, and exclaimed: make paha e ora
paha-i runa te ars? i raro te aro. “To die, perhaps to live, perhaps
upward the face!” If the king did not speak, or said “The face down,”
it was sentence of death, and some one in attendance either
despatched the poor captive in his presence or led him away to be
slaughtered. But if the king said, “Upward the face,” they were
spared only to be slaves or to be sacrificed when the priests should
require human victims.
When the king, or any chief of high rank, was known to be
humane, or any of the vanquished had formerly been on terms of
friendship with him, avoiding carefully the warriors, an individual
risking his life on the conqueror’s clemency would lie in wait for him
in his walks, and prostrating himself in the path, supplicate his
compassion, or rush into his house and throw himself on the ground
before him. Though anyone might have killed him while on his way
thither, none dared touch him within the king’s enclosure without his
orders. When the king did not speak, or directed the fugitive to be
carried from his presence, which was very unusual, he was taken out
and slain. Generally the prince spoke to the individual who had thus
thrown himself into his power; and if he did but speak, or only
recognise him, he was secure. He might either join the retinue of the
sovereign, or return to his own house. No one would molest him, as
he was under maru shade or the screening protection of the king.
These individuals, influenced by feelings of gratitude, generally
attached themselves to the person or interest of the prince by whom
they had been saved, and frequently proved through subsequent life
the most faithful attendants on his person and steady adherents to
his cause.
The gentleman just mentioned furnishes us with an account of the
massacre of the teachers which some few years since took place at
the Isle of Pines. There were three of them. They were blamed for
causing sickness. Mantungu, the chief, ordered them away, and as
Captain Ebrill, of the brig “Star,” was there at the time and offered to
take them to Samoa, they left in his vessel. Captain Ebrill first went
to Sydney, came back, was on his way to Samoa with the teachers,
but touched at the Isle of Pines to procure some more sandal-wood.
He anchored at Uao, some little distance from the residence of the
chief. The natives went off to the vessel. “Where are Mantungu and
his sons?” said a person on board. “Dead,” replied the natives in a
joke. “Dead, dead; that is good,” said the same person; “let such
chiefs be dead, and let the common folk live, and help us cut sandal-
wood.” For some reason which we cannot ascertain, Captain Ebrill
and his crew were angry with the old chief, and as a further proof of
it, when he sent a present of food to the teachers, who he heard
were in the vessel, it was not allowed to be received on board.
Those who took it had pieces of wood thrown at them and two
musket shots fired at them. None were killed, but one man was
wounded in the knee. “What can this mean,” said Mantungu,
“wishing me and my sons dead in our own land? and why commit
such outrages upon my people who went with a present?” Whether
he had any intentions previously to take the vessel we know not; but
any one who knows the old despot can imagine how such treatment
would make his savage heart flame with revenge. Next morning
thirty select men were off, determined to kill all on board. They took
some sandal-wood with them to sell; and as a further trick did not
arm themselves with clubs or axes, but with the adzes which they
use in dressing off the bark and sap from the wood. They reached
the vessel. The sandal-wood pleased all on board, was immediately
bought, and the natives were allowed to go up on deck to grind their
adzes on pretence that they were going off for more wood. One of
the crew was turning the handle of the grindstone, a native grinding
an adze, and the captain close by. Seizing a favourable moment the
native swung his adze and hit the captain in the face between the
eyes,—this was instant death to Captain Ebrill, and the signal for
attack all over the vessel. In a few minutes seventeen of the crew
were killed—viz., ten white men, including the captain, two
Marquesans, two Mangarans, one Aitutakian, one New Zealander,
and a Karotongan teacher. The cook fought desperately for awhile
with an axe and killed one man, but was at length overpowered and
fell. This occurred on the afternoon of the 1st of November, 1842. A
young man named Henry, two Samoan teachers, and a native of the
New Hebrides made their escape below. Henry loaded muskets and
fired up the companion, but without effect. It only exasperated the
natives on deck, who threw down upon them billets of sandal-wood.
The teachers then collected their property, six red shirts, eight axes,
etc., called up and offered all for their lives, but there was no mercy.
Night came on. The natives divided; a party went on shore in the
boat, and the rest remained on deck to guard those below. In the
morning the natives called down to Henry and the Samoans to come
up, take the vessel further in, and then go on shore, as Mantungu
had come and declared they were to live. The poor fellows felt they
were entirely in the hands of the natives, came up, ran close in
shore, and again dropped anchor. They were then taken to the
shore. A son of Mantungu, with a tomahawk in his right hand, met
Henry as he stepped out of the boat, held out his left hand with a
feigned grin of friendship to shake hands; but the moment he got
hold of Henry’s right hand, the villain up with his axe and laid the
poor fellow dead at his feet. Others were up and at the remaining
three. Lengolo, the New Hebrides native, and the Samoan Taniela,
were killed at once. Mantangu and a party of natives were sitting
under the shade of the cocoa-nuts looking on. Lasalo, the other
Samoan teacher, escaped streaming with blood, threw himself at the
feet of the old chief and begged for life. Mantungu was silent for a
minute or two, but soon gave the wink to a Lifu man. Lasalo was now
dragged away to be killed, but he sprang from the fellow as he lifted
his axe and darted off to sea. The savages were at his heels, he was
hit repeatedly, but escaped to the deep water, struck out and swam
off to a little island. Four men jumped into a canoe and after him; he
climbed a pine tree and talked for awhile with them; they assured
him Mantungu had determined to spare him, and at last he came
down. It was treachery again. They sprang upon him like tigers; but
again he extricated himself, and rushed to the canoe; there,
however, at length the poor fellow was overpowered and fell.
After the massacre the bodies were divided. There were people
there from Caledonia, Mare, and Lifu, and each had a share. Then
followed the plundering of the vessel; deck, cabins, and forecastle
were stripped of everything. They cut down the masts to get at the
sails and rigging, and then set fire to her without opening the hold.
As the fire reached the powder there was a terrific explosion, but no
lives lost. She burned to the water’s edge and then sank.
Another curious story is related of these people in connexion with
their warlike disposition. On one occasion they captured a European
ship called the “Sisters,” and having massacred the crew, proceeded
to rifle the vessel of everything portable. Some kegs of gunpowder
came under this category, and being unacquainted with its nature,
after conveying it ashore, they amused themselves by sprinkling
pinches of it in the fire to “make sparks.” The result may be easily
imagined; the whole bulk of powder became ignited and scattered
the amazed savages right and left; many were maimed and a few
killed, and among the latter was a chief of some renown. The
calamity was of course attributed to the evil spirits of the murdered
crew of the “Sisters,” and the Samoans vowed to take dire revenge
on the first batch of white men who fell into their clutches. They had
not long to wait. A large boat with seven men in her put in not long
after near the same place. This was a party of runaway convicts from
Norfolk Island. Five of them were killed and the boat broken to
pieces. The other two had gone off to forage in the bush, and happily
met with old Jeni (the chief) and his sons, who were travelling there
that very day about some war affairs. The murderers of the five who
were in search of the other two found them with Jeni and his sons
and proposed to kill them. Jeni refused and took them home with
him. They lived for two months under the wing of the old chief and
our teachers, and were kindly treated. But the fellows were out-and-
out Norfolk Islanders. One night they got up and robbed old Jeni of
four muskets, ten hatchets, four felling axes, and a saw. They went
to the teachers’ house, took four shirts, two knives, and an axe, and
off they set in the teachers’ canoe to join some white men reported
to be at Lifu. At daylight the things were missed and the place in an
uproar. Suspicion fell on the teachers. Their canoe is away—they
must have helped the fellow to lift it into the water. “No,” said Tataio,
“how can that be? We are robbed too, and our canoe gone to boot.
But I’ll tell you they cannot be far away, let us be off after them: I go
for one, who will join me?” A party was made up in a twinkling, and
off they went, hard drive at their paddles, out to sea in the direction
of Lifu. Soon they sighted something rising now and then on the top
of the waves. Two men in it—just the fellows. A little further and they
were in sight of each other. The thieves loaded their muskets and
fired two or three shots. No one was hurt. Their pursuers paddle
steadily on and are determined to be at them. Then they threw the
stolen property into the sea towards them, but who could pick up
sinking axes? All were lost. The two scoundrels knew what they
deserved, thought it was a choice of deaths, and jumped into the sea
to drown themselves. “Poor fellows,” said Tataio, “they think we are
going to kill them. Let us save them if we can.” He got his hand into
the mouth of one of them when he had but almost sunk, and pulled
him up. The other was also secured and laid flat in the bottom of the
canoe half dead. The sea was running high, the outrigger broke, and
all had to jump out except the two vagabonds who were lying
senseless in the bottom of the canoe. But it was hard work to swim
and drag the disabled canoe through a heavy sea. “What are we
doing?” said the natives to each other. “By and by we shall be all
dead. Why should we be drowned in trying to save these fellows? It
is their own doing. Let us tilt the canoe over, pitch them out and save
ourselves.” “No,” said Tataio; “see the current is drifting us fast to
that little island. Let us try it a little longer.”
They reached the little island, landed, and rested, and scolded the
two scoundrels as they recovered and were able to listen to what
was going on. Some natives of the island, when they heard the tale,
would have them killed, but the votes with Tataio carried it for their
lives. “Well then spare their lives, but we must punish them.” They
stripped them naked, besmeared them from head to foot with a
mixture of mud and ashes, and then said, “Now you must go about
so.” Native like, however, they repented next day, washed the fellows
clean, and gave them back their clothes. After resting a day or two
the party returned to Mare.
The Mare people were delighted to see the party return, but when
they heard the story, and knew that all the property was thrown
away, they could hardly keep their clubs off the vagabonds. But old
Jeni united with the teachers and forbade. “What good,” said he, “will
it do to kill them? It won’t bring back my property.” Here again they
were allowed to live, and were fed too by the people, as if nothing
had happened, until they had an opportunity of leaving in a vessel
which touched at the place some time after.
Justice demands some few words of explanation concerning the
reputed “wanton massacres” by the natives of these islands. Without
doubt they set but little value on human life, and are treacherous in
the extreme; naturally, they are suspicious and likely to regard the
actions of men so totally different in manner and habit from
themselves, as are white men, with constant uneasiness; added to
this, it is an ascertained fact that in numerous instances European
and American ships trading to this part of the world have not
scrupled to cheat and ill-use the ignorant savages with whom they
had to deal, and though the aggressors have succeeded in sailing off
with impunity, such behaviour could not fail to plant seeds of ill-
feeling, the crop of which would certainly be garnered for the next
batch of “white cheats” who touched their shores.
The following little story of this South Sea traffic, related by a
traveller named Coulter (who relates it rather as a joke than a
disgrace) will illustrate what the above lines are meant to convey:
“There was some firewood collected on the beach which had yet
to be got off, as we were in actual want of it. The natives were
offered some trifling presents to bring it to the schooner, but acted so
slowly that the captain got out of patience and dispatched his boat
with four men and the interpreter to effect the desired object; he
gave them every caution not to mix with the natives, but work quick
and get off the wood at once, and if there should be any attempt to
attack them on the part of the natives, to run to the water’s edge and
the guns of the schooner would cover them.
“I may here remark, that it is a usual plan with almost all the
islanders in the Pacific, who are treacherously disposed, to obtain
first as much as they can by fair trade, and if the suspicions of the
captain, or any vessel trading with them, should be lulled so as to
throw him off his guard by this apparent honesty and safety, to take
advantage of such a state of things and either cut off a boat’s crew
or attempt to board and plunder the ship, if possible.
“Trainer, the mate, who knew these people well, had no
confidence in any of them; though he seemed to take matters easy
enough he was well prepared for any surprise that might be
attempted, and he was doubly particular in his means of defence, as
the interpreter informed him that the natives were laying plans to
board the schooner, thinking as she was small the capture of her
would be an easy matter. Two boat’s load of the firewood was gone
off and the boats sent for the third and last. The wood was about
forty yards from the beach and had to be carried down by the men to
the boat. A number of canoes were rapidly shoved into the water and
filled with men. This was the critical time, and we all kept ready and
an anxious watch on the boat.
“In a few minutes the four men on shore were observed to run
with all their might down to the water’s edge followed by a crowd of
armed natives. They had scarcely time to get into the boat and push
her off from the beach when the natives were close on, throwing a
number of spears at them, one of which took effect on one of the
men. However, the remaining three got her off into deep water. The
interpreter, who could not get into the boat, stole into the water at
another point unperceived by the natives and swam off. They were
all taken quickly on board; but there was no time to hoist the boat up
as the canoes filled with armed men were fast approaching.
“The seaman who was wounded in the boat died in a few minutes
after reaching the deck—the spear had passed right through his
chest. The men, who were all enraged at the loss of an excellent
man and an esteemed messmate, were burning for revenge, and
were waiting with impatient eagerness for the orders to slap at them.
Trainer was at the gangway and his eye on the advancing fleet of
canoes; I was with him. We were well prepared. The short
carronades were the most useful articles on the present occasion
and were loaded with grape. The crew were also armed. ‘Well,’ said
the captain, ‘I have been here several times, and have always
treated them fairly and kindly, and now, without cause, they have
killed one of our best men and want to take my vessel and murder us
all. They shall catch it.’ Thus spoke a really humane man, but he was
irritated beyond all patience by the treachery of the natives and loss
of his man. ‘Now, my lads, are you ready?’ ‘Ay, ay, sir,’ ‘Remember, if
we let these savages board us not a man will be alive in ten
minutes.’ ‘Never fear, sir; we’ll pay them.’ On the canoes came; they
separated into two divisions, one advancing to the bows the other
towards the stern.
“Trainer keenly eyed them, whilst he made frequent exclamations,
such as ‘Well, you want the schooner, I suppose,’ etc. The natives in

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Identification and Quantification of

Drugs, Metabolites, Drug Metabolizing

Library of Congress Cataloging-in-Publication Data

For information on all Elsevier publications

Publisher: Susan Dennis

Typeset by SPi Global, India

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

be universally applied to data acquired on instruments from different vendors. Alternatively,

10.1.1 LC-MS bioanalysis of mAb

I. Techniques for identifying and quantifying drugs and metabolites

10.1.2 LC-MS bioanalysis of ADC

10.1.3 LC-MS bioanalysis of PDC

I. Techniques for identifying and quantifying drugs and metabolites

FIG. 2 (A) The workflow to quantify total and

10.1.4 LC-MS bioanalysis of protein biomarkers

10.1.5 LC-MS bioanalysis of drug metabolizing enzymes and transporters

I. Techniques for identifying and quantifying drugs and metabolites

10.1.6 LC-MS bioanalysis of half-life extended biotherapeutics

I. Techniques for identifying and quantifying drugs and metabolites

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