Table Of ContentMass Spectrometry for the
Clinical Laboratory
Edited by
Hari Nair, PhD, DABCC, FACB
Boston Heart Diagnostics, Framingham, MA, United States
William Clarke, PhD, MBA, DABCC
Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, United States
AMSTERDAM • BOSTON • HEIDELBERG • LONDON
NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier
125 London Wall, London EC2Y 5AS, United Kingdom
525 B Street, Suite 1800, San Diego, CA 92101-4495, United States
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
Copyright © 2017 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical,
including photocopying, recording, or any information storage and retrieval system, without permission in writing
from the publisher. Details on how to seek permission, further information about the Publisher’s permissions poli-
cies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing
Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher (other
than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our
understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
information, methods, compounds, or experiments described herein. In using such information or methods they
should be mindful of their own safety and the safety of others, including parties for whom they have a professional
responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability
for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or
from any use or operation of any methods, products, instructions, or ideas contained in the material herein.
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
ISBN: 978-0-12-800871-3
For information on all Academic Press publications
visit our website at https://www.elsevier.com/
Publisher: Mica Haley
Acquisition Editor: Tari Broderick
Editorial Project Manager: Pat Gonzalez
Production Project Manager: Julia Haynes
Designer: Maria Inês Cruz
Typeset by Thomson Digital
List of Contributors
L.M. Bachmann, PhD, DABCC
Department of Pathology, Virginia Commonwealth University, Richmond, VA, United States
W. Clarke, PhD, MBA, DABCC
Johns Hopkins University School of Medicine, Baltimore, MD, United States
J.C. Cook-Botelho, PhD
Clinical Chemistry Branch, Division of Laboratory Sciences, Centers for Disease Control and
Prevention, Atlanta, GA, United States
C.A. Crutchfield, PhD
Johns Hopkins University School of Medicine, Baltimore, MD, United States
D. French, PhD, DABCC, FACB
Department of Laboratory Medicine, University of California San Francisco, San Francisco,
CA, United States
U. Garg, PhD
Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City,
MO, United States
D.A. Herold, MD, PhD
Department of Pathology, University of California San Diego, La Jolla;
VAMC-San Diego, San Diego, CA, United States
P.J. Jannetto, PhD, DABCC, FACB, MT(ASCP)
Mayo Clinic, Department of Laboratory Medicine and Pathology, Toxicology and Drug Monitoring
Laboratory, Metals Laboratory, Rochester, MN, United States
H. Ketha, PhD, NRCC
Department of Pathology, University of Michigan Hospital and Health Systems, Ann Arbor,
MI, United States
P.B. Kyle, PhD, DABCC
University of Mississippi Medical Center, Jackson, MS, United States
K.L. Lynch, PhD, DABCC, FACB
Department of Laboratory Medicine, University of California, San Francisco, CA, United States
I.W. Martin, MD
Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH, United States of
America
H. Nair, PhD, DABCC, FACB
Boston Heart Diagnostics, Framingham, MA, United States
B. Rappold
Essential Testing, LLC, Collinsville, IL, United States
R.J. Singh, PhD, DABCC
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States
xiii
xiv List of Contributors
D.F. Stickle, PhD
Department of Pathology, Jefferson University Hospital, Philadelphia, PA, United States
J. Stone, MT(ASCP), PhD, DABCC
Center for Advanced Laboratory Medicine, University of California San Diego Health System,
San Diego, CA, United States
J.Y. Yang, PhD
Department of Pathology, University of California San Diego, La Jolla, CA, United States
Preface
This book was born out of an idea that a select compilation of the illustrated experiences of a panel of
expert practitioners of clinical mass spectrometry might be beneficial to those of us who are consider-
ing implementation of the art in our own laboratories perhaps for the first time. I would like to thank
my mentors and colleagues from the Department of Lab Medicine at the University of Washington as
well as those friends that I get to meet at AACC and other venues for their valuable insights that, in part,
helped shape the idea for this book.
Personally, I saw this project as an opportunity to learn. I feel extremely lucky to have Bill Clarke as
my coeditor and mentor in this pursuit. I am grateful for his kind and effective mentorship, encourage-
ments, collaboration, and vast technical and professional insights.
First we listed the topics that we felt might be of interest to most clinical labs and then requested
some of the best known practitioners in the field to tell their stories on those topics. For their belief
in this project, their willingness to contribute, and for their expertise that is so valuable to the clinical
chemistry community. I reserve my utmost gratitude to the authors of this book.
This project has been in the works for nearly 3 years. Patience and goodwill gestures from many
individuals along the way lit its path. Foremost, I would like to thank my wife Rekha and my boys
Shreyas and Shree for their patience and for being my inspiration. I am thankful to Ruthi Breazeale, Sr.
VP and Dr. Ernst Scheafer, co-founder and medical director at Boston Heart Diagnostics for their en-
couragements and accommodation.
What a pleasure it has been to work with the incredibly professional and pleasant Team Elsevier!
Thank you!!
My hope is that this book will add at least a drop to the ever growing number of resources that we as
a community will need to perfect the art of implementing mass spectrometry in the clinical lab.
Hari Nair, PhD, DABCC, FACB
xv
CHAPTER
1
MASS SPECTROMETRY IN
THE CLINICAL LABORATORY:
DETERMINING THE NEED
AND AVOIDING PITFALLS
W. Clarke
Johns Hopkins University School of Medicine, Baltimore, MD, United States
1 CLINICAL MASS SPECTROMETRY
Historically, the complexity of instrumentation and sample preparation has relegated LC-MS based
assays to specialized laboratories with extensive technical expertize. Until recently, applications of MS
in the clinical laboratory were limited to gas chromatography (GC)-MS for toxicology confirmation
testing and testing for inborn errors of metabolism, some GC-MS applications for steroid analysis in
specialty laboratories, and inductively coupled plasma (ICP)-MS for elemental analysis. In most cases,
this testing has been restricted to specialized laboratories within a hospital, or to large reference labora-
tories. However, with the simplification of MS instrumentation and introduction of atmospheric spray
ion sources along with the emergence of routine liquid-chromatography tandem MS (LC-MS/MS), MS
has become a viable option for routine testing in clinical laboratories.
1.1 BASIC MASS SPECTROMETRY CONCEPTS
MS is a powerful analytical technology that can be used to identify unknown organic or inorganic
compounds, determine the structure of complex molecules, or quantitate extremely low concentrations
of known analytes (down to one part in 1012). For MS-based analysis, molecules must be ionized, or
electrically charged, to produce individual ions. Thus, MS analysis requires that the atom or molecule
of interest has the ability to be ionized and be present in the gas phase. MS instruments analyze mol-
ecules by relating the mass of each molecule to the charge; this identifying characteristic is specific
to each molecule and is referred to as the mass-to-charge ratio (m/z). Therefore, if the molecule has a
single charge (z = 1), the m/z ratio will be equal to the molecular mass.
The analytical power of the mass spectrometer lies in its resolution, or the ability to discern one
molecular mass from another. The resolution can be determined by examining the width of an m/z peak
or the separation between adjacent peaks; a narrow peak with little overlap indicates greater resolution.
For two adjacent peaks of masses m and m, the resolving power is defined as m/(m – m). The expres-
1 2 1 1 2
sion (m − m may also be referred to as ∆m. Higher instrument resolution results in increased mass
1 2)
accuracy and the ability to avoid interference from compounds of similar mass that may also be present
Mass Spectrometry for the Clinical Laboratory. http://dx.doi.org/10.1016/B978-0-12-800871-3.00001-8 1
Copyright © 2017 Elsevier Inc. All rights reserved.
2 CHAPTER 1 MASS SPECTROMETRY IN THE CLINICAL LABORATORY
FIGURE 1.1 Schematic Diagram of Mass Spectrometry (MS)
in the sample. Mass accuracy is defined as the mass difference that can be detected by the analyzer
divided by the observed, or true mass.
Although there are numerous instrument configurations available, MS system operation can be
organized into three main segments: (1) generation of ions; (2) separation of ions based on mass and
charge in a mass analyzer; (3) detection of ions and instrument output (Fig. 1.1). Depending upon the
type of ionization used, these steps fully or partially occur under vacuum pressure to drive ion move-
ment forward through the instrument.
1.2 COMMON ION SOURCES FOR CLINICAL MASS SPECTROMETRY
There are a variety of ion sources available for mass spectrometers. Some of these ion sources are “di-
rect ionization sources,” in which analytes are directly ionized from a surface or from a solution. Other
sources, such as atmospheric pressure ionization sources, produce ions from analytes in solution and
these are more commonly used in clinical assays due to their compatibility with liquid chromatography.
Common atmospheric pressure ion sources include:
• electrospray ionization (ESI)
• atmospheric pressure chemical ionization (APCI)
• atmospheric pressure photoionization (APPI)
A summary of the strengths and weaknesses for these sources can be found in Table 1.1.
1.2.1 Electrospray Ionization (ESI)
ESI is perhaps the most commonly used ionization technique in clinical MS. It is a sensitive ionization
technique for analytes that exist as ions in the LC eluent. In ESI, a solvent spray is formed by the ap-
plication of a high voltage potential held between a stainless steel capillary and the instrument orifice,
coupled with an axial flow of a nebulizing gas (typically nitrogen). Solvent droplets from the spray
evaporate in the ion source of the mass spectrometer, releasing ions to the gas phase for analysis in the
mass spectrometer. In some ESI sources, heat is used to increase the efficiency of desolvation. While
ESI is widely used, it is subject to matrix effects, particularly ion suppression, which must be taken into
consideration during method development.
1.2.2 Atmospheric Pressure Chemical Ionization (APCI)
APCI uses heat and a nebulization gas to form an aerosol of the eluent from an LC system. In contrast to
ESI, ions are not formed in solution or liquid phase. Instead, ions are formed in the gas phase using a co-
rona discharge (high voltage applied to a needle in the source) to ionize solvent molecules and analytes in
the aerosol. Ions released to the gas phase are then analyzed by the mass spectrometer. During ionization
1 CLINICAL MASS SPECTROMETRY 3
Table 1.1 Overview of Three Ionization Techniques Used in Clinical Mass Spectrometry (MS)
Ionization Technique Advantages Limitations
ESI • Sensitive ionization technique for polar • May be more sensitive to matrix effects
analytes or ions generated in solution compared to APCI
• Has broad applicability for relevant
analytes in clinical MS
• May yield multiply charged ions,
which allows for analysis of larger
molecules (i.e., >1000 Da)
APCI • Typically less sensitive to matrix • Typically only singly charged ions are
effects than ESI formed, limiting the effective mass range,
• May provide better sensitivity for less • May be unsuitable for thermally labile
polar analytes analytes
• May yield less absolute signal relative to
ESI
APPI • Works well with nonpolar analytes • Demonstrates limited applicability in
• In some cases will ionize analytes that clinical MS to date.
do not ionize by either ESI or APCI.
APCI, Atmospheric pressure chemical ionization; APPI, atmospheric pressure photoionization; ESI, electrospray ionization.
in the APCI source, some thermal degradation may occur, which can lead to a greater degree of fragmen-
tation in electrospray ionization. For analysis using APCI, the analytes of interest should be heat stable
and volatile for best results. APCI is often less susceptible to matrix effects (including ion suppression)
as compared to ESI, and may be considered for a wide range of applications, including measurement of
nonpolar analytes.
1.2.3 Atmospheric Pressure Photoionization (APPI)
APPI is an alternative mechanism to ionize analytes eluting from a chromatography system, although
it is much less frequently used than ESI or APCI. In APPI, the solvent is first vaporized in the presence
of a nebulizing gas (e.g., nitrogen) and then enters the instrument ion source at atmospheric pressure.
Once the aerosol is generated, the mixture of solvent and analyte molecules is exposed to a UV light
source that emits photons with energy level that is sufficient to ionize the target molecules, but not high
enough to ionize unwanted background molecules. Often, an additive to the LC eluent (commonly
toluene) is used to increase ionization efficiency in APPI.
1.3 COMMONLY USED MASS ANALYZERS
When coupled to an LC system, the mass spectrometer functions as powerful multiplex detector for
chromatography. The analyte of interest is ionized in the source of the mass spectrometer by any of a
variety of mechanisms as previously discussed. The ions are then directed to the mass analyzer com-
ponent of the mass spectrometer, where individual ions are selected according to their m/z. Ions pro-
duced from small molecule analytes (<1000 Da) usually possess a single charge; therefore, their m/z is
4 CHAPTER 1 MASS SPECTROMETRY IN THE CLINICAL LABORATORY
equivalent to the mass of the ion. This is commonly designated as [M + H]+ for protonated (positive)
and [M − H]− for deprotonated (negative) ions. A chromatogram can then be generated in which rela-
tive ion abundance of a specific m/z is plotted versus time. Multiple overlapping chromatograms can be
extracted from the same analytical run, as the mass analyzers are able to detect multiple ions indepen-
dently within the time scale of a chromatographic peak.
1.3.1 Quadrupole Mass Analyzers
A quadrupole mass analyzer consists of a set of four conducting rods arranged in parallel, with a space
in the middle; the opposing pairs of rods are electrically connected to each other. This type of mass
analyzer separates ions based on the stability of their flight trajectories through an oscillating electric
field in the quadrupole. The field is generated when a radio frequency (RF) voltage is applied between
one pair of opposing rods within the quadrupole. A DC offset voltage is then applied to the other pair
of opposing rods. Only ions of a certain m/z will have a stable flight path through the quadrupole
in the resulting electric field; all other ions will have unstable trajectories and will not reach the detec-
tor. The RF and direct current voltages can be fixed in a way that the quadrupole acts as a mass filter or
analyte-specific detector for ions of a particular m/z. Alternatively, the analyst can scan for a range of
m/z values by continuously varying the applied voltages.
Single quadrupole mass spectrometers contain a single mass analyzer and can only measure ions
formed in the instrument source; these can be intact molecular ions or fragment ions formed by in-
source fragmentation. Based on this limitation, single quadrupole mass spectrometers do not provide
a large amount of structural information and specificity is limited when compared to tandem quadru-
pole mass spectrometers. A tandem quadrupole mass spectrometer, often called a triple quadrupole,
consists of two quadrupole mass analyzers separated by a collision cell. Precursor ions are selected by
the first quadrupole mass analyzer. The selected precursor ion is then fragmented in the collision cell
by a process known as collision-induced dissociation (CID). CID results from collisions of the analyte
of interest with an inert gas, such as nitrogen or argon. The specific product ions produced by CID are
a function of the bond energies inherent in the molecular structure of the precursor ion, as well as the
collision gas and energy used. Product ion patterns and relative ion abundance can be highly repro-
ducible if the CID conditions are stable and robust. The product ions are analyzed or selected by the
final quadrupole mass analyzer, and then passed to the detector. These pairs of precursor and product
ions are called a mass transitions. When the electric fields and collision energy are held constant,
only analyte ions having a specified mass transition (precursor/product ion pair) are able to reach the
detector, which results the high specificity of tandem quadrupole mass spectrometric methods. This
mode of data acquisition is referred to as selected-reaction monitoring (SRM). When multiple transi-
tions are monitored during a chromatographic run, the data acquisition is called multiple-reaction
monitoring (MRM).
1.3.2 Time-of-Flight Mass Analyzers
Time-of-flight (TOF) mass analyzers separate ions based on their different flight times over a defined
distance or flight path. After generation in the source, ions are accelerated by an electric field into a
flight tube, such that ions of like charge have equal kinetic energy. Because kinetic energy is equal to
1/2 mv2, where m is the mass of the ion and v is the ion velocity; the lower the ion’s mass, the greater
the velocity and shorter its flight time. The travel time from the source of the ion pulse through the
flight tube to the detector can be calibrated to the m/z value based on the relationship described earlier.
1 CLINICAL MASS SPECTROMETRY 5
Unlike a quadrupole mass analyzer, TOF analyzers are not scanning—all ion masses are measured
for each ion pulse. Because of this, TOF mass spectrometers offer high sensitivity and a high duty
cycle. Historically, TOF mass spectrometers have been used for qualitative experiments in which high
resolving power and exact mass measurements are necessary (e.g., metabolite identification or protein
sequencing). However, modern TOF mass spectrometers are capable of accurate and precise quantita-
tive measurements as well.
1.3.3 Ion Trap Mass Analyzers
Ion trap mass analyzers use a combination of electric or magnetic fields to capture or “trap” ions inside
the mass analyzer. There are multiple configurations of ion traps including 3D ion traps (the Paul ion
trap), a linear ion trap (2D trap), and electrostatic trap (Orbitrap), or a magnetic field-based trap (ion
cyclotron resonance). The 3D ion trap basically works on the same principle as a quadrupole mass
analyzer, using static DC current and RF oscillating electric fields, but the hardware is configured dif-
ferently, where the parallel rods are replaced with two hyperbolic metal electrodes (end caps) facing
each other, and a ring electrode placed halfway between the end cap electrodes; ions are trapped in a
circular flight path based on the applied electric field. A linear ion trap uses a set of quadrupole rods
coupled with electrodes on each end to facilitate the ion trapping. This configuration gives the linear
ion trap a dual functionality—it can be used as a quadrupole mass filter or an ion trap. An orbitrap mass
spectrometer consists of an inner spindle-like electrode and an outer barrel-like electrode. The orbitrap
stores ions in a stable flight path (orbit around the inner spindle) by balancing their electrostatic attrac-
tion by their inertia coming from an RF only trap. The frequency of the axial motion around the inner
electrode is related to the m/z of the ion. Last, ion cyclotron resonance (ICR) traps use a strong mag-
netic field to induce a radial orbit of ions, where the frequency of orbit in the magnetic field is a function
m/z for the ion. For both orbitrap and ICR traps, their strength is the ability to trap all ions at once and
detect them on the basis of their detected frequencies—a Fourier transform algorithm is required for
this signal processing. Mass spectrometers that include an ion trap analyzer are most commonly used
for qualitative work (e.g., metabolite identification, protein identification, and screening applications).
Although examples of ion trap mass analyzers for quantitative analysis do exist, their use in quantita-
tive clinical MS to date is limited.
1.3.4 Hybrid Tandem Mass Analyzers
There are now many instrument configurations that combine two or more mass analyzers of different
design, and are therefore called “hybrid instruments.” One such hybrid combines a quadrupole with a
linear ion trap; this instrument is referred to as a Q-Trap and has found a niche in toxicology screening
for unknown agents, and also can be used for quantitative analyses when the linear trap is operated as
a quadrupole. Another type of hybrid combines quadrupole and TOF mass analyzers to form a Q-TOF.
In this instrument, the quadrupole mass analyzer performs precursor ion selection, while a TOF mass
analyzer performs the product ion analysis. The same strategy has been applied using a quadrupole
coupled with the electrostatic orbitrap analyzer. With both Q-TOF and Q-Orbitrap instruments, having
a high-resolution instrument as the second mass analyzer quantitative analyses in addition to their use
for protein sequencing and proteomic or metabolomics screening. High-resolution mass spectrometry
will be covered further in Chapter 12.
The choice of mass spectrometer really depends on its intended use. While tandem quadrupole mass
spectrometers are by far the most common type of mass spectrometer used in the clinical laboratory, the
