This edition first published 2018
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Library of Congress Cataloging‐in‐Publication Data
Names: Mallet, A. I. (Anthony I.), author.
Title: Quantitative biological and clinical mass spectrometry : an introduction / by Anthony I. Mallet.
Description: First edition. | Hoboken, NJ : John Wiley & Sons, 2018. | Includes bibliographical references and index. |
Identifiers: LCCN 2017054863 (print) | LCCN 2018003549 (ebook) | ISBN 9781119281221 (pdf) | ISBN 9781119281214 (epub) | ISBN 9781119281207 (cloth)
Subjects: LCSH: Mass spectrometry. | Biochemical engineering. | Clinical chemistry.
Classification: LCC QP519.9.M3 (ebook) | LCC QP519.9.M3 M3147 2018 (print) | DDC 610.28–dc23
LC record available at https://lccn.loc.gov/2017054863
Cover Design: Wiley
Cover Image: (Background) © NatchaS/Gettyimages; (Image inset) © snvv/Gettyimages
I must express my gratitude to all of those persons and all my colleagues in St John’s Institute of Dermatology and other Departments of St Thomas’ Hospital and Guy’s Hospital for all they have taught me about the mechanisms and pathology of disease. Thank you too for the post doctoral students from whom I have learnt far more than I ever gave. I also wish to thank my mass spectrometry colleagues and the British Mass Spectrometry Society as well as the instrument equipment industry for their help, support and assistance over the past 40 years.
Finally, I must express my gratitude to my wife for the forbearance she has shown to my continued interest in the subject and the ever growing boxes of papers from the scientific literature.
This book is designed to provide information and help to new users of mass spectrometry (MS) working in clinical or biochemical fields who are faced with implementing and designing quantitative mass spectrometric assays for molecules of biological interest. While a working knowledge of basic and physical chemistry and some experience of MS is assumed, there are simple explanations and further sources of information included of the techniques and basic chemistry involved. These will be clearly separately indicated and can be avoided by the majority of the expected readership.
While MS has been used for the quantitative analysis of trace biological molecules since the late 1960s, it has been the rapid development of compact instruments, automation, efficient ionisation methods, modern ion optics, electronics and digital control, and data manipulation methods that has led to a rapid growth of novel applications in the field of clinical and biochemical analysis in recent years. Up until the 1970s MS, developed to assist the oil industry, had principally been used for analysis of small‐molecule gaseous samples. The pharmaceutical industry took up the technique in order to permit the qualitative and quantitative analysis of biologically relevant substances, and in the early 1980s it was the development of methods for handling solutions, liquids, and solids and the ability to begin to examine really large molecules and polymers that precipitated the technique into acceptance in a wide variety of fields. In the early 2000s, a couple of reviews1,2 discussed the relevance of MS to clinical practice. The advent of reliable tandem MS (MS/MS) in the 1990s soon made its appearance in clinical analyses on account of the extra dimension of analyte specificity that it provided, but even in 2012 one author still warned that fully automated analysis based on MS/MS combined with liquid chromatography (LC) would take a decade or more to match the current immunoassays in use in hospital laboratories.3
In 2016, an issue of Clinical Chemistry4 was dedicated to current views on the state of use of MS in clinical settings. From an article by Cooks and others on this subject, it is clear to see that much progress has been made in the past decade. The articles in the latter reference cover a number of specific analytical reports, but also include helpful guidelines and indications of future developments.
MS is inherently a method for sensitive and specific analysis, but its use in clinical areas has been slow to develop, principally on account of the equally efficient and manipulatively simple immunological‐based assay methods. The latter methods are especially good where automation with very large sample numbers and speed of producing results are involved, and the MS instrument industry is now responding to the competition.
A necessary consequence of these developments has been that modern instruments are presented to the operator as a ‘black box’, which makes invisible everything that takes place between the presentation of the sample and the appearance on a computer screen of a result. It can be difficult for an inexperienced operator to recognise that the apparent production of a stream of results may be hiding some serious failure of the basic system. This text is designed to show how the presence of false results can be detected and understood.
MS for use in quantitative biochemistry with small‐molecule drugs, in the pharmaceutical industry, has been the principal driving force for instrumental and method development in recent years. The introduction of biopolymer drugs interacting with the immune system has now propelled the interest in analysis of large biopolymers, and their quantitative analysis is a growing area of research publications.
In the fields of clinical chemistry, as well as in forensic science and sports medicine, a different perspective is found. Two different types of need for a quantitative analysis are present: one is for a precise and validated figure for the concentration in a defined matrix, and the other, while still needing precision, requires a knowledge of whether the concentration exceeds a predetermined permitted or safe level, such as applies in drug misuse or water or food safety considerations.
Included in ‘quantitative analysis’ are those experiments designed to discover the absence or presence of a defined analyte. The quantitative aspect lies in knowing the limits of detection that are available in the designed protocol and the confidence in the precise specificity of the assay.
The ‘parts’ of modern instruments from sample introduction through ionisation, mass analysis and detection and the variety of techniques of MS/MS will be described and compared. Modern MS has available a wide variety of configurations, but the methods optimally suited for quantitative analysis of a variety of compound classes will follow. It is sometimes not fully recognised that, unlike true spectroscopic analytical instruments, the measurements that are made in MS are metric, and these are not of the same nature as those spectroscopic measurements such as ultraviolet (UV), infrared (IR) and nuclear magnetic resonance, which measure the interactions of the atomic structures of the molecules being examined with electromagnetic radiation. Mass spectrometers measure the mass of an ion, by inducing its movement in electrical or magnetic fields. While all instruments will come to the same overall result in the ion mass determined, different mass spectrometers can produce significantly different results in their overall responses from identical samples in the manner in which they handle the ions, especially in regard to tandem mass spectra, and this has often led to the complaint that it is very hard to reproduce published data in a different laboratory. The reasons for this will be discussed.
The first two chapters describe the mass spectrometer instrumentation. Chapter 1 discusses the methods in use to create ions from the analyte, and Chapter 2 the means for determining the mass‐to‐charge ratio, and hence the molecular weight of these ions.
Chapter 3 discusses the interpretation of the mass spectrum and different forms of data output. The influence of stable isotopes on a spectrum is shown, as are methods for extracting elemental compositions. The identification of the true signal from the ionised molecule is explained, as well as the interpretation of fragmentation from electron ionisation sources and MS/MS.
This is followed in Chapter 4 by a short discussion of the optimum methods for sample introduction, principally using chromatography. The emphasis is on the best method that permits good quantitative analysis from the mass spectrometer.
While the emphasis will be on quantitative analysis, the requirement for specificity in an assay method is discussed in Chapter 5 on qualitative analysis. The mass spectrometric methods used for determining molecular structure are precisely those which provide the necessary specificity in a quantitative assay. The scale of the difficulty of the task was well illustrated in 2010 by Kushnir and Rockwood.5 Figure 1 shows a range of concentrations of biologically relevant molecules covering over ten decades in value.
It is now accepted that a validated MS assay is probably the optimum method for cross‐checking the specificity of any immunoassay and provides a ‘gold standard’ procedure for that analyte. Recent developments have questioned the need for a method in which full calibrations are performed with each batch of analyte samples. This is not an efficient method for the analysis of one‐off samples, such as those from clinical situations where rapid results are essential. Novel approaches to quantitative mass spectrometric analyses will be described in Chapter 6. A detailed discussion is given on how to optimise the parameters important for a candidate reference quantitative analysis, including calibration procedures, sensitivity, reproducibility, speed of assay and compliance with regulatory authorities.
Chapter 7 contains illustrations of the aforementioned procedures with examples of a variety of small and medium‐size, primarily endogenous, molecules from the literature, including, acids, lipids, amino acids, vitamins, small peptides and carbohydrates, especially from those in which unexpected difficulties have arisen and how they have been overcome. The need for understanding of the basic chemistry, biochemistry, pharmacology and clinical management involved will be emphasised. Quantitative analyses of large biopolymers have their own specific difficulties; while much work is in progress to achieve satisfactory quantitative results in this field, only outline descriptions of experiments will be discussed.
Advances in addressing the very large numbers of clinical samples that arise on routine screening programmes, such as those involved in inborn errors of metabolism studies, are discussed in Chapter 8. Direct mass‐spectrometric‐based analyses applicable to point‐of‐care testing situations will also be covered. Apart from one‐by‐one assay methods, often without a chromatographic inlet system, mixture analysis and experiments carried out directly from the sample in the open air will be discussed.
A short section with appendices, bibliography, a glossary of terms and an index will conclude the book.