Quantitative Biological and Clinical Mass Spectrometry E-Book

Anthony I. Mallet

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 290

Veröffentlichungsjahr: 2018

Beschreibung

A basic overview of mass spectrometry relevant to life and health science applications, illustrated throughout with relevant case studies

This introductory text provides information and assistance 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 a variety of classes of molecules of biological interest. It presents a detailed discussion on how to optimize measurement parameters for a candidate reference quantitative analysis, including calibration procedures, sensitivity, reproducibility, speed of assay and compliance with regulatory authorities. 

Quantitative Biological and Clinical Mass Spectrometry uses examples where development has not been immediately successful but where unforeseen problems have arisen and describes the strategies used to solve these. Advances in addressing the very large numbers of clinical samples that arise on routine screening programs such as those involved in inborn errors of metabolism studies are discussed. Direct mass spectrometric based analyses applicable to point of care testing (POCT) situations are also covered. The book concludes with a short section on possible novel developments, bibliography, references, and a glossary of terms.

• Shows how the presence of false results can be detected and understood
• Describes the ‘parts’ of modern instruments from sample introduction through ionization, mass analysis and detection, and the variety of techniques of tandem mass spectrometry
• Discusses the requirement for specificity in an assay method
• Fully illustrated throughout
• Highly relevant to all key areas of mass spectrometric analysis

Quantitative Biological and Clinical Mass Spectrometry appeals to those newly exposed to the use of combined chromatography and mass spectrometry for analysis of biological material and to scientists experienced in automated clinical analysis using immunoassays or who are new to mass spectrometry.

Leseprobe

Table of Contents

Cover

Introduction

References

1 The Instrument: Ion Creation

1.1 Introduction

1.2 Sample handling

1.3 Vacuum ion sources

1.4 Atmospheric pressure ion sources

1.5 Ambient ionisation methods

References

2 The Instrument

2.1 The analyser

2.2 Tandem mass spectrometry

2.3 The detector

2.4 Control and data handling

2.5 Ambient ionisation

2.6 Summary

References

3 The Mass Spectrum

3.1 Spectral output

3.2 Electron ionisation/chemical ionisation spectra

3.3 Stable isotopes and accurate m/z determinations

3.4 Chemical ionisation

3.5 Atmospheric‐pressure spray ionisation

3.6 Tandem mass spectra, MS/MS

3.7 Manipulating chromatographic data output

3.8 Fragmentation of even‐ and odd‐electron ions

3.9 Spectra of peptides proteins and other biopolymers

3.10 Summary

References

4 Sample Handling Prior to Ionisation

4.1 Gas chromatography

4.2 Liquid chromatography: HPLC/UHPLC

4.3 Alternative sample purification methods

4.4 Theory of chromatography relevant to clinical MS ion sources

4.5 Avoiding chromatography: flow injection analysis

4.6 Summary

References

5 Establishing Optimum Specificity

5.1 Structure from the molecular ion or its derivative

5.2 Structure from fragmentation

5.3 Spectra of peptides and proteins

5.4 Example of the deduction of the identity of an unknown

5.5 Potential problems with MS/MS for quantitative analysis

5.6 Conclusions

References

6 Quantitative Analysis with Mass Spectrometry

6.1 Introduction

6.2 Calibration with internal standards

6.3 Creation of a calibration curve

6.4 Assay validation

6.5 Matrix interference

6.6 Immediate calibrations

6.7 Selected or multiple ion recording

6.8 Summary

References

7 Examples of Quantitative Analysis

7.1 Vitamin D metabolite analysis

7.2 Testosterone/epitestosterone

7.3 Oxygenated neural sterols

7.4 Cholic acids

7.5 Phospholipids

7.6 8‐iso‐Prostaglandin F2α

7.7 Metanephrine and normetanephrine

7.8 Isotopic internal calibration assay for clozapine and norclozapine

7.9 Glycolipids and carbohydrates

7.10 Matrix‐assisted laser desorption ionisation analysis of simple carbohydrates

7.11 LC–MS/MS ceramides in Fabry disease

7.12 N‐Tetrasaccharides from protein glycosylation defects

7.13 Peptides

7.14 Hepcidin

7.15 Thyroglobulin

7.16 Quantitative proteomics

7.17 Summary

References

8 Rapid Clinical Analysis

8.1 Flow injection analysis

8.2 Dried blood spots and neonate inborn errors of metabolism analysis

8.3 Haemoglobin analyses

8.4 Application of ambient ionisation methods

8.5 Conclusions

References

A Simple Mass Spectrometry Fragmentation Mechanisms

Odd‐electron molecular ions

Even‐electron molecular ions

B Some Simple Derivatisation Methods

C Acronyms and Glossary of Common Terms

Acronyms commonly used in the literature

Glossary of terms commonly used in the literature

D Simple Statistics

E Helpful Web Links

Bibliography

Index

End User License Agreement

List of Tables

Chapter 01

Table 1.1 ESI compatible solvents.

Table 1.2 Volatile buffer solutions.

Table 1.3 Ion sources compared. An outline comparison of the specifications of different analysers and some tandem mass spectrometry analyser combinations.

Chapter 02

Table 2.1 Comparison of analysers.

Chapter 03

Table 3.1 Stable isotope composition of common biological elements.

Chapter 04

Table 4.1 Solvent flows for different HPLC column dimensions.

Chapter 06

Table 6.1 Scoring specificity LC–MS assay summary of EU agency suggestions.

Chapter 08

Table 8.1 Ambient ionisation methods.

List of Illustrations

Introduction

Figure 1 The concentrations (medians and ranges) of small endogenous molecules in adult serum.

5

Chapter 01

Figure 1.1 Schematic of a modern chromatography/MS instrument.

Figure 1.2 An EI source; the sample from a GC column eluent is introduced into the plane of the diagram to intersect with the electron beam. The whole source is inside a high vacuum.

Figure 1.3 Production of cations in CI.

Figure 1.4 M

−*

is the activated molecular anion that then dissociates rapidly into two fragments.

Figure 1.5 A MALDI ion source. A laser beam with a wavelength that will be absorbed by the matrix impinges on a co‐crystallised matrix and analyte. Cations, anions and radical species can be formed.

Figure 1.6 (a) ESI nano needle spray. (b) Electrospray source. The initial spray forms a stable conical shape (Taylor cone) and from this successive evaporation steps eventually lead to solvent‐free molecular ions.

Figure 1.7 An ESI ion source. The spray is orthogonal to the entrance cone of the analyser.

Figure 1.8 Nano flow ESI can be achieved from fine needles with the application of a high voltage to the tip.

Figure 1.9 APCI schematic.

Figure 1.10 An APCI ion source. The sample spray is heated to evaporate the solvents and is ionised by the plasma from the high‐voltage needle.

Figure 1.11 Atmospheric pressure photoionisation (APPI) source schematic.

Chapter 02

Figure 2.1 The four parallel rods of a quadrupole analyser.

Figure 2.2 The Mathieu equation defines the

m

/

z

of the ions which can be transmitted through a quadrupolar field. The areas defined by the triangles are calculated for three

m

/

z

values. The peaks of these will lie along the scan line.

Figure 2.3 A cut‐away view of a Paul ion trap.

Figure 2.4 The schematic of an ion trap analyser. The ion path through the instrument, is from left to right

Figure 2.5 Axial LIT.

2

Figure 2.6 Orbitrap. In order to achieve the efficient injection of a packet of ions into the Orbitrap they are corralled in the C‐trap, which is so shaped that on being sent out they will be focused in a tight group to pass into the trap as a small cloud of ions.

Figure 2.7 Schematic of a TOF analyser.

Figure 2.8 The four modes of MS/MS scans.

Figure 2.9 Triple quad. Showing the ESI source and in the main part the two analysing quadrupoles with a collision chamber between. This latter is not an RF‐only quadrupole but a series of ring electrodes.

Figure 2.10 The arrangement of a Q‐TOF mass spectrometer. The collision cell can also be operated as an ion mobility device.

Figure 2.11 Sciex 6500 series system.

Figure 2.12 Q‐Orbitrap Exactive schematic. The initial set of ions is stored in an LIT (HCD Cell), and then transferred to the Orbitrap. In the former, CID can be performed.

7

Figure 2.13 Electron multiplier with discrete electrodes.

Figure 2.14 Horn style of EM.

Figure 2.15 Channeltron multiplier.

Figure 2.16 The time of rise and fall of the ion intensity signal is recorded and the time of detection of the centroid

X

of the peak is saved.

Figure 2.17 On the left is a peak which is asymmetrical perhaps due to contamination in the instrument. On the right is a peak which is clearly not of a homogeneous signal.

Figure 2.18 A calibration process, over a limited mass range, for a quadrupole analyser using a mixture of sodium and rubidium ions.

Figure 2.19 A common definition of the resolving power is the full width at half‐maximum (FWHM) height.

Chapter 03

Figure 3.1 EI ‘stick’ spectrum of toluene.

Figure 3.2 ESI + ve 3‐chlorotyrosine stick and full analogue spectrum.

Figure 3.3 Theoretical spectrum showing that the expected 100% abundant peak of a mycolic acid sample, C

118

H

230

O

5

, is not the

12

C molecular ion but the ion with one

13

C substituent.

Figure 3.4 The EI spectrum of a fatty acid methyl ester.

Figure 3.5 Printout from a request to calculate an elemental formula from an

m

/

z

determination.

Figure 3.6 The isobutane CI spectrum of glycerol, molecular weight 92.

Figure 3.7 NICI spectrum of derivatised estriol.

2

Figure 3.8 The ESI spectrum of a peptide molecule C

43

H

74

N

8

O

10

accurate molecular weight 862.5527, when protonated leads to a rounded‐up integral

m

/

z

of 864.

Figure 3.9 ESI negative ion of flurbiprofen C

15

H

18

FO

2

, accurate molecular weight 244.09.

Figure 3.10 An example of a radical cation in ESI.

Figure 3.11 Comparison of ESI signals for Bradykinin at 1000 and 5000 resolving powers.

Figure 3.12 (a) ESI spectrum of reserpine. (b) Spectrum taken with elevated source or cone voltage and is called in‐source fragmentation. (c) True MS/MS product ion spectrum using two analysers; note the signal intensity magnification after

m

/

z

200.

Figure 3.13 Traces C and B are TIC and base peak intensity extracted values respectively, while trace A clearly shows the expected analyte signal at

m

/

z

341.

Figure 3.14 Peptide p1025.

Figure 3.15 ESI spectrum of equine myoglobin. On the right is shown the family of multi‐protonated ions and on the left is the deconvoluted spectrum.

Chapter 04

Figure 4.1 A GC column mounted in its oven.

Figure 4.2 The relationship between column diameter, solvent flow and ESI sensitivity improvement for a number of small drug molecules. The base sensitivity is for a 2.1 mm column and solvent flow ~500 µl/min.

2

Figure 4.3 SPE cartridges for purification of analyte solutions.

Figure 4.4 The procedure for separating analyte from excess protein.

Figure 4.5 Analyte trap. The use of two linked six‐way valves to trap an analyte, wash it and then elute it into the mass spectrometer.

Figure 4.6 Van Deemter plot. The plate height is a theoretical concept such that the lower its value the more efficient the separation will be.

Figure 4.7 A hypothetical chromatogram to illustrate the common terms used in discussing the theory of separation.

Figure 4.8 Nano‐flow 2 to 3 micron ESI with chip‐mounted emitters.

Chapter 05

Figure 5.1 Showing the emergence of the true analyte signal by increasing the resolving power.

1

Figure 5.2 The three peptide bonds that can break in an MS/MS experiment with three fragments keeping the carboxylic acid end and three the amino terminus.

Figure 5.3 The ESI MS/MS spectrum of a doubly charged toxic peptide ion at

m

/

z

1156.22. The y ion series are shown in blue and the b series in red. The two complementary series can be read from the spectrum. Note, too, that the doubly charged parent ion has product ions with a single charge at higher

m

/

z

values.

Figure 5.4 ESI spectrum of the fake drug.

2

Figure 5.5 MS/MS spectrum of the

m

/

z

279 ion.

2

Figure 5.6 Isotope pattern of the protonated molecule compared for C

12

H

12

N

4

O

3

F (top), C

12

H

15

N

4

O

2

S (middle) and the counterfeit Halfan sample (bottom). Insets show the relative isotope abundance for [M + H]

+

, [M + H + 1]

+

, and [M + H + 2]

+

.

2

Figure 5.7 Structure of sulphamethazine.

Figure 5.8 Structure of difloxacin showing two possible positions for protonation.

Figure 5.9 Difloxacin MS/MS with ion mobility analysis showing the presence of two ionised species.

3

Figure 5.10 The three alternative pathways for MS/MS fragmentation of the two single and one double charged species.

3

Chapter 06

Figure 6.1 A hypothetical internal standard calibration curve (

).

Figure 6.2 The molecular region of cysteine, C

3

H

7

NO

2

S, which has a mono‐isotopic molecular weight of 121.019.

Figure 6.3 Illustration of the residuals on

y

‐axis.

Figure 6.4 (a)Plot is truly random and indicates a true linear relationship. (b) Plot is bowed and will indicate no linear regression exists.

Figure 6.5 Illustration of the differences between accuracy and precision.

Figure 6.6 (a) An instrumental arrangement to detect matrix effects. (b) Panel (b) shows the authentic mass spectrum of the analyte at points 1 and 3 in panel (a). Panel (c) shows the recorded spectrum at point 2 in panel (a). Panel (d) shows the suppression of the TIC signal.

6

Chapter 07

Figure 7.1 The metabolism of vitamin D3.

Figure 7.2 Vitamins D3 and D2.

Figure 7.3 A possible complication of this derivatisation is that the new ring can be formed in two epimeric configurations: one in the plane of ring A and the other across it. Luckily, these two isomers co‐elute on RP C‐18 HPLC columns but will separate on diol‐bonded columns. PTAD: 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione.

Figure 7.4 The multiplexed PTAD derivative point of MS/MS fission and the position of alkyl and aryl substituents for multiplexing.

Figure 7.5 The use of multiple versions of derivative being analysed in a time of less than 1 min. A single injection of five samples analysed for 24‐hydroxyvitamin‐D

3

and ‐D

2

and the corresponding internal standards (a total of 20 peaks).

10

Figure 7.6 Testosterone trimethylsilyl ether derivative.

Figure 7.7 Derivative testosterone.

12

Figure 7.8 Girard‐P derivative. The Girard reagent can be a chloride or bromide and will react with keto groups to form the hydrazone. The final derivative is already a cation improving the ESI performance.

Figure 7.9 Set of cholic acids.

Figure 7.10 A phosphatidylcholine molecule. R

1

and R

2

are the long‐chain fatty acid residues usually expressed as C

n

:

u

, with

n

carbon atoms and

u

unsaturations.

Figure 7.11 Structure of 8‐

iso

‐prostaglandin F2α.

Figure 7.12 Separating the prostaglandin F2α analyte together with the internal standard from closely related prostaglandins.

Figure 7.13 The two plasma metanephrines: metanephrine (left) and normetanephrine (right).

Figure 7.14 MS/MS scans for the two metanephrines. The parent ion scans were optimised for the [M − H

2

O]

+

ion intensity, and these were chosen for the MS/MS assay.

27

Figure 7.15 Structures of clozapine (left) and norclozapine (right).

Figure 7.16 (a) The five MRM channels using the adjusted collision energies.

29

(b) The resulting calibration plot.

29

Figure 7.17 A ceramide marker for Fabry disease.

Figure 7.18 An

N

‐glycan. The green circles are mannose residues, the blue squares are

N

‐acetylglucosamine, the yellow circle is a galactose and the red lozenge is a fucose.

Figure 7.19 Peptide structure with the cys–cys bridges.

Figure 7.20 ESI spectrum. Molecular ions with two, three, four and five protons.

34

Figure 7.21 LIT MS/MS of the [M + 4H]

4+

ion which produced product ions 10 times more intense than any other precursor, showing the (y

23

)

4+

and (y

24

)

4+

ions together with the other qualifier ions used in the assay.

34

Figure 7.22 Ideal, in silico, filtering without and with immunoaffinity, to isolate a single target peptide. This was distinguished from the other seven which had same

m

/

z

and similar MS/MS, by HPLC. If an immunological enrichment can be performed, the single product peptide will be found after step 3.

35

Chapter 08

Figure 8.1 An HPLC injection valve can be used to permit the maintenance of solvent flow to the ion source and to insert a volume of analyte sample into that flow from a sample loop: (a) loading; (b) injecting.

Figure 8.2 Neonate dried blood spot (DBS) Guthrie card.

Figure 8.3 The fragmentation of an acylcarnitine showing the common product ion at

m

/

z

85, which is ideal for an MRM precursor scanning mode of operation.

Figure 8.4 The patterns of acylcarnitines detected using an MS/MS procedure with a normal DBS sample compared with one from an MCAD patient. The internal standards are indicated with an asterisk.

Figure 8.5 The MS/MS product ion spectra from which the most appropriate signals for MRM analysis were chosen.

Figure 8.6 The MS/MS product ion scans of Hb normal and Hb sickle. The two panels show the product ion scans for the [M + 2H]

2+

doubly charged parent ions

m

/

z

476.8 and 461.8 for (A) normal and (B) sickle Hb and the corresponding y

4

sequence singly charged product ions at

m

/

z

502.3 and 472.5.

9

Figure 8.7 The MRM traces for detection of the peptides for control, sickle cell trait and sickle cell disease.

9

Figure 8.8 The microsampling system directly linked to an on‐chip ESI emitter.

Figure 8.9 (a) Spectrum of Hb B‐chain variant. (b–d) Isotopic distributions of CID products of the 15

+

precursor b

21

, b

22

and b

23

fragments.

10

Figure 8.10 DESI from a surface.

13

Figure 8.11 EESI: two sprays arranged to intersect immediately in front of the analyser acceptance aperture.

Figure 8.12 The paper sample presented to the analyser entrance cone.

Figure 8.13 A DART ionisation source analysing liquids on glass rods.

Figure 8.14 Formation of protonating species in a DART device.

Figure 8.15 Using an autosampler to process an array of samples for analysis by DART.

24

Figure 8.16 MALIDI‐TOF scans of five different pathogens.

Figure 8.17 A three‐dimensional scatter plot from the first three principal components of six different

Pseudomonas

species.

Figure 8.18 A commercially available REIMS unit that can be operated inside a clinical operating theatre. The cloud of vapour containing lipids from the iKnife is sampled by a gentle vacuum created by a Venturi effect immediately in front of the analyser ion aperture.

Appendix D

Figure D.1 Flow chart used to choose an appropriate statistical test.

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Quantitative Biological and Clinical Mass Spectrometry

An Introduction

ANTHONY I. MALLET

This edition first published 2018© 2018 John Wiley & Sons Ltd

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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–dc23LC record available at https://lccn.loc.gov/2017054863

Cover Design: WileyCover Image: (Background) © NatchaS/Gettyimages; (Image inset) © snvv/Gettyimages

Acknowledgements

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.

Tony Mallet

Introduction

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.5Figure 1 shows a range of concentrations of biologically relevant molecules covering over ten decades in value.

Figure 1 The concentrations (medians and ranges) of small endogenous molecules in adult serum.5

Source: Reproduced with permission of John Wiley & Sons.

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.

References

1 Vogeser, M. and Seger, C. (2008) A decade of HPLC–MS/MS in the routine clinical laboratory – goals for further developments.

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2 Mechtler, T. P., Stary, S., Metz, T. F.,

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(2012) Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria.

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3 Van den Ouweland, J. M. and Kema, I. P. (2012) The role of liquid chromatography–tandem mass spectrometry in the clinical laboratory.

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4 Annesley, T. M., Cooks, R. G., Herold, D. A. and Hoofnagle, A. N. (2016) Clinical mass spectrometry – achieving prominence in laboratory medicine.

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5 Kushnir, M. M. and Rockwood, A. L. (2010) Liquid chromatography–tandem mass spectrometry applications in endocrinology.

Mass Spectrom Rev

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: 480–502. doi: 10.1002/mas.20264

1The Instrument: Ion Creation

1.1 Introduction

The modern mass spectrometer is made up of a number of distinct units. Figure 1.1 shows the disposition of these.

Figure 1.1 Schematic of a modern chromatography/MS instrument.

Modern instruments are more closely integrated in the sample introduction and mass analysis sectors. The whole system should, ideally, be controlled by a single data system.

A sample introduction device.