Über die Autorin bzw. den Autor

Professor Pietro Traldi, Research Executive, National Council of Research, CNR, ISTM Corso Stati Uniti 4 I35127 Padova, Italy

Fields of interest include structure and reactivity of gaseous ions; -MS application in environmental and biomedical fields; Development of new analytical methods; Instrumental developments. Pietro Traldi is author of over 500 publications on international journals; he is member of the advisory boards of JMS, MSR, RCM and EMS. He is the promoter and the chairman of the Informal Meeting of Mass Spectrometry.

Professor Irma Lavagnini, Associate Professor in Analytical Chemistry, Faculty of Pharmacy. Scientific research focus is Chemical data handling

Professor Franco Magno, Padua University, Italy, Full Professor of Analytical Chemistry since 1980. Main research areas in electroanalytical techniques; Development and application of the digital simulation techniques to rationalize voltammetric responses; Development and optimization of analytical procedures to determine species of relevant enviromental and/or industrial interest.

Dr Roberta Seraglia, National Council of Research, Padova, Italy, Researcher of CNR, Institute of Molecular Sciences and Technologies, Padova. 130 papers published in International Journals and 160 communications at International and National congresses.

Von der hinteren Coverseite

What is usually required of an analytical technique? Specificity, selectivity and sensitivity -or, more precisely, a detection limit as low as possible. These criteria are fulfilled by mass spectrometry is an essential tool for qualitative and quantitative investigations in many applications, in particular in environmental science and biomedicine,with positive benefits to mankind.

The problem that has arisen from this large expansion is that mass spectrometry is mainly considered as a "magic box" technique, in which on one side a sample is introduced and on the other side the analytical data comes out. Thus, the basic aspects of the technique are often not well known. To overcome this, the authors present within this text some basic information for scientists and technicians working in the field of quantitative organic mass spectrometry, in particular for those who, for the first time, face all the problems arising from the development and use of a quantitative procedure.

This informative text presents basic information on:

• What instrumental approaches are available
• How to design a quantitative analysis
• How to improve specificity
• Some thoughts on calibration and data analysis

This book is mainly aimed at analytical chemists working in academic, environmental, pharmaceutical, biochemical, forensic, clinical and industrial laboratories, but is also appropriate for researchers new to the technique, e.g. biochemists, biologist, physicians and workers in pharmaceutical, food and the health sciences.

Aus dem Klappentext

What is usually required of an analytical technique? Specificity, selectivity and sensitivity -or, more precisely, a detection limit as low as possible. These criteria are fulfilled by mass spectrometry is an essential tool for qualitative and quantitative investigations in many applications, in particular in environmental science and biomedicine,with positive benefits to mankind.

The problem that has arisen from this large expansion is that mass spectrometry is mainly considered as a "magic box" technique, in which on one side a sample is introduced and on the other side the analytical data comes out. Thus, the basic aspects of the technique are often not well known. To overcome this, the authors present within this text some basic information for scientists and technicians working in the field of quantitative organic mass spectrometry, in particular for those who, for the first time, face all the problems arising from the development and use of a quantitative procedure.

This informative text presents basic information on:

• What instrumental approaches are available
• How to design a quantitative analysis
• How to improve specificity
• Some thoughts on calibration and data analysis

This book is mainly aimed at analytical chemists working in academic, environmental, pharmaceutical, biochemical, forensic, clinical and industrial laboratories, but is also appropriate for researchers new to the technique, e.g. biochemists, biologist, physicians and workers in pharmaceutical, food and the health sciences.

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Quantitative Applications of Mass Spectrometry

John Wiley & Sons

Chapter One

The fantastic development of mass spectrometry (MS) in the last 30 years has led this technique to be applied practically in all analytical fields. We focus our attention on the application in the organic, biological and medical fields which nowadays represent the environment in which MS finds the widest application. This chapter is devoted to a short description of the different instrumental approaches currently in use and commercially available.

MS is based on the production of ions from the analyte, their analysis with respect to their mass to charge ratio (m/z) values and their detection. Consequently, at instrumental level three components are essential to perform mass spectrometric experiment: (i) ion source; (ii) mass analyser; and (iii) detector (Figure 1.1). Of course, the performances of these three components reflect on the quality of both quantitative and qualitative data. It must be emphasized that generally these three components are spatially separated (Figure 1.1a) and only in two cases [Paul ion trap and Fourier transform mass spectrometer without external source(s)] can they occupy the same physical space and, consequently, the ionization and mass analysis must be separated in time (Figure 1.1b).

1.1 ION SOURCES

The ion production is the phenomenon which highly affects the quality of the mass spectrometric data obtained. The choice of the ionization method to be employed is addressed by the physico-chemical properties of the analyte(s) of interest (volatility, molecular weight, thermolability, complexity of the matrix in which the analyte is contained).

Actually the ion sources usually employed can be subdivided into two main classes: those requiring sample in the gas phase prior to ionization; and those able to manage low volatility and high molecular weight samples.

The first class includes electron ionization (EI) and chemical ionization (CI) sources which represent those worldwide most diffused, due to their extensive use in GC/MS systems. The other ones can be further divided into those operating with sample solutions [electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI)] and those based on the contemporary sample desorption and ionization from a solid substrate [matrix - assisted laser desorption/ionization (MALDI) and LDI].

1.1.1 Electron Ionization

EI is based on the interaction of an energetic (70 eV) electron beam with the sample vapour (at a pressure in the range [10.sup.-7]-[10.sup.-5] Torr) (Figure 1.2). This interaction leads to the production of a series of ions related to the chemical properties of the compound(s) under study. The theoretical treatment of EI is beyond the scope of the present book and it is possible to find it in many publications. For the present discussion it is enough to consider that EI generally leads to a molecular ion [M.sup.+], originating by the loss of an electron from the neutral molecule:

M + [e.sup.-] -> [M.sup.+] + 2[e.sup.-]

and to a series of fragments, generally highly diagnostic from the structural point of view:

Some of them originate from simple bond cleavages, while some others are produced through rearrangement processes. What must be emphasized is that EI leads towell reproduciblemass spectra. In other words, by different EI sources spectra practically superimposable are obtained and this is the reason for which the only spectrum libraries available are those based on EI data.

The main efforts done in the last decade in the EI field are due to the development of ion sources with the highest possible ion yield. To reach this aim, on the one hand an optimization of ion source geometry has been performed (this has been achieved by the development of suitable ion optics to increase either the ion production or the ion extraction), on the other, to make inert the ion source walls (originally in stainless steel) so as to avoid the sample loss due to its pyrolysis on the hot metallic surface.

The quantitative data obtained by EI can be strongly affected mainly by two parameters: the first related to sample loss (due to problems related to sample injection lines and to 'open' source configuration as well as to thermal decompositions occurring in injection lines and/or source), while the second can be related to a decreased efficiency of ion extraction (nonoptimized extraction field, field modification due to the presence of polluted surfaces). These two aspects reflect not only on the limit of detection (LOD) of the system but also on the linearity of the quantitative response.

The ion most diagnostic from the qualitative point of view is usually considered the molecular one ([M.sup.+]). However, wide classes of compounds, easily vaporized, do not lead to the production of [M.sup.+]. This is due to the energetics of EI induced decomposition processes. In other words if a decomposition process is energetically favoured (with a particularly low critical energy) it takes place immediately, due to the internal energy content of [M.sup.+]. To overcome this problem in the 1960s a new ionization method was developed, based on gas-phase chemical reactions.

1.1.2 Chemical Ionization

To obtain a lower energy deposition in the molecule of interest, reflecting in the privileged formation of charged molecular species, in the 1960s CI methods were proposed. They are based on the production in the gas phase of acidic or basic species, which further react with a neutral molecule of analyte leading to [[M + H].sup.+] or [[M - H].sup.-] ions, respectively. Generally, protonation reactions of the analyte are those more widely employed; the occurrence of such reactions is related to the proton affinity (PA) of M and the reactant gas, and the internal energy of the obtained species are related to the difference between these proton affinities. Thus, as an example, considering an experiment performed on an organic molecule with PA value of 180 kcal/mol (P[A.sub.M]), it can be protonated by reaction with [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII = 127 kcal/mol), [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII = 165 kcal/mol), but not with [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII = 205 kcal/mol). This example shows an important point about CI: it can be effectively employed to select species of interest in complex matrices. In other words, by a suitable selection of a reacting ion [[AH].sup.+] one could produce [[MH].sup.+] species of molecules with PA higher than that of A. Furthermore the extension of fragmentation can be modified in terms of the difference of [P[A.sub.M] - P[A.sub.A]].

From the operative point of view CI is simply obtained by introducing the neutral reactant species inside an EI ion source in a 'close' configuration, by which quite high reactant pressure can be obtained (Figure 1.3). If the operative conditions are properly set the formation of abundant [[AH].sup.+] species (or, in the case of negative ions [B.sup.-]) is observed in high yield. Of course, attention must be paid in particular in the case of quantitative analysis to reproduce carefully these experimental conditions, because they reflect substantially on the LOD values.

CI, as well as EI, requires the presence of samples in vapour phase and...