Mass Spectrometry and Nutrition Research

By Laurent B. Fay, Martin Kussmann

The Royal Society of Chemistry

Copyright © 2010 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-036-5

Contents

Abbreviations, xv,
About the Editors, xviii,
SECTION 1: MASS SPECTROMETRY TECHNOLOGIES,
Chapter 1 Mass Spectrometry Technologies Laurent B. Fay and Martin Kussmann, 3,
SECTION 2: MASS SPECTROMETRY ANALYSIS OF FOOD INGREDIENTS,
Chapter 2 Mass Spectrometry for Food Analysis: The Example of Fat Soluble Vitamins A and K Gregory G. Dolnikowski, 51,
Chapter 3 Mass Spectrometry for the Analysis of Milk Oligosaccharides Daniel Kolarich and Nicolle H. Packer, 59,
Chapter 4 Mass Spectrometry in Protein, Peptide and Amino Acid Analysis Claudio Corradini, Lisa Elviri and Antonella Cavazza, 78,
Chapter 5 Lipidomics and Metabolomics of Dietary Lipid Peroxidation Arnis Kuksis, 102,
Chapter 6 Mass Spectrometry in Phytonutrient Research Jean-Luc Wolfender, Aude Violette and Laurent B. Fay, 163,
SECTION 3: ADDRESSING THE HEALTH ASPECTS OF NUTRITION,
Chapter 7 Addressing the Health Beneficial Aspects of Nutrition — The Example of the Obesity Epidemic Maria Lankinen and Matej Oresic, 237,
Chapter 8 Mass Spectrometry, Diet and Cardiovascular Disease: What will They Mean for Food? J. Bruce German, 244,
Chapter 9 Nutrition and Immunity Martin Kussmann, 268,
Chapter 10 Mass spectrometry, Nutrition and Protein Turnover Michael Affolter, 310,
SECTION 4: CONCLUSION,
Chapter 11 Conclusion Laurent B. Fay and Martin Kussmann, 329,
Subject Index, 332,

CHAPTER 1

Mass Spectrometry Technologies

LAURENT B. FAY AND MARTIN KUSSMANN

1.1 Introduction

Mass spectrometers are molecular balances. They can determine the size, quantity and structure of inorganic and organic compounds. Traditionally, these measures had been limited to volatile organic compounds, but for 20 years mass spectrometers have generated such information also on large, fragile and non-volatile molecules such as vitamins, peptides, proteins, oligo- and poly-saccharides, and even DNA and RNA.

Mass spectrometry (MS) has become an essential analytical tool in modern life sciences, not only thanks to its sensitivity but also to the large amount of information delivered by this technique from a structural and a quantitative viewpoint. Typically, mass spectrometers enable structure elucidation of organic molecules via the determination of molecular weight and the study of fragmentation patterns. Moreover, and increasingly importantly, such instruments can quantify these organic molecules. Additionally, in scientific areas other than the life sciences (geochemistry, ecology, food chemistry, forensic and sport science), mass spectrometry is widely deployed to precisely determine stable isotope ratios of exogenous or endogenous molecules. 13C labelled compounds are mainly used in mass spectrometry as internal standards or to generate metabolite information. Whereas hydrogen/deuterium exchange experiments are used to distinguish between isomeric structures of analytes.

In 1910, J. J. Thomson was the first to build a so-called “parabola spectrograph” meant for the determination of mass-to-charge (m/z) ratios of ions. Following this pioneering work, A. J. Dempster and F. W. Aston developed the first mass spectrometric instrument. Dempster constructed a magnetic analyzer that focused ions into an electrical collector, while Aston utilized both electrostatic and magnetic fields to focus ions onto a photographic plate. From the late 1930s to the early 1950s, A. Nier in collaboration with J. H. E. Mattauch, R. F. K. Herzog and K. T. Bainbridge (amongst others) incorporated many developments in vacuum technologies and electronics for power supplies and ion detection. Their work significantly improved magnetic focusing instruments leading to better performance, convenience and lower costs. Double-focusing machines, attaining greater precision by adding an electrostatic analyzer, were also greatly refined. These instruments were built for the purpose of accurately determining the exact atomic weights of the elements and their isotopes; they made use of Faraday cups to convert particle impacts into an electric current for signal recording. Tremendous progress has been made since this pioneering work in, for example, ion generation, ion transmission, ion detection, signal amplification and, last but not least, computing technologies to control the instrument and record the data.

Since the 1980s, mass spectrometry has become one of the most popular analytical platforms for the identification and/or quantification of organic molecules in complex samples such as body fluids, tissues and food matrices. During less than two decades, we have witnessed the transformation of mass spectrometers from multi-purpose research grade instruments operated only by instrumental experts into user-friendly computer-embedded solutions dedicated to specific measurements such as nutrient/metabolite and peptide/protein identification and quantification. Even the detailed analysis of genetic and genomic material is now being addressed by mass spectrometry. A Google® search with the term “mass spectrometry” results in more than six million entries. In 2007, the mass spectrometry market was estimated at $2 billion with an expected 8% annual growth rate through 2010 (www.allbusiness.com/ instrument-business-outlook/1179913-1.html).

The performance of mass spectrometers in combination with ionization techniques can be defined by several intrinsic parameters, i.e. mass resolving power (or resolution), mass accuracy, sensitivity and linear dynamic range (Figure 1.1).

The mass resolving power or resolution is defined as the ratio m/Δm, with the mass (m) at the apex of the mass signal and Δm the width at x% height (typically 50%) of this mass signal, designated by the full width at half height maximum (FWHM). The mass accuracy is described by the ratio between the mass error (difference between measured and real mass) and the theoretical mass, often represented as parts per million (ppm), e.g. a mass accuracy of 100 ppm corresponds to a theoretical mass of 1000 with a measured mass at 999.9. The sensitivity is described by the ratio between the intensity level of the mass signal and the intensity level of the noise. The linear dynamic range is described as the range of linearity of the ion signal measured as a function of the analyte concentration.

The enormously broad scope of nutritional research (e.g. organic and inorganic nature of the analytes, their volatility or thermal instability, the wide polarity range from water soluble compounds to lipophilic molecules), and the need to measure isotopic abundance to investigate the metabolic fate of nutrients require the deployment of virtually all the mass spectrometric instrumentations available today.

There are many books describing in great detail various mass spectrometric technologies either from a purely instrumental perspective or from a more applicative viewpoint. Below we briefly describe each instrumentation starting with the ionization sources, and then describe the various mass analyzers and ion detection devices commonly available on the market. The hyphenation with different chromatographic systems is covered at the end of the chapter.

1.2 Ionization Sources

Any species — be it an organic molecule or an inorganic element — to be analyzed in a mass spectrometer needs to be ionized unless already present in an ionic form. Ionisation of an analyte (M) takes place by removing or adding an electron to yield an M+• or M-•, respectively. Both these species have the same mass as the original molecule, with the mass of the electron being negligibly small. The analyte may also be ionized by addition or subtraction of charged species (e.g. H+) to give [M + H]+ or [M – H]- with masses that are different from that of the starting analyte.

Ionization is a key process in mass spectrometry and much instrumental and theoretical work has been devoted to understanding the processes that convert a neutral molecule into an ionized species (cation or anion). This process takes place in the so-called ionization source, which is also responsible for the transfer of the newly produced ions into the gas phase prior to their introduction into the analyzer of the mass spectrometer. There are several ionization sources namely:

• electron ionization;

• chemical ionization;

• electrospray ionization;

• atmospheric pressure chemical ionization;

• atmospheric pressure photoionization; and

• matrix-assisted laser desorption ionization.

1.2.1 Electron Ionization

Electron ionization (EI) — formerly also called electron impact ionization — is the oldest method of ionization. It originates from the work of J. J. Thompson (1856–1940) who won the Nobel Prize in physics in 1906 for having generated ionized species following a discharge of electricity into gases. The unit for the mass-to-charge (m/z) values in mass spectrometry, the Thompson (Th), is the recognition of his groundbreaking work.

Energetic electrons from a heated filament are accelerated by an electric field through the high vacuum ion chamber containing the gaseous sample. Such energetic electrons will transfer part of their energy to the neutral volatilized analyte, causing an electron to be ejected from the molecule forming a molecular ion through the reaction:

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In most electron ionization conditions, only one molecule out of a million will be ionized. The energy of the electron beam is approximately 70 eV; keeping this energy constant between instruments allows standardized spectra to be obtained and the assembly of EI spectra libraries for identification of compounds with so far unknown EI spectra.

Most of the organic analytes have an ionization potential (i.e. energy required to liberate an electron from the molecule) in the range 6–10 eV. Therefore, the excess of energy remaining in the molecular ion produces fragmentation, which is often pronounced enough to decompose the molecular ion. Indeed, the process of electron ionization is considered a “hard” ionization process resulting in considerable fragmentation of the molecular ion due to the excess of energy imparted to the analyte (see, for example, Figure 1.2). Fragmentation of the molecular ion produces both even and odd electron fragment ions.

1.2.2 Chemical Ionization

Chemical ionization (CI) relies on gas phase ion-molecule reactions to ionize the neutral analyte. First, the collision of an ion with a reactant gas such as methane gives rise to CH+·4, which reacts with another methane molecule yielding a stable CH+5 ions. This so-called reactant gas ion collides with the analyte, ionizing the latter usually by proton transfer and giving a protonated molecule:

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These ion-molecule reactions need to take place at a source pressure of about 0.1–1.0 Torr, which is a much higher pressure than the one used for EI (10-5 to 10-6 Torr). In the CI ion source, the pressure of 0.1–1.0 Torr favours ionmolecule reactions before the ions are repelled from the ion chamber. Moreover, under this high pressure, electron capture is very e?cient making the generation of negative ions just as likely as generation of positive ions. Negative CI is, therefore, a frequently used technique. The sensitivity of positive CI is similar to the sensitivity of EI. However, for highly electrophilic analytes, negative CI is many times more sensitive than positive CI enabling, for example, the detection of 200 zeptomol (10-21 mol) of methyl uracil as its pentafluoro derivative.

Chemical ionization is a softer ionization method compared with electron ionization as a greater abundance of ionized analyte can be detected (see, for example, Figure 1.3), making CI useful for determining the molecular weight of unknown analytes. The most popular reactant gases for chemical ionization are methane, isobutane and ammonia.

1.2.3 Atmospheric Pressure Chemical Ionization

Atmospheric pressure chemical ionization (APCI) resembles CI in the sense that ionization is conferred by a reactive reagent. In APCI, the ions are formed at atmospheric pressure using electrons emitted either by a 63Ni foil or, more commonly today, by a corona discharge needle. In such a corona discharge mode, a needle is located near the ion source and a very high negative voltage is applied to it. The analyte solution is sprayed into a heated nebulizer and converted into a fine mist which is carried by a flow of nitrogen and passes through the corona discharge. Primary ions (N+·2, O+·2 or H2O+·) are formed by electron ionization via electrons from the corona. They collide with the solvent molecules forming secondary clusters of reactant gas ions, e.g. H3O+(H2O)n. Analyte ionization takes place via gas phase ion-molecule reactions such as proton transfer and charge exchange. Proton transfer occurs if the analyte has a high proton affinity, whereas for charge exchange, the analyte should possess low ionization energy. Only relatively small and stable compounds up to about 1000–1500 Da can be analyzed using APCI because of the heat required in the vaporization process. In addition, APCI often suffers from a high background due to efficient ionization of gases, solvents and impurities that have high proton affinities.

1.2.4 Atmospheric Pressure Photoionization (APPI)

Atmospheric pressure photoionization (APPI) involves the absorption of energy from an ultraviolet (UV) source. Ionization is induced with vacuum-ultraviolet 10 eV photons emitted by a krypton discharge lamp. The photons can ionize compounds that possess ionization energy (IE) below their own energy (10 eV); this includes most analytes, but excludes most of the typically used gases and solvents. Single-photon ionization occurs according to following the reaction:

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An alternative route may also take place if a carrier gas such as nitrogen is used which strongly absorbs the UV radiation:

[FORMULA NOT REPRODUCIBLE IN ASCII]

Typical analytes possess ionization energies in the range 7–10 eV, whereas the common carrier gases have higher IE values (Table 1.1). Therefore, the analytes can be selectively ionized without interference from ionized carrier gas molecules. Moreover, APPI allows the ionization of less polar analytes because the ionization depends on the ionization energy of the analyte rather than its proton a?nity, as in the case of ESI (see below) and APCI.

1.2.5 Electrospray Ionization

The development of electrospray ionization (ESI) for the analysis of biological macromolecules was rewarded with the attribution in 2002 of the Nobel Prize in chemistry to J. B. Fenn (together with Koichi Tanaka for “the development of methods for identification and structure analyses of biological macromolecules” and K. Wüthrich for “his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution”). Electrospray ionization revolutionized the way ions were transferred from a solution to the gas phase.

There is ample literature debating the mechanism of electrospray ionization. In a nutshell, analytes are introduced to the source in an ionic solution at a low flow rate (a few µl min-1). This solution passes through an electrospray needle with a high potential difference between its tip and a nearby counter electrode (typically in the range 2.5–4 kV). The positive charges in the solution are repelled by each other and by the positively charged needle walls, so that a cone-shaped flow is formed on the tip of the needle. In a similar way, negatively charged ions can be formed by setting a negative voltage on the needle wall.

As the solvent evaporates, droplets shrink with a resulting increase of the charge density on their surface. Eventually, the droplets reach a state called “Rayleigh’s instability limit”, which causes them to undergo a series of coulombic fissions until gas-phase ions are left. There is another theory, by Iribarne and Thomson, stating that the droplets start emitting ions directly to the gas phase when the repulsion forces on the droplet surface become high enough to break the surface tension (“ion evaporation”).

ESI is a very soft ionisation method as the analytes retain very little residual energy upon ionization. It is applicable to small polar molecules for which almost no fragmentations are observed, and to large macromolecules (e.g. proteins or polysaccharides) as multicharged ions are produced (see, for example, Figure 1.4). Multicharged ions have proved extremely useful in determining the molecular weight of large macromolecules more precisely. Mathematical deconvolution of the series of peaks allows calculating their molecular weights with an accuracy of 10 ppm.

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