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spectrometry with a much longer pedigree than ICP-

MS. It is based on the production of positive or

negative atomic or molecular ions at the surface of a

resistively heated metal filament. It has been used to

conduct stable studies of nutrient mineral metabolism

and is a ‘gold standard’ analytical technique for quan-

titative analysis of nutrients and toxicants in foods

by isotope dilution mass spectrometry, in which

measurements are conducted by adding a predeter-

mined quantity (the ‘spike’) of an enriched stable

isotope (of known isotopic composition) to the target

analyte. Isotope ratio measurement of the mineral of

interest then yields the amount of analyte precisely

and accurately.

0028 Mass analyzer The mass analyzer of the mass spec-

trometer separates ionized atoms and molecules

according to their mass-to-charge ratio, m/z. Several

different types of mass analyzer are available. The

main types of relevance to food and nutrition research

are described below.

0029 The maximum practical resolving power, R,ofa

mass spectrometer is an important instrumental char-

acteristic. It is effectively a measure of the instru-

ment’s ability to separate ions. Several different

methods can be used to define R, but the two most

common are the ‘10% valley’ definition (eqn (7)),

which is based on the separation between two peaks

of equal height and mass m and m þ Dm,

R ¼

m þ m

, ð7Þ

when the peaks are separated by a valley of 10% peak

height, and the full width at half height definition

(eqn (8)),

R ¼

m

, ð8Þ

where Dm is the width of a peak of mass m at 50% of

its height.

0030Some instruments, magnetic sector-based mass

spectrometers, for example, yield constant resolution

over the entire mass range (i.e., m/Dm is constant). In

contrast, quadrupole mass analyzers yield a constant

Dm over the entire mass range. In this case, resolution

is mass-dependent, usually some multiple of the mass,

e.g., twice the mass.

0031Magnetic sector mass spectrometers Ions formed in

the ion source region are accelerated through a po-

tential and are injected into a sector magnetic field

(See Figure 8). The motion of the ions in the magnetic

field is described by eqn (9),

m=z ¼

, ð9Þ

where z is the number of charges on an ion of mass m,

e is the electronic charge, B is the magnetic field

strength, and r is the radius of the ion path. The

mass scale is generally scanned by varying, B rather

than V, as this yields the most sensitive and reprodu-

cible data. Voltage scanning is used to perform

selected ion monitoring measurements, in which

a small number of mass channels are monitored con-

secutively and repetitively. This mode is used to

record quantitative data with high sensitivity

(selected ion monitoring measurements may also be

performed using other types of mass analyzer).

0032Single-focusing magnetic sector mass spectrom-

eters (now something of a rarity) are capable of a

maximum resolution of about 5000. Increased resolv-

ing power is attainable by adding an energy resolving

sector, a pair of curved plates held at a defined elec-

trical potential, to yield a ‘double-focusing’ instru-

ment. These help to increase resolving power by

Temperature K⫾10%

6200

Sampling

region

6500 8000

Distance from coil (mm)

Induction coil

Sample aerosol

Body of torch

Induction

region

20 15 10

fig0004 Figure 4 Schematic diagram of an ICP-MS ion source. Redrawn from a diagram supplied by VG Elemental, with permission.

3744 MASS SPECTROMETRY/Principles and Instrumentation

correcting for the spread in ion velocities that most

ionization techniques produce. The electric sector can

be placed before or after the magnetic sector. Figure 5

shows a design of ‘Nier–Johnson’ geometry, in which

an electric sector deflects the ion beam through p/2

radians, before it enters a magnetic sector with a

deflection of p/3 radians.

0033 Double focusing mass spectrometers are available

in a number of designs and can attain maximum

resolving powers from 10 000 to over 200 000, de-

pending on their design.

0034High-resolution mass spectrometry is commonly

used to measure ‘accurate masses’, i.e., masses deter-

mined to three or four decimal places. These data may

then be converted into the empirical formulae of ions

in the mass spectrum. High-resolution mass spec-

trometry may also be used to increase the specificity

of quantitative measurements, or to resolve isotopic

clusters of multiply charged peaks formed by electro-

spray so that the charge state can be measured unam-

biguously.

0035Isotope ratio mass spectrometers Although isotope

ratios may be measured successfully using conven-

tional mass spectrometers, some applications much

require higher precision measurements than can be

attained by using scanning instruments. In such cases,

specialized high precision isotope ratio mass spec-

trometers are available. An example of this type of

instrument is the gas isotope ratio mass spectrometer

(GIRMS), used to determine the isotope ratios of

gases such as CO

,SO

, and H

to a very high

degree of precision and accuracy.

0036The analyte is combusted or reduced to generate

the gases that are ionized in a gas tight EI source. A

magnetic sector separates the ion beam, with the

difference (from a conventional scanning instru-

ment) that each mass channel is collected simultane-

ously, i.e., the mass spectrometer uses a multicollector

ion detection system. This yields high-precision

isotope ratios because any fluctuations in ion beam

Ion source Collector

Magnetic

analyzer

1FFR

2FFR

Electrostatic

analyzer

−

fig0005 Figure 5 Schematic diagram of double-focusing magnetic

sector ion optics of Nier–Johnson geometry. FFR, first field-free

region. From Chapman JR (1985) Practical Organic Mass Spec-

trometry, 2nd edn. Chichester, UK: Wiley-Interscience with per-

mission.

Computer

Amplification

and

recording

Ion

Source

Combustion

system

Sample

Standard

Faraday

collectors

Magnet

beam

fig0006 Figure 6 Schematic diagram of a gas isotope ratio mass spectrometer. From Mellon F, Self R and Startin JR (2000) Mass Spec-

trometry of Natural Substances in Food. London: The Royal Society of Chemistry with permission.

MASS SPECTROMETRY/Principles and Instrumentation 3745

intensity occur simultaneously at each detector.

A diagram of a GIRMS instrument is shown in

Figure 6.

0037 The main uses of GIRMS in food and nutrition

science are in determining the authenticity of foods

and in human studies of nutrient metabolism. High-

precision multicollector mass spectrometers are also

available for use with TIMS or ICP-MS.

0038 Quadrupole mass analyzers Quadrupole mass ana-

lyzers comprise four parallel rods of circular or hyper-

bolic cross-section. These are connected to radio-

frequency (V

cos ot) and direct-current (U) power

supplies, as shown in Figure 7.

0039 The equations of motion governing the path of

ions between the rods describe a complex series

of oscillations and will not be discussed here. These

oscillations are stable for particular values of the

equations and unstable for other values. By operating

the quadrupole in a stable region, the ions will be

constrained to follow a path between the rods until

they reach the detector. The mass spectrum is scanned

by varying V

and U so that the ratio U/V

remains

constant. The quadrupole mass analyzer acts as a

mass filter, allowing one mass channel at a time to

reach the detector as the mass range is scanned.

Quadrupole mass spectrometers are intrinsically

low-resolving-power instruments but are relatively

cheap and robust.

0040 Ion traps The Paul ion trap is related to quadrupole

mass analyzers. The term ‘Ion Trap’ is, strictly speak-

ing, generic and can encompass the Penning ion traps

used in Fourier transform ion cyclotron resonance

(FTICR) instruments (see below). However, ion

traps are generally used in the scientific literature to

refer to Paul ion traps, thanks to the great success of

these instruments in compact, bench-top designs of

mass spectrometer.

0041An ion trap may be described as a quadrupole that

has undergone a solid of rotation. A typical ion trap

comprises two endcap electrodes and a ring electrode,

all of hyperbolic or hemispherical cross-section

(Figure 8).

0042The end-cap electrodes contain small-diameter

holes for allowing ions to enter and leave the trap.

Ions are confined inside the trap by a radio-frequency

field of constant frequency but variable power. The

ions may be detected, according to their m/z ratio, by

applying voltages sufficient to eject them from the

trapping field. Ion traps, despite their simplicity and

cheapness of construction, can be used to perform

sophisticated tandem mass spectrometry experiments

in addition to fulfilling a role as conventional scan-

ning mass spectrometers.

0043FTICR mass spectrometry FTICR (sometimes

simply known as Fourier transform mass spectrom-

etry) is a Penning trap in which ions confined by a

strong magnetic field, B, move in circular orbits of

characteristic frequency, o (Figure 9).

0044The motion of the ions is governed by the cyclotron

equation (eqn. (10)),

! ¼ z

: ð10Þ

0045A broad-band ‘chirp’ of electromagnetic radiation

is applied to excite all ions in the trapping cell to their

cyclotron frequency. The orbiting ions induce an

alternating ‘image current’ in the walls of the cell

Detector

lons in

−(U+Vcos ωt)

(U+Vcos ωt)

fig0007 Figure 7 Schematic diagram of a quadrupole mass spectrom-

eter.

Amplifier and

RF generator,

fundamental

RF voltage

Amplifier and

RF generator

supplementary

RF voltage

Scan acquisition

processor

(computer)

To preamplifier

(Ion signal)

Electron multiplier

detector

End cap

Filament

Ring

electrode

fig0008Figure 8 Schematic diagram of an ion trap (Paul trap) mass

spectrometer. From Andrews DL (1990) Perspectives in Modern

Chemical Spectroscopy. Berlin: Springer-Verlag with permission.

3746 MASS SPECTROMETRY/Principles and Instrumentation

receiver plates. This signal is complex because each

m/z value generates its own characteristic frequency.

A Fourier transform of this complex signal yields the

masses of all ions in the spectrum. Frequencies can

be measured with great accuracy, and so very high

resolving powers, over 1 000 000 in some cases, are

attainable. However, resolution does fall off with

increasing m/z.

0046 FTICR instruments can be used to perform se-

quential tandem mass spectrometry experiments and

are compatible with pulse ionization techniques.

Although they are expensive to purchase, and running

costs are high, they have undergone something of

a renaissance in recent years. This is partly due to

their compatibility with newer pulse ionisation

techniques like MALDI. Perhaps more importantly,

the multiple charging observed in ESI of proteins

yields a mass envelope in the region where full advan-

tage can be taken of FTICR’s high-resolution

performance (i.e., 600–2000 Th). This allows unam-

biguous determination of the charge state of product

ions formed in tandem mass spectrometry experi-

ments.

0047 Time of flight (ToF) mass analyzers The ToF mass

analyzer is now becoming widespread for several

reasons. First, it is compatible with MALDI, a pulsed

ionization technique that is a perfect match for ToF.

Second, improvements in design and performance

have yielded major advances in performance, particu-

larly resolution and sensitivity. Third, ToF mass ana-

lyzers have, theoretically, an unlimited mass range.

0048 ToF analyzers are based on the property that the

drift velocities of ions are dependent on their m/z

ratio. Ions formed in the source region are accelerated

through a potential, V, and then enter a field-free drift

region (Figure 10).

0049According to eqn (11), the ions will all have the

same kinetic energy.

¼ zeV, ð11Þ

where v is the ion velocity. The ToF, t, of the ions is

governed by eqn (12),

t ¼

2zeV



L: ð12Þ

0050Early ToF instruments had limited mass resolution,

but ‘delayed extraction’ techniques and the introduc-

tion of electrostatic mirrors (reflectrons) have

increased their resolution dramatically. ToF instru-

ments are capable of a very high sensitivity because

they record all the ions generated in a single ioniza-

tion pulse.

0051Ion detection and recording The most common

detector in mass spectrometry is the electron multi-

plier; this amplifies the weak ion beam signal greatly,

yielding gains of up to the order of 10

. Multipliers

can be either a discrete dynode type or a continuous

dynode, generally known as a channel electron multi-

plier or channeltron. The discrete dynode electron

multiplier comprises a series of Be–Cu alloy dynodes

arranged as shown in Figure 11.

0052The first dynode is held at a negative potential of

several kilovolts relative to the (positive ion) beam.

The beam strikes the first dynode and generates a

shower of secondary electrons. These electrons are

then attracted to the second dynode, generating

more electrons, and so on, in a cascade through the

device. Negative ions may be detected by using a

conversion dynode held at a high positive potential.

Channeltron detectors operate according to similar

Drift tube

(length L)

Acceleration

grids

Detector

Ion drift velocity (v)

Recording

electronics

and computer

Ionization region

(pulsed EI, FAB, SIMS,

laser or

252

Cf)

fig0010Figure 10 Schematic diagram of a ToF mass spectrometer.

From Andrews DL (1990) Perspectives in Modern Chemical Spec-

troscopy. Berlin: Springer-Verlag with permission.

Receiver plate

Transmitter

plate

Transmitter

plate

Magnetic

field B

Ion

motion

fig0009 Figure 9 Schematic diagram of a Fourier transform ion cyclo-

tron resonance mass spectrometer. From Andrews DL (1990)

Perspectives in Modern Chemical Spectroscopy. Berlin: Springer-

Verlag with permission.

MASS SPECTROMETRY/Principles and Instrumentation 3747

principles as discrete dynode electron multipliers. The

main difference is that individual dynodes are re-

placed by a tapered, coiled tube, and electrons bounce

from along the tube towards the earthed terminus.

Array detectors comprise a series of miniaturized

channeltrons that are arranged adjacent to each

other. They can be used to detect ions simultaneously

over a range of masses and are used to increase

sensitivity in some types of magnetic sector mass

spectrometer.

0053 The scintillation detector is an alternative ion beam

amplification device in quite widespread use. The ion

beam strikes a surface that emits electrons towards a

phosphor screen, generating photons. The photons

are amplified using a device akin to the image inten-

sifiers used in night-vision binoculars and similar

devices. Scintillation-based photomultiplier arrays

are also available.

0054 Faraday cup detectors are used mainly in isotope

ratio mass spectrometers. The Faraday detector is a

very simple device, comprising a conducting ‘bucket’

that collects the ion beam, yielding an electrical

signal. The minute electrical signal generated is then

amplified and recorded. Faraday cups are used in

applications where accurate measurement is more

important than sensitivity, for example in high-

precision isotope ratio measurements conducted by

GIRMS, ICP-MS, or TIMS.

0055 Tandem mass spectrometry (mass spectrometry/mass

spectrometry (MS/MS)) Tandem mass spectrom-

etry, or mass spectrometry/mass spectrometry

(MS/MS), experiments are conducted classically by

combining two mass analyzers in a single instrument.

For example, two double focusing magnetic sector or

two quadrupole mass analyzers may be combined in

series. Tandem quadrupole mass spectrometers are

generally known as ‘triple quadrupoles.’ However,

only two of the quadrupoles are involved in mass

analysis: the third (middle) quadrupole transmits all

ions and is used to ‘activate,’ i.e., transfer energy to,

the ions of interest.

0056MS/MS has been described as ‘taking the

mass spectrum of an ion in a mass spectrum.’ The

principle of MS/MS is that a sample is ionized

and mass-analyzed in the first mass analyzer. A

particular m/z value of interest is selected from

the mass spectrum and is directed into a collision

cell that contains a neutral gas (argon, for example).

The ion is vibrationally excited by collision with

the target gas, a process known as collision induced

dissociation. This generates fragment ions that

are separated and recorded by the second mass

analyzer. The process is shown schematically in

Figure 12.

0057The example given is of a product ion scan and is

used as a general technique for analyzing target com-

ponents in a mixture, or for generating characteristic

fragment ions from a sample that yields stable mo-

lecular ions under the ionization conditions used.

Other scan modes are available, including precursor

ion scans, constant neutral loss, and selected reaction

monitoring.

0058Tandem mass spectrometers are available in several

different configurations. ‘Hybrid’ instruments, for

example magnetic sector/quadrupole or quadrupole

ToF, are also found in many laboratories. ToF mass

spectrometers may also be used in MS/MS mode. MS/

MS experiments may also be performed using ion

trap or FTICR instruments. In this case, MS/MS is

conducted by temporal, rather than spatial, separ-

ation of ions. For example, an ensemble of ions in

an ion trap is ‘swept’ using a scan function that expels

all ions from the trap except the m/z value of interest.

These ions are activated by collision with a gas

admitted to the ion trap, and the ionic products of

Electrons

Amplifier

Ion beam

fig0011 Figure 11 Schematic of a discrete dynode electron multiplier ion detector. From McFadden WH (1973) Techniques of Combined Gas

Chromatography/Mass Spectrometry. Chichester, UK: Wiley-Interscience with permission.

3748 MASS SPECTROMETRY/Principles and Instrumentation

this activation are measured by a conventional

mass scan. This process of isolation can be repeated,

i.e., a particular product ion can, in turn, be isolated

and activated, and so on in a sequence of reactions.

This allows MS/MS/MS or MS

experiments to be

carried out.

0059 MS/MS has many analytical applications. These

include the analysis of complex mixtures without

any chromatographic separation, generation of char-

acteristic fragment ions from molecular ions, and

improving the selectivity of quantitative measure-

ments.

0060 Data acquisition and processing Computers, typic-

ally PCs, are now used routinely both to control

mass spectrometers and to acquire and process

mass-spectrometric data. Parameters such as focus-

ing, resolution, and scan speed may be adjusted and

set via a computer. The same computer may be used

to control mass spectrometer inlet systems such as gas

chromatography, high-performance liquid chroma-

tography, or automated direct insertion probes. The

computer is also the final link in the signal-recording

chain. It forms part of the system used to digitize

the mass spectrum, calibrate the mass scale,

enhance the quality of data, and, ultimately, to aid

in the interpretation of data. All these functions may

be carried out by a single computer coupled to a

sophisticated data digitization device.

0061Library searching of a large database of the

mass spectra of known compounds is now routine

as an aid to interpreting EI spectra and identifying

components in (for example) a complex gas chroma-

tography – mass spectrometry chromatogram. More

sophisticated, compound class-specific algorithms are

available for particular types of analysis, such as

identification of proteins in proteomics experiments.

Computer systems are now an integral component of

mass spectrometers and have greatly simplified the

process of instrument control, data acquisition, and

interpretation.

See also: Chromatography: Gas Chromatography; Mass

Spectrometry: Applications

Further Reading

Adams F, Gijbels R and van Grieken R (1988) Inor-

ganic Mass Spectrometry. Chichester, UK: Wiley-Inter-

science.

Chapman JR (1995) Practical Organic Mass Spectrometry,

2nd edn. Chichester, UK: Wiley-Interscience.

McLafferty FW and Turecek F (1993) Interpretation of

Mass Spectra, 4th edn. Sausalito, USA: University

Science Books.

Rose ME and Johnstone RAW (1996) Mass Spectrometry

for Chemists and Biochemists, 2nd edn. Cambridge:

Cambridge University Press.

Collision cell

Mass spectrum : MS 1

600 700 800

300 500 700 800

MS/MS spectrum : MS 2

Collision

induced

dissociation

900

fig0012 Figure 12 Schematic representation of tandem mass spectrometry (product ion analysis).

MASS SPECTROMETRY/Principles and Instrumentation 3749

Applications

F A Mellon, AFRC Institute of Food Research,

Norwich, UK

Background

0001 Food science, food technology, and nutrition present

the scientist with a very wide range of analytical

problems. Consequently, many different mass spec-

trometric techniques, alone or in combination with

chromatographic or other methods, have been used to

address specific analytical problems in food and nu-

trition research. It is impossible to give a comprehen-

sive account of applications of mass spectrometry in

food science in a short article. Instead, some represen-

tative examples are chosen from a range of topics that

illustrate the diversity of techniques and applications

in selected areas.

Major Food Components

Carbohydrates

0002 Carbohydrate analysis has a long history in the food

sciences because of the importance of carbohydrates

as macronutrients, as major constituents of dietary

fiber, and as food structure components and food

additives. A classic, still widely used, method for

determining the composition and linkages of polysac-

charides involves gas chromatography/mass spec-

trometry (GC/MS) analysis of partially methylated

alditol acetates (PMAAs). The polysaccharides are

methylated, hydrolyzed, and reduced to O-methyl

alditols. These are acetylated and analyzed (both

qualitatively and quantitatively) by GC/MS. Partially

methylated alditol acetates yield characteristic mass

spectra that allow determination of the nature and

linkage position of the monosaccharides comprising

the polysaccharide. This technique has been used

to analyze cell wall polysaccharides in many fruits,

vegetables, and cereals.

0003 The molecular weights of large polysaccharides can

be measured with the aid of electrospray ionization

(ESI) (in liquid chromatography/mass spectrometry

(LC/MS) or direct injection modes) and matrix-

assisted laser desorption ionization (MALDI) mass

spectrometry (MS). Both techniques may also be

used to obtain partial or (for smaller polysaccharides)

complete sequence information in MS/MS mode.

Pectins, for example, are widely used in the food

industry as stabilizers or to form gels. Nanospray

MS/MS of an unseparated digest of partially methyl-

esterified (DE (degrees of esterification) 31%) pectin

has been used to obtain useful structural information.

The tandem mass spectrum of the doubly deproto-

nated decamer with two methyl esterified residues is

shown in Figure 1.

O labeling is used to distinguish

the reducing and nonreducing ends of the molecule.

Complete Foods

0004Many mass-spectrometric methods used in food an-

alysis and research focus on the determination of

specific components or of mixtures of structurally

related compounds. However, pyrolysis mass spec-

trometry (Py/MS) is capable of yielding analyti-

cal information on microgram or submicrogram

amounts of whole foodstuffs. Py/MS is based on the

rapid, controlled thermal degradation of samples in

an inert atmosphere. This produces mixtures of vola-

tile compounds whose electron ionization (EI) or

chemical ionization (CI) mass spectra are recorded.

This generates a complex ‘fingerprint’ of the sample

under investigation. Py/MS data are generally pro-

cessed by chemometric methods to obtain optimal

information from the analyte. The technique has

been used in the quality assurance of foods and

drinks, in the study of food plants, and in the identifi-

cation of food pathogens.

Flavors and Taints

0005Because most flavor components are highly volatile,

combined GC/MS (using EI and/or CI) has been a

primary technique in flavor analysis and research since

the early 1960s. For example, almost 1000 discrete

compounds have been identified in coffee volatiles.

0006Direct injection of headspace samples on to a GC

column reflects flavor composition more accurately

than extraction or trapping methods and is less likely

to generate artefacts. However, the ease with which

solid-phase microextraction GC/MS studies may be

conducted has made this an important and useful

technique for sampling flavors and taints. GC/MS

has been supplemented by LC/MS techniques for

studying involatile flavor precursors (glucosinolates,

for example) or semivolatile or involatile food com-

ponents that have important flavor characteristics.

0007The availability of a large knowledge base of flavor

profiles (largely defined by GC/MS analysis) and recent

instrumental advances have resulted in a recent shift in

emphasis of mass spectrometric applications. Instead

of characterizing complex mixtures of volatiles, several

3750 MASS SPECTROMETRY/Applications

researchers are now focusing on flavor release and,

more specifically, on sampling volatiles released into

the mouth and nose. By using a special atmospheric-

pressure chemical-ionization (APCI) probe coupled to

a mouth or nose piece, it is possible to conduct dy-

namic, breath-by-breath analyses of air expired during

eating. An example of the type of data that can be

acquired is illustrated in Figure 2, which shows volatile

release curves for menthol, menthone, and limonene

released while eating a mint-flavored sweet. The APCI

technique represents the ‘state of the art’ in modern

flavor-release studies.

0008 Mass-spectrometric methods are also useful for au-

thenticating flavor components. Flavor concentrates

from natural sources are usually sold at a premium

price compared with their synthetic analogs. Mass

spectrometry has an important role in detecting fraudu-

lent representation of synthetic flavor compounds as

natural. Microcombustion of flavor samples yields

that is then swept into the ion source of an isotope

ratio mass spectrometer where

ratios are

measured to a very high precision. These isotope ratios

are characteristic of the source of the sample and can be

used to authenticate the material. The technique can be

used directly on the sample without prior pretreatment.

If a more sophisticated analysis is required, GC/com-

bustion/isotope ratio mass spectrometry will yield

accurate isotope ratios on the individual components

of a flavor sample.

Lipids

0009Although complex mixtures of lipids cannot be char-

acterized by any single analytical technique, mass

spectrometry is very useful in the qualitative and

quantitative analysis of many different lipid types,

including long-chain fatty acids and their esters,

mono-, di-, and triacylglycerols, and phospholipids.

0010Branched-chain fatty acids and their esters exhibit

characteristic bond scissions adjacent to the branch-

ing point. The degree of unsaturation of fatty acids

esters can be determined readily from EI mass spectra.

However, double-bond location presents consider-

able difficulties because long-chain unsaturated

compounds rearrange extensively under EI, and

consequently, fragment ions do not generally supply

reliable information about double-bond position. A

number of strategies have been devised to overcome

these problems: one of the most popular is derivatiza-

tion, followed by GC/MS. Several different proced-

ures are available, including oxidation followed by

silylation, epoxidation and the analysis of methoxy or

dimethyldisulfide derivatives. An alternative strategy

is to generate chemical derivatives that localize

50 100 150

Time (s)

Percentage peak area

200 250 300

Limonene

Menthone

Menthol

fig0002 Figure 2 Average volatile release curves for menthol,

menthone, and limonene released while eating mint-flavored

sweets. From Linforth RST, Ingham KE and Taylor AJ (1996) Fla -

vour Science: Recent Developments, pp. 361–368. Cambridge:

Royal Society of Chemistry with permission.

200

500

1000

1500

Intens. Intens.

Intens.

400 600 800 1000 1200 1400 1600 1800 m/z

360

380 m/z

200

400

600

200

400

600

1Me

3Me

4Me

2Me

4Me

3Me

4Me

1Me

2Me)2−

3Me)2−

4Me)2−

(10mer

4Me−60Da−2H)2−

(10mer

4Me−2H)

2−

1Me

2Me

1450 1460 1470 1480 m/z

fig0001 Figure 1 Negative ion MS/MS spectrum of the doubly deprotonated decamer with two methyl esterified residues and

O-labeled

reducing end from an unseparated PG II digest of 31% methyl esterified pectin. From Korner R, Limberg G, Christensen TMIE,

Mikkelsen JD and Roepstorff P (1999) Sequencing of partially methyl-esterified oligogalacturonates by tandem mass spectrometry and

its use to determine pectinase specificities. Analytical Chemistry 71: 1421 with permission.

MASS SPECTROMETRY/Applications 3751

charge, preventing charge-induced double-bond mi-

gration. Pyrrolidide derivatives have been used for this

purpose but are insufficiently volatile for GC/MS. A

useful alternative that generates derivatives suitable

for GC/MS is provided by 2-substituted 4,4-dimethy-

loxazoline formed by condensation of the fatty

acid with 2-amino-2-methylpropanol. The ‘charge-

localizing’ derivatives yield clusters of ions 14 mass

units apart, reducing to 12 mass units when a double

bond interrupts the chain. MS/MS techniques for

locating double bonds include ‘charge-remote’ frag-

mentation of molecules ionized by negative ion CI. If

high collision energies are used, free fatty acids yield

ions characteristic of the double-bond position.

0011 Capillary GC/MS is useful for identifying the carbon

and unsaturation number of acylglycerols. However,

APCI LC/MS has the ability to analyze less volatile

acylglycerols (particularly-triacylglycerols) that may

be unsuitable for GC/MS. It is fast becoming the

technique of choice for qualitative and quantitative

analysis of acylglycerols and, when combined with

MS/MS, is capable of distinguishing fatty acid chains

in the sn-2 position from those in the sn-1/3 positions.

0012 The sensitivity, selectivity, and convenience of posi-

tive and negative ion ES LC/MS make this the current

benchmark technique for analyzing phospholipid

mixtures. Additional structural information can be

obtained by MS/MS. Where these advanced tech-

niques are unavailable, a more laborious approach

of chromatographic class separation, chemical deg-

radation, and GC/MS analysis of fatty acid can be

used to generate useful structural information.

Proteins, Oligopeptides, and Amino Acids

0013 Amino acids, peptides, and proteins are of interest in

several areas of food and nutrition science, and only a

small sample of current applications can be described

here. For example, GC/MS, and LC/MS are particu-

larly useful for analyzing the products of amino acid/

sugar reactions (Maillard or Amadori products) that

have important flavor characteristics. Free amino acids

themselves may be analyzed by electrospray ionization

(ESI) LC/MS. However, alternative (and cheaper) tech-

niques to mass spectrometry are available, and mass

spectrometric methods are generally confined to spe-

cialist applications (for example, metabolic studies or

the identification of unusual amino acids).

0014 The mass-spectrometric study of proteins and

oligopeptides was once largely conducted by fast-

atom bombardment (FAB) MS, but this methodology

has now been supplanted by the more robust and

sensitive methods of ESI and MALDI time of flight

(ToF). An instructive example is provided by a rapid

screening system, based on MALDI-ToF, developed

to determine the presence of gliadins in food

samples. Direct observation of the characteristic glia-

din mass pattern in processed and unprocessed

gluten-containing foods has yielded an nonimmuno-

logical alternative for the quantitative estimation of

gluten gliadins in foods. Figure 3 shows the MALDI-

ToF spectrum of an unfractionated ethanol extract of

wheat gliadins.

0015Tandem mass spectrometry of oligopeptides, usu-

ally following ESI ionization, yields characteristic

ions that allow full or partial amino acid sequencing

of the analyte molecule. These techniques can be used

to confirm the structures of individual oligopeptides,

for example the food preservative nisin and genetic-

ally engineered analogs of this molecule.

0016One of the most powerful applications of mass-

spectrometric peptide sequencing methodology is in

the rapidly developing field of proteomics. Two main

mass-spectrometric techniques have been developed

for proteomic studies. The first involves enzymatic

digestion of individual spots excised from a two-

dimensional gel. The resulting peptide mixture is ana-

lyzed, without prior separation, by MALDI-ToF mass

spectrometry. This yields a unique pattern of molecu-

lar weights that are searchable against protein data-

bases. If this fails to yield a positive identification, a

backup technique, involving ultrasensitive MS/MS

sequencing of peptides generated by specific digestion

of a protein two-dimentional spot, yields a ‘sequence

tag,’ again searchable against a database to identify

the unknown protein. Finally, a third level in this

hierarchy of mass spectrometric techniques may be

used to identify posttranslational modifications of

proteins, where required. Proteomics has consider-

able potential in food and nutrition research, for

example in the investigation of environmental re-

sponses in food-poisoning microorganisms, in the

study of diet–health relationships, and in the assess-

ment of genetically modified food safety.

Nutritional Studies

Stable Isotope Methods

0017Interest in the use of stable-isotope mass spectrometry

for studying the nutritional value of foods and diets

has increased considerably. Enriched stable isotopes,

in contrast to radioisotope methods, provide safe,

scientifically rigorous techniques for determining nu-

trient bioavailability and metabolism in all human

population groups.

0018The only major drawbacks of stable isotope

studies are associated with the presence of endogen-

ous isotopes of the elements under investigation.

Sufficient label must be administered to generate a

measurable increase in isotope ratio above natural

3752 MASS SPECTROMETRY/Applications

abundance. However, care is needed to prevent ad-

ministration of excessive amounts of label, as this

would yield unrepresentative data and might even

have toxic effects. However, administration of small

quantities of label requires very accurate measure-

ment of isotope ratios because the enriched material

is diluted by the endogenous nutrients already present

in the body. In the case of mineral metabolism studies,

additional precautions are necessary when studying

trace elements because of the possibility of contamin-

ation with environmental minerals during sample

processing.

0019 Stable isotope nutritional studies usually involve

administration of the enriched stable isotope of an

element or isotopically labeled compound in, or

with, a meal. The method of labeling depends on the

type of study undertaken. For elements such as selen-

ium, where absorption and metabolism are highly

dependent on chemical form, an intrinsic label (i.e.,

one incorporated biosynthetically in the food) should

be used in most cases. However, minerals believed to

form a ‘common pool’ in the digestive system may be

mixed directly with the food and administered

following equilibration (extrinsic labeling). In some

cases, a second isotope is injected or infused intraven-

ously to correct for endogenous losses. Samples of

breath, blood, urine, saliva, or feces are then collected

for an appropriate period and subjected to isotopic

analysis. Isotope ratio measurement often requires

specialized instrumentation such as high-precision

gas isotope ratio mass spectrometers or specialized

inorganic mass spectrometers (e.g., thermal ioniza-

tion mass spectrometry (TIMS) ) or inductively

coupled plasma mass spectrometry (ICP-MS).

Mineral Nutrients

0020Although GC/MS, FAB TIMS, and ICP-MS have

all been used to measure enriched stable isotopes

of nutrient minerals (in the case of GC/MS, after

derivatization to generate volatile metal–chelate

complexes), the dedicated inorganic methods of

TIMS and, especially, ICP-MS are now used almost

exclusively. ICP-MS has distinct advantages over

alternative techniques because it is rapid and

sensitive, and sample preparation can be minimized.

Furthermore, because ICP-MS can be used with con-

tinuous flow sample introduction methods, it is suit-

able for coupling directly to separation techniques

such as size-exclusion chromatography or HPLC.

This means that speciation studies, i.e., determination

of the chemical form of particular elements (oxida-

tion state, protein binding, etc.) and the effect of this

on metabolism may also be studied. The two major

drawbacks of ICP-MS, low precision relative to TIMS

and interference effects from polyatomic ions in the

argon plasma, have largely been overcome by new

generations of instruments equipped with multiple

collectors and collision cells, respectively.

20 000

5000

10 000

15 000

20 000

25 000

30 000

a.i

40 000 60 000 80 000 100000 m/z

13407*

16213

17722

13968*

15773*

17275

15453*

30895

31539

34616

33998

32414

38510

WHEAT

48458

65353

69620

73196

95962

94238

62238

50934

fig0003 Figure 3 MALDI-ToF spectrum of the unfractionated 70% ethanol extract of wheat endosperm. A box indicates the characteristic

gliadin region. Asterisked peaks are doubly charged ions. From Mendez E, Camfieta E, Sansebastian J et al. (1995) Direct identification

of wheat gliadins and related cereal prolamins by matrix assisted laser desorption ionization mass spectrometry. Journal of Mass

Spectrometry and Rapid Communications in Mass Spectrometry (Combined Issue) S123 with permission.

MASS SPECTROMETRY/Applications 3753