COMMONLY USED TECHNIQUES
Introduction to Mass Spectrometry for Food
Chemistry
Almost a century ago, the first mass spec-
trometers were used to prove the existence of
isotopes of the elements. During the first half
of the 20th century, physicists and physical
chemists used mass spectrometers to help char-
acterize new elements and the fission products
of radioactive elements as they were created or
discovered. Other applications included the
analysis of isotopic enrichment of elements and
their inorganic derivatives. As this era of mass
spectrometry reached maturity, by the 1940s,
the analysis of organic molecules emerged as a
new application of mass spectrometry. Begin-
ning in 1945, organic mass spectrometers using
electron impact (EI) ionization became com-
mercially available and were used primarily by
the petroleum industry. Toward the late 1950s,
organic mass spectrometers began to be used
for the analysis of a wider variety of organic
molecules, and gradually became a fundamen-
tal analytical tool for the characterization of
synthetic organic compounds.
During the 1960s, high-resolution, double-
focusing magnetic sector instruments became
available from multiple manufacturers and
were widely used in organic chemistry for exact
mass measurements and elemental composi-
tion analysis. EI was used for generating struc-
sample
mol. wt. < 1000
volatile nonvolatile
El, Cl, APCI FAB, MALDI,
APCI electrospray
Figure A.3A.1 Flow chart illustrating the selection of a suitable ionization technique for the mass
spectrometric analysis of a sample. Abbreviations: APCI, atmospheric pressure chemical ionization;
CI, chemical ionization; EI, electron impact; FAB, fast atom bombardment; MALDI, matrix-assisted
laser desorption/ionization.
Contributed by Richard B. van Breemen
Current Protocols in Food Analytical Chemistry (2001) A.3A.1-A.3A.7
Copyright © 2001 by John Wiley & Sons, Inc.
APPENDIX 3
APPENDIX 3A
turally significant fragment ions for compound
identification, and rules for structure elucida-
tion using mass spectrometry were developed
(for a thorough review of EI and ion fragmen-
tation pathways, see McLafferty and Turecek,
1993). Biomedical and food chemistry appli-
cations of mass spectrometry were developed
during this time. Chemical ionization (CI),
which was developed by researchers in the
petroleum industry (Field, 1990), was quickly
adopted as a softer ionization alternative to EI,
useful in reducing fragmentation so that mo-
lecular weights could be confirmed more easily.
CI became another standard ionization tech-
nique for mass spectrometry (see Figure
A.3A.1 for a guide to the selection of ionization
techniques in mass spectrometry).
GAS CHROMATOGRAPHY/MASS
SPECTROMETRY (GC/MS)
With the introduction of computerized data
systems for data acquisition, reduction, and
storage during the 1960s, the efficiency of mass
spectrometric analysis grew rapidly and con-
tinues to grow to this day. The use of computers
for data reduction and analysis helped gas chro-
matography/mass spectrometry (GC/MS) be-
come a practical and powerful tool for qualita-
mol. wt. > 1000
mol. wt. < 5000 mol. wt. > 5000
MALDI,
electrospray
Commonly Used
Techniques
A.3A.1
Supplement 2
 tive and quantitative analysis of compounds in
mixtures. Both EI and CI were immediately
useful for GC/MS, since both of these ioniza-
tion methods require that the analytes be in the
gas phase. When capillary GC was incorpo-
rated into GC/MS, this technique reached ma-
turity. The advantages of GC/MS include
speed, selectivity, and sensitivity. Typically,
GC/MS may be used to select, identify, and
quantify organic compounds in complex mix-
tures at the femtomole level. Compounds are
selected using a combination of chroma-
tographic separation and mass selection, and
when using tandem mass spectrometry
(MS/MS; see discussion below), the fragmen-
tation pathway may be used for additional se-
lectivity. The speed of GC/MS is determined
by the chromatography step, which typically
requires from several minutes to one hour per
analysis. Although GC/MS remains important
for the analysis of many organic compounds,
this technique is limited to volatile and ther-
mally stable compounds (see chromatogra-
phy/MS selection flow chart in Fig. A.3A.2).
Therefore, thermally unstable compounds—
including food pigments such as carotenoids
and chlorophylls and biomolecules such as pro-
teins, carbohydrates, and nucleic acids—can-
not be analyzed in their native forms using
GC/MS (for more details regarding GC/MS
and its applications, see Watson, 1997).
DESORPTION IONIZATION MASS
SPECTROMETRY
During the 1970s and early 1980s, desorp-
tion ionization techniques such as field desorp-
tion (FD), desorption EI, desorption CI (DCI),
and laser desorption were developed to extend
the utility of mass spectrometry towards the
analysis of more polar and less volatile com-
pounds (see Watson, 1997, for more informa-
tion regarding desorption ionization techniques
including DCI and FD). Although these tech-
niques helped extend the mass range of mass
spectrometry beyond a traditional limit of m/z
1000 and toward ions of m/z 5000 (Fig.
A.3A.1), the first breakthrough in the analysis
of polar, nonvolatile compounds occurred in
1982 with the invention of fast atom bombard-
ment (FAB; Barber et al., 1982). FAB and its
counterpart, liquid secondary ion mass spec-
trometry (LSIMS), facilitate the formation of
abundant molecular ions, protonated mole-
cules, and deprotonated molecules of nonvola-
tile and thermally labile compounds such as
peptides, chlorophylls, and complex lipids up
to approximately m/z 12,000. FAB and LSIMS
use energetic particle bombardment (fast atoms
or ions from 3,000 to 20,000 V of energy) to
ionize compounds dissolved in nonvolatile ma-
trices such as glycerol or 3-nitrobenzyl alcohol
and desorb them from this condensed phase
into the gas phase for mass spectrometric analy-
sis. Molecular ions and/or protonated mole-
cules are usually abundant and fragmentation
is minimal.

sample

mol. wt. < 1000 mol. wt. > 1000

GC/MS LC/MS MW > 5000 LC/MS

El, Cl APCI, electrospray CF-FAB,

electrospray, electrospray
particle beam,
CF-FAB

Introduction to Figure A.3A.2 Selection of chromatography-mass spectrometry system for the analysis of a
Mass sample. Abbreviations: APCI, atmospheric pressure chemical ionization; CF, continuous flow; CI,
Spectrometry for chemical ionization; EI, electron impact; FAB, fast atom bombardment; GC/MS, gas chromatogra-
Food Chemistry
phy/mass spectrometry; LC/MS, liquid chromatography/mass spectrometry.
A.3A.2
Supplement 2 Current Protocols in Food Analytical Chemistry
 Introduced in the late 1980s, matrix-assisted
laser desorption/ionization (MALDI) has
helped solve the mass-limit barriers of laser
desorption mass spectrometry so that singly
charged ions may be obtained up to m/z 500,000
and sometimes higher (Hillenkamp et al.,
1991). For most commercially available
MALDI mass spectrometers, ions up to m/z
200,000 are readily obtained. Like FAB and
LSIMS, MALDI samples are mixed with a
matrix to form a solution that is loaded onto the
sample stage for analysis. Unlike the other
matrix-mediated techniques, the solvent is
evaporated prior to MALDI analysis, leaving
sample molecules trapped in crystals of solid
phase matrix. The MALDI matrix is selected
to absorb the pulse of laser light directed at the
sample. Most MALDI mass spectrometers are
equipped with a pulsed UV laser, although IR
lasers are available as an option on some com-
mercial instruments. Therefore, matrices are
often substituted benzenes or benzoic acids
with strong UV absorption properties. During
MALDI, the energy of the short but intense UV
laser pulse obliterates the matrix and in the
process desorbs and ionizes the sample. Like
FAB and LSIMS, MALDI typically produces
abundant protonated or deprotonated mole-
cules with little fragmentation.
LIQUID
CHROMATOGRAPHY/MASS
SPECTROMETRY (LC/MS)
By the time that GC/MS had become a
standard technique in the late 1960s, LC/MS
was still in the developmental stages. Produc-
ing gas-phase sample ions for analysis in a
vacuum system while removing the HPLC mo-
bile phase proved to be a challenging task. Early
LC/MS techniques included a moving belt in-
terface to desolvate and transport the HPLC
eluate into a CI or EI ion source, or a direct inlet
system in which the eluate was pumped at a low
flow rate of 1 to 3 µl/min into a CI source.
However, neither of these systems was robust
enough or suitable for a broad enough range of
samples to gain widespread acceptance.
Since FAB (or LSIMS) requires that the
analyte be dissolved in a liquid matrix, this
ionization technique was easily adapted for
infusion of solution-phase samples into the
FAB ionization source, in an approach known
as continuous-flow FAB. Continuous-flow
FAB was connected to microbore HPLC col-
umns for LC/MS applications (Ito et al., 1985).
Since this method is limited to microbore
HPLC applications at flow rates of <10 µl/min
Current Protocols in Food Analytical Chemistry
and requires considerable operator interven-
tion, it is not ideal for the analysis of large
sample sets. Instead, more robust techniques
have been developed to fulfill this requirement.
However, continuous-flow FAB is still in use
in some laboratories.
Like continuous-flow FAB, the popularity
of particle beam interfaces is diminishing, but
systems are still available from commercial
sources. During particle beam LC/MS, the
HPLC eluate is sprayed into a heated chamber
connected to a vacuum pump. As the droplets
evaporate, aggregates of analyte (particles)
form and pass through a momentum separator
that removes the lower-molecular-weight sol-
vent molecules. Finally, the particle beam en-
ters the mass spectrometer ion source where the
aggregates strike a heated plate from which the
analyte molecules evaporate and are ionized
using conventional EI or CI ionization. Particle
beam LC/MS is limited to the analysis of vola-
tile and thermally stable compounds that are
amenable to flash evaporation and EI or CI
mass spectrometry. Therefore, this approach is
not used for polar compounds in food chemistry
such as carbohydrates, sugars, peptides, pro-
teins, or nucleic acids (Fig. A.3A.2).
Since thermospray became the first widely
utilized LC/MS technique (during the late
1970s and early 1980s), this technique should
be mentioned here. Thermospray facilitates the
interfacing of standard analytical HPLC sys-
tems at flow rates up to 1 ml/min with mass
spectrometers. Although the interface between
the HPLC and mass spectrometer is inefficient
and exhibits low sensitivity for most analytes,
thermospray has been useful for the LC/MS
analysis of many types of small molecules.
During thermospray, the HPLC eluate is
sprayed through a heated capillary into a heated
desolvation chamber at reduced pressure. Gas
phase ions remaining after desolvation of the
droplets are extracted through a skimmer into
the mass spectrometer for analysis. The sensi-
tivity of thermospray is poor since there is no
mechanism or driving force to enhance the
number of sample ions entering the gas phase
from the spray during desolvation. Also, ther-
mally labile compounds tend to decompose in
the heated source. These problems were solved
when thermospray was replaced by elec-
trospray during the late 1980s.
During the 1990s, electrospray ionization
(ESI) and atmospheric pressure chemical ioni-
zation (APCI) became the standard interfaces
for LC/MS. Unlike thermospray, particle beam, Commonly Used
or continuous-flow FAB, ESI and APCI inter- Techniques
A.3A.3
Supplement 2
 faces operate at atmospheric pressure and do
not depend upon vacuum pumps to remove
solvent vapor. As a result, they are compatible
with a wide range of HPLC flow rates. Also, no
matrix is required. Both APCI and ESI are
compatible with a wide range of HPLC col-
umns and solvent systems. Like all LC/MS
systems, the solvent system should contain
only volatile solvents, buffers, or ion-pair
agents, to reduce fouling of the mass spec-
trometer ion source. In general, APCI and ESI
form abundant molecular ion species (Figures
A.3A.1 and A.3A.2). When fragment ions are

100

computer-reconstructed
mass chromatogram of m /z 269
%

500

m/z

150
8 12 16 20 24 28
Retention time (min)
100
269 [M−H]−
Relative abundance

mass spectrum
at 12.4 min
%

189
0
100 140 180 220 260 300 340
m /z

Figure A.3A.3 LC/MS analysis of a dietary supplement consisting of extract of Trifolium pratense
Introduction to (red clover). Reversed-phase C18 HPLC and negative ion electrospray ionization mass spectrome-
Mass try were used with a quadrupole mass spectrometer analyzer (Agilent; also see Table A.3A.1). The
Spectrometry for map illustrates the abundance of information provided by this hyphenated technique with HPLC
Food Chemistry
mass chromatograms in one dimension and mass spectra in another dimension.
A.3A.4
Supplement 2 Current Protocols in Food Analytical Chemistry
formed, they are usually more abundant in
APCI than ESI mass spectra.
The APCI interface uses a heated nebulizer
to form a fine spray of the HPLC eluate, which
is much finer than the particle beam system but
similar to that formed during thermospray. A
cross-flow of heated nitrogen gas is used to
facilitate the evaporation of solvent from the
droplets. The resulting gas-phase sample mole-
cules are ionized by collisions with solvent
ions, which are formed by a corona discharge
in the atmospheric pressure chamber. Molecu-
lar ions, M+. or M−., and/or protonated or de-
protonated molecules can be formed. The rela-
tive abundance of each type of ion depends
upon the sample itself, the HPLC solvent, and
the ion source parameters. Next, ions are drawn
into the mass spectrometer analyzer for meas-
urement through a narrow opening or skimmer,
which helps the vacuum pumps to maintain
very low pressure inside the analyzer while the
APCI source remains at atmospheric pressure.
During ESI, the HPLC eluate is sprayed
through a capillary electrode at high potential
(usually 2000 to 7000 V) to form a fine mist of
charged droplets at atmospheric pressure. As
the charged droplets migrate towards the open-
ing of the mass spectrometer due to electrostatic
attraction, they encounter a cross-flow of
heated nitrogen that increases solvent evapora-
tion and prevents most of the solvent molecules
from entering the mass spectrometer. Molecu-
lar ions, protonated or deprotonated molecules,
and cationized species such as [M+Na]+ and
a
ion b MS
source analyzer 1
c
sample
compounds a and
b and impurity c
Figure A.3A.4 Scheme illustrating the selectivity of MS/MS and the process by which collision-
induced dissociation (CID) facilitates fragmentation of preselected ions.
Current Protocols in Food Analytical Chemistry
[M+K]+ can be formed (for additional informa-
tion on ESI, see Cole, 1997). In addition to
singly charged ions, ESI is unique as an ioni-
zation technique in that multiply charged spe-
cies are common and often constitute the ma-
jority of the sample ion abundance. The relative
abundance of each of these species depends
upon the chemistry of the analyte, the pH, the
presence of proton-donating or -accepting spe-
cies, and the levels of trace amounts of sodium
or potassium salts in the mobile phase. In con-
trast, APCI, MALDI, EI, CI, and FAB/LSIMS
usually produce singly charged species. A con-
sequence of forming multiply charged ions is
that they are detected at lower m/z values (i.e.,
|z| >1) than the corresponding singly charged
species. This has the benefit of allowing mass
spectrometers with modest m/z ranges to detect
and measure ions of molecules with very high
masses. For example, ESI has been used to
measure ions with molecular weights of hun-
dreds of thousands or even millions of daltons
on mass spectrometers with m/z ranges of only
a few thousand (for a review of LC/MS tech-
niques, see Niessen, 1999).
An example of the LC/MS analysis of a plant
extract is shown in Figure A.3A.3. In this case,
negative ion ESI-MS was used in combination
with C18 reversed-phase HPLC separation. Ex-
tracts of the botanical Trifolium pratense (red
clover) are used as dietary supplements by
menopausal and post-menopausal women (Liu
et al., 2001). The two-dimensional map illus-
trates the amount of information that may be
b
CID
MS
analyzer 2
M+⋅
m/z
Commonly Used
Techniques
A.3A.5
Supplement 2
 acquired using hyphenated techniques such as
LC/MS. In the time dimension, chromatograms
are obtained and a sample computer-recon-
structed mass chromatogram is shown for the
signal at m/z 269. One intense chromatographic
peak was detected in this chromatogram eluting
at 12.4 min. In the m/z dimension, the negative
ion electrospray mass spectrum recorded at
12.4 min shows a base peak at m/z 269. Based
on comparison to authentic standards (data not
shown), the ion of m/z 269 was shown to cor-
respond to the deprotonated molecule of
genistein, which is an estrogenic isoflavone
(Liu et al., 2001). Since almost no fragmenta-
tion of the genistein ion was observed, addi-
tional characterization would require collision-
induced dissociation (CID) and tandem mass
spectrometry as discussed in the next section.
TANDEM MASS SPECTROMETRY
(MS/MS) AND HIGH RESOLUTION
Desorption ionization techniques like FAB
and MALDI and LC/MS ionization techniques
like ESI and APCI facilitate the molecular
weight determination of a wide range of polar
and nonpolar, low- and high-molecular-weight
compounds. However, the “soft” ionization
character of these techniques means that most
of the ion current is concentrated in molecular
ions and few structurally significant fragment
ions are formed. In order to enhance the amount
of structural information in these mass spectra,
collision-induced dissociation (CID) may be
used to produce abundant fragment ions from
molecular ion precursors formed and isolated
during the first stage of mass spectrometry.
Then, a second mass spectrometry analysis may
be used to characterize the resulting product
ions. This process is called tandem mass spec-
trometry or MS/MS and is illustrated in Figure
A.3A.4.
Another advantage of the use of tandem
mass spectrometry is the ability to isolate a
particular ion such as the molecular ion of the
Table A.3A.1 Types of Mass Spectrometers and Tandem Mass Spectrometersa
Instrument Resolution
Magnetic sector 100,000
Quadrupole <4,000
Triple quadrupole <4,000
TOF 15,000
FTICR >200,000
Introduction to QTOF 12,000
Mass
Spectrometry for TOF-TOF 15,000
Food Chemistry aFTICR, Fourier transform ion cyclotron resonance; QTOF, quadropole time-of-flight; TOF, time-of-flight.
A.3A.6
Supplement 2
analyte of interest during the first mass spec-
trometry stage. This precursor ion is essentially
purified in the gas phase and is free of impuri-
ties such as solvent ions, matrix ions, or other
analytes. Finally, the selected ion is fragmented
using CID and analyzed using a second mass
spectrometry stage. In this manner, the result-
ing tandem mass spectrum contains exclusively
analyte ions without impurities that might in-
terfere with the interpretation of the fragmen-
tation patterns. In summary, CID may be used
with LC/MS/MS or desorption ionization and
MS/MS to obtain structural information such
as amino acid sequences of peptides and sites
of alkylation of nucleic acids, or to distinguish
structural isomers such as β-carotene and ly-
copene.
The most common types of MS/MS instru-
ments available to researchers in food chemis-
try include triple quadrupole mass spectrome-
ters and ion traps. Less common but commer-
cially produced tandem mass spectrometers
include magnetic sector instruments, Fourier
transform ion cyclotron resonance (FTICR)
mass spectrometers, and quadrupole time-of-
flight (QTOF) hybrid instruments (Table
A.3A.1). Beginning in 2001, TOF-TOF tandem
mass spectrometers became available from in-
strument manufacturers. These instruments
have the potential to deliver high-resolution
tandem mass spectra with high speed and
should be compatible with the chip-based chro-
matography systems now under development.
In addition to MS/MS with CID to obtain
structural information, it is also useful to use
high-resolution exact mass measurements to
confirm the elemental compositions of ions.
Essentially, exact mass measurements permit
the unambiguous composition analysis of low-
molecular-weight compounds (mol. wt. <500)
through precise and accurate m/z measure-
ments. The types of mass spectrometers capa-
ble of exact mass measurements include mag-
netic sector mass spectrometers, QTOF hybrid
m/z Range Tandem MS
12,000 Low resolution
4,000 None
4,000 Low resolution
>200,000 None
<10,000 High resolution
4,000 High resolution
>10,000 High resolution
Current Protocols in Food Analytical Chemistry
mass spectrometers, reflectron TOF instru-
ments, and FTICR mass spectrometers (Table
A.3A.1). Some of these instruments permit the
simultaneous use of tandem mass spectrometry
and exact mass measurement of fragment ions.
These include FTICR instruments, QTOF, and
the TOF-TOF.
CONCLUSION
Mass spectrometry has become an essential
analytical tool for a wide variety of biomedical
applications such as food chemistry and food
analysis. Mass spectrometry is highly sensitive,
fast, and selective. By combining mass spec-
trometry with HPLC, GC, or an additional stage
of mass spectrometry (MS/MS), the selectivity
increases considerably. As a result, mass spec-
trometry may be used for quantitative as well
as qualitative analyses. In this manual, mass
spectrometry is mentioned frequently, and ex-
tensive discussions of mass spectrometry ap-
pear, for example, in units describing the analy-
ses of carotenoids (UNIT F2.4) and chlorophylls
(UNIT F4.5). In particular, these units include
examples of LC/MS and MS/MS and the use
of various ionization methods.
LITERATURE CITED
Barber, M., Bordoli, R.S., Elliott, G.J., Sedgwick
R.D., and Tyler, A.N. 1982. Fast atom bombard-
ment mass spectrometry. Anal. Chem. 54:645A-
657A.
Cole, R.B. (ed.). 1997. Electrospray Ionization
Mass Spectrometry. John Wiley & Sons, New
York.
Field, F. 1990. Early days of chemical ionization. J.
Am. Soc. Mass Spectrom. 1:277-283.
Hillenkamp, F., Karas, M., Beavis, R.C., and Chait,
B.T. 1991. Matrix-assisted laser desorption/ioni-
zation mass spectrometry of biopolymers. Anal.
Chem. 63:1193A-1203A.
Current Protocols in Food Analytical Chemistry
Ito, Y., Takeuchi, T., Ishii, D., and Goto, M. 1985.
Direct coupling of micro high-performance liq-
uid chromatography with fast atom bombard-
ment mass spectrometry. J. Chromatogr.
346:161-166.
Liu, J., Burdette, J.E., Xu, H., Gu, C., van Breemen,
R.B., Bhat, K.P.L., Booth, N., Constantinou,
A.I., Pezzuto, J.M., Fong, H.H.S., Farnsworth,
N.R., and Bolton, J.L. 2001. Evaluation of estro-
genic activity of plant extracts for the potential
treatment of menopausal symptoms. J. Agric.
Food Chem. 49:2472-2479.
McLafferty, F.W. and Turecek, F. 1993. Interpreta-
tion of Mass Spectra, 4th ed. University Science
Books, Mill Valley, Calif.
Niessen, W.M. 1999. State-of-the-art in liquid chro-
matography-mass spectrometry. J. Chromatogr.
A 856:179-189.
Watson, J.T. 1997. Introduction to Mass Spectrome-
try, 3rd ed. Lippincott-Raven, Philadelphia, Pa.
KEY REFERENCES
McLafferty and Turecek, 1993. See above.
This classic text describes fragmentation pathways
and mechanisms for ions formed using electron
impact (EI) ionization. In addition, this edition con-
tains additional information regarding desorption
ionization and the corresponding related fragmen-
tation mechanisms.
Watson, 1997. See above.
This textbook provides an overview of biomedical
mass spectrometry with particular emphasis on
GC/MS and quantitative methods. In addition, de-
scriptions are provided of the various types of mass
spectrometers and ionization techniques that are
used for biomedical applications.
Contributed by Richard B. van Breemen
University of Illinois at Chicago
Chicago, Illinois
Commonly Used
Techniques
A.3A.7
Supplement 2