Anal Bioanal Chem (2012) 403:12331249
DOI 10.1007/s00216-011-5629-4
REVIEW
The current role of high-resolution mass spectrometry
in food analysis
Anton Kaufmann
Received: 18 October 2011 / Revised: 28 November 2011 / Accepted: 29 November 2011 / Published online: 17 December 2011
# Springer-Verlag 2011
Abstract High-resolution mass spectrometry (HRMS),
which is used for residue analysis in food, has gained wider
acceptance in the last few years. This development is due to
the availability of more rugged, sensitive, and selective instru-
mentation. The benefits provided by HRMS over classical
unit-mass-resolution tandem mass spectrometry are consider-
able. These benefits include the collection of full-scan spectra,
which provides greater insight into the composition of a
sample. Consequently, the analyst has the freedom to measure
compounds without previous compound-specific tuning, the
possibility of retrospective data analysis, and the capability of
performing structural elucidations of unknown or suspected
compounds. HRMS strongly competes with classical tandem
mass spectrometry in the field of quantitative multiresidue
methods (e.g., pesticides and veterinary drugs). It is one of
the most promising tools when moving towards nontargeted
approaches. Certain hardware and software issues still have to
be addressed by the instrument manufacturers for it to dis-
lodge tandem mass spectrometry from its position as the
standard trace analysis tool.
Keywords High-resolution mass spectrometry . Food
analysis . Oribitrap . Time-of-flight mass spectrometry .
Residue analysis
Published in the special issue High-Resolution Mass Spectrometry with
guest editors Hans H. Maurer and David C. Muddiman.
A. Kaufmann (*)
Official Food Control Authority,
Fehrenstrasse 15,
8032 Zurich, Switzerland
e-mail: anton.kaufmann@klzh.ch
A. Kaufmann
Kantonales Labor Zrich,
Fehrenstrasse 15,
8032 Zurich, Switzerland
Introduction
History of high-resolution mass spectrometry in food
analysis
High-resolution mass spectrometry (HRMS) has long been
used in food analysis, but until recently it was associated
with high instrumental complexity. Therefore, HRMS has
been limited to the most demanding applications, such as the
analysis of natural organic matter or dioxin-related com-
pounds. The appearance of modern HRMS instruments such
as time-of-flight (TOF) and Orbitrap instruments has funda-
mentally changed this situation. Consequently, the capabil-
ities and limitations of current HRMS techniques in the field
of food analysis must be reevaluated.
Coupled with gas chromatography (GC), HRMS has been
the core technology utilized for the analysis of halogenated
dioxin residues in food and the environment [1, 2]. It is inter-
esting to note that for some 30 years the same type of mass
spectrometer (sector-field instrument) has been utilized. This
choice of instrumentation was obvious during the early days of
dioxin analysis. The complexity of matrices and the required
low detection limits (around 1 ppt) forced analysts to utilize this
capital-, operator-, and know-how-intensive mass spectrometry
(MS) technology. The high mass defect of ions containing
halogen atoms relative to endogenous nonhalogenated ions
produced a very selective detection even in complex and chal-
lenging samples. The complexity of the sampling, extraction,
cleanup, separation, and detection processes involved led to the
definition and international acceptance of norms such as EPA
method 1613. Considering the technological progress in ana-
lytical chemistry, it is remarkable that the community of dioxin
analysts is still using the same type of instrument it accepted
three decades earlier. On the other hand, analysts not involved
in dioxin analysis were not easily convinced of the suitability
and ruggedness of HRMS in general and sector-field MS in
1234
particular for their analytical problems. Until recently, the
HRMS technique was probably considered too cumbersome
for non-dioxin-related work.
The analysis of persistent halogenated pesticide residues in
food and in the environment was initially performed with GC
and an electron capture detector. The widening range of com-
pounds included in analysis led to the use of nitrogen- and
phosphorous-specific detectors and then to single-stage low-
resolution MS. Electron ionization (EI) was and still is the
most commonly used type of GCMS ionization. Additional
selectivity was obtained by chemical ionization techniques.
The importance of monitoring very polar and thermolabile
pesticides as well as derived degradation products led to the
investigation of liquid chromatography (LC). Researchers
used single-stage ion trap or single-stage quadrupole instru-
ments or even non-MS-based detection [36]. Single-stage
LCMS was utilized for relatively simple matrices such as
water, but seldom for interference-prone samples such as fruits
and vegetables. Currently, analysis of more and more analytes
has been transferred from the GC-based to the LC-based meth-
ods. It seems that only analytes that cannot be ionized by
electrospray ionization (ESI) or atmospheric pressure chemical
ionization (APCI) are now analyzed by GC methods. On the
other hand, overlapping analytes which can be determined by
GC and LC add a greater degree of dependability to the results.
Because LCMS/MS was extensively accepted within a few
years, many analysts welcomed the switch from the GCMS to
the GCMS/MS method as well. The acceptance of the latter
technology can be understood by the need for additional selec-
tivity (caused by the lowering of detection limits and the
reluctance to employ an extensive sample cleanup) as well as
the familiarity with LCMS/MS. Users of the latter are familiar
with selecting precursor and product ion masses. However, the
precursor ion mass in LCMS most often corresponds to the
molecular mass of the analyte molecule plus a proton (positive
ionization) or minus a proton (negative ionization). This guide-
line no longer exists for GCMS/MS, since the commonly
utilized EI is a hard ionization technique causing extensive
fragmentation during the ionization process. This aspect might
be less relevant if chemical or cold ionization techniques are
used. Still, more often than not, the user has to select one of the
fragments produced as a precursor and must optimize the
conditions for a suitable product ion as a second fragmentation.
The straightforwardness of such an approach is somewhat
questionable. There is a loss of absolute signal intensity (sensi-
tivity) that is normally more than compensated by the higher
selectivity provided by MS/MS (signal to noise improves). The
process of fragmenting a relatively small molecule during
ionization and then fragmenting again the resulting EI ions in
the collision chamber produces even smaller ions with limited
diagnostic quality. Hence, the overall selectivity is lower than
that derived from ESI-based LCMS/MS. Although until very
recently GCHRMS was not used to any relevant extent by
A. Kaufmann
pesticide analysts [7, 8], attempts were made to introduce TOF
spectrometry in GC [7, 8]. However, TOF spectrometry be-
came more popular in the field of GC because of the fast scan
speed rather than the mass resolution. Higher than unit mass
resolution GCMS detectors are yet to become commercially
successful. As a consequence, only a few articles have focused
on this otherwise promising technology [9]. This situation has
changed with the very recent commercial introduction of sev-
eral new types of HRMS-based GCTOF instrumentation.
They will likely have a relevant impact in the field of GC
MS. Generally, there is a much wider choice of commercially
available HRMS detectors which can be readily coupled to LC
systems than to GC systems. This is clearly reflected by the
number of published articles.
Current use of high-resolution mass spectrometry in different
fields of food analysis
LCHRMS has become increasingly popular in the field of
pesticide analysis [7, 8] within the last few years. This
development was initiated by an ever-increasing number
of active compounds and derived degradation products to
be monitored within a single analytical method.
The analysis of marine biotoxinssuch as paralytic shell-
fish poisoning toxins, diarrheic shellfish poisoning toxins,
domoic acid, tetrodotoxins, ciguatera, and azaspiracidshas
a unique history in that it relied until very recently on biolog-
ical tests. The mice assay utilized is based on the survival rate
of mice into which shell extracts have been injected. The high
number of false-positive and false-negative findings as well as
the questionable ethics of such an assay resulted in pressure to
switch to alternative detection principles. However, this process
was not straightforward since reference substances are not only
very expensive but also often consist of a number of isomeric
analogues. Their limited availability as reference material and
the assumption that toxicity varies significantly from analogue
to analogue made the development of LCMS/MS methods
difficult. This is probably the reason why HRMS seems to be
the preferred technique for the analysis of marine toxins
[1012].
Many mycotoxins have to be detected at a very low level,
requiring a good cleanup and high-sensitivity detection. The
use of very specific but expensive and time-consuming immu-
noassasy steps can be circumvented by employing LCMS/
MS instead of classical LC-based detection (e.g., fluorescence
or COBRA cell). HRMS is not yet widely used for the
analysis of mycotoxins. However, the limited availability of
many mycotoxin reference materials will make this technique
attractive soon [1315]. Furthermore, HRMS facilitates the
monitoring of the so-called masked mycotoxins [16].
Veterinary drug residues have long been analyzed by immu-
noassay tests or by LCfluorescence detection. The relatively
high molecular weight of these compounds and their high
The current role of high-resolution mass spectrometry in food analysis
polarity required a derivatization step prior to their injection
into the gas chromatograph, which led to the early use of LC
MS technology. Ion trap instruments were known for their
relatively low speed and their limited quantification capabilities
when utilized for trace level analysis of difficult matrices such
as liver and kidney. On the other hand, single-stage quadrupole-
based LCMS instrumentation produced insufficient selectivity
and correspondingly poor sensitivity. As a consequence, LC
MS/MS was investigated earlier than in many other analytical
fields. This process was accelerated by the extensive use of
LCMS/MS in the pharmaceutical industry, which led to the
availability of suitable hardware and software. HRMS was only
recently introduced by analysts to cope with the large number
of active compounds and derived metabolites that required
detection and quantification. This can be partially explained
by the compelling use of validation and confirmation protocols
(2002/657/EC) that were written with a strong focus on MS/
MS technology [5, 17].
The analysis of organic contaminants originating from
the environment has an extensive history of using GC as the
separation technique. The initial electron capture detectors
were replaced by single-stage and then triple quadrupole
(QqQ) detectors. Probably more than any other analytical
discipline, comprehensive GC (GCGC) has gained accep-
tance, which is due to the complexity of the matrix and the
attempted limits of detection. However, GCGC is currently
not widely used as a routine technique. Again, LCHRMS
and to a certain degree GCHRMS have been used by a
number of authors [1, 2, 9]. There is increasing interest in
the field of water utility in using the latest generation of
single-stage LCHRMS technology [18] because of its suf-
ficient full-scan sensitivity. This permits monitoring of un-
expected contaminants (chemical spill) at relevant
concentrations.
Large, biologically active molecules such proteins with
allergenic potential are most often analyzed by bioassays
such as the enzyme-linked immunosorbent assay. There is a
clear trend towards nonbioassays such as LCMS/MS and
LCHRMS. This development is clearly observable for the
detection of allergens in food [19].
The plethora of HRMS articles published in recent years
was motivated by several factors. The increasing number of
analytes covered by a single analytical method (multiresidue
method) makes the use of a full-scan method an attractive
option. In addition, the lack of commercial availability of
some reference substances (e.g., active compounds, degra-
dation products, and metabolites) requires instrumentation
that does not need previous individual compound-specific
instrument tuning. Finally, HRMS is a powerful tool for
semitargeted or nontargeted screening while not only en-
abling the analyst to detect suspected peaks, but also helping
in identifying and confirming the underlying chemical
structure.
1235
Screening techniques
Advantages of high-resolution mass spectrometry
for screening techniques
Screening is intended to produce a yesno answer, whether a
single or a set of different compounds is present in a large
number of samples. The focus is on speed and often also on
cost-effectiveness. Although quantification capabilities might
be desired, the focus is on a manageable number of false-
positive findings at the relevant concentrations and the absence
of false-negative results. Screening techniques can be either
targeted or nontargeted. Targeted approaches focus on a pre-
defined list of compounds to be tested for. The ideal nontar-
geted approaches should be capable of detecting any unknown
exogenous compound in a given sample. Given the complexity
of such an approach, the vast majority of published screening
approaches are still targeted techniques.
The high level of selectivity provided by MS (as compared
with LCUV detection or GCflame ionization detection)
permitted the monitoring of lower analyte concentrations in
complex matrices without producing an excessive number of
false-positive findings. The search for even higher selectivity
led to MS/MS-based targeted multiresidue screening techni-
ques. Such approaches became feasible with modern MS/MS
instruments capable of monitoring selected reaction monitor-
ing (SRM) traces with very short dwell times. Still, analysts
have to strike a compromise between the number of transitions
to be monitored, the length of the dwell times, and the number
of data points across a chromatographic peak. The number of
compounds to be monitored could be increased up to 1,000 by
using high-end instruments and the application of retention-
time-window-based SRM traces. It is obvious that this tech-
nique requires knowledge of the exact retention time of every
screened analyte. This becomes a serious limitation when the
number of compounds extends beyond 200. The user is then
forced to test the correctness of the expected retention time by
injecting standard solutions. Retention times often show nei-
ther an absolute nor a consistent relative stability. Retention
times of acid or alkaline compounds may drift relative to those
of neutral compounds. Hence, SRM-based retention time
windows not only must be set up carefully but also might
have to be readjusted during a long sample series. The com-
bined difficulties associated with managing and monitoring a
large number of SRM traces motivated the shift towards full-
scan-based HRMS screening. Furthermore, a positive finding
very often raises the question of additional information. Typ-
ical examples are the tentative identification of a suspected
drug that is known to be often co-administered with another
drug (e.g., trimethoprim, which is used in combination with
sulfonamides) or the detection of a pesticide that is known to
produce a degradation product at a relevant concentration.
Furthermore, there are occasions where it might be of interest
1236
to know if a newly found residue was already present in
similar samples analyzed a year ago. Such information is
stored within the full-scan data on the hard disk. Access to
existing HRMS data files can quickly provide answers to such
questions and permit the formulation of a posteriori hypothe-
ses. This is a significant advantage over MS/MS-based
approaches, in which the analyst normally needs to have
physical access to reference material before determining the
correct MS/MS settings, which finally enables the analyst to
build an acquisition method.
Selectivity issues
HRMS permits selectivity for the monitoring of compounds
that share the same nominal mass but differ in their elemental
composition and therefore have a different exact mass. Many
reports in the literature described the use of a narrow mass
window to improve selectivity [20, 21]. Figure 1 shows the
chromatogram of a bovine liver extract spiked with marboflox-
acin [21]. The detection relied on monitoring the [M+H]+ion by
HRMS (m 10,000 full width at half maximum, FWHM).
Different mass window widths were applied. A first look at
Fig. 1 might lead to the conclusion that the best results (selec-
tivity) appear to be obtained when the narrowest mass window
width is used. Yet selectivity is not simply the product of the
narrowness of the defined mass window. Selectivity of detec-
tion in complex matrices is only ensured when the HRMS
instrument can physically resolve the analyte mass from that
of other coeluted isobaric matrix compounds. Figure 1 should
therefore not be interpreted as demonstrating that selectivity
improves when reducing the mass extraction window width.
This will be more clearly illustrated by another compound
(norfloxacin), which was monitored in the same spiked extract
[21]. The observed accurate mass deviated slightly from the
calculated exact mass when an intermediate mass window and
a resolution of 10,000 FWHM were used. Figure 2 shows the
observed chromatogram of marbofloxacin in the liver extract
where a 2-mDa mass window was applied. Even such a very
narrow mass window did not reduce the number of interfering
peaks as extensively (chromatogram at the top) as observed for
norfloxacin in Fig. 1. Furthermore, there is a clear distortion of
the mass signal corresponding to marbofloxacin (amplified
chromatogram at the bottom). The splitting of the peak was
caused by a single scan which recorded the mass peak of
marbofloxacin outside the defined mass window. Use of a
significantly higher resolution of 100,000 FWHM revealed
the presence of an isobaric matrix peak (Fig. 3). Close inspec-
tion of a deviating single scan, obtained at 10,000 FWHM
(Fig. 2), showed an accurate mass of 320.13855m/z, which
deviated by 6 ppm from the theoretical value of 320.14047m/z.
This can be traced to the presence of a coeluted interfering
endogenous compound. The observed mass error is also de-
pendent on the relative mass peak intensity and the centroiding
A. Kaufmann
algorithm utilized [22]. Applying too narrow a mass window
around the exact mass of norfloxacin could even cause the
complete disappearance of this signal when the measured
signal lies outside the defined mass range. Hence, there is a
risk of reporting false-negative findings if mass window widths
narrower than the resolving capability of the instrument utilized
are defined [2024].
Some studies have addressed the issue of the mass resolu-
tion required for trace analysis in food matrices [20, 21, 23].
Resolutions of 50,000 or more FWHM were suggested for
low levels of analytes in complex matrices such as animal feed
[20]. The same authors concluded that a resolving power of
25,000 FWHM is sufficient for less complex matrices (e.g.,
honey). Their conclusion was based on experiments that in-
volved analyzing matrix samples spiked at low levels with
various veterinary drugs, mycotoxins, and pesticides. The
absence of isobaric interferences was concluded when the
detected analytes showed mass deviations below 2 ppm at a
concentration of some 10 ng/g [20]. On the basis of experi-
ments with synthetic hormones, screening resolutions of
10,000 or more FWHM with mass windows of 50 mDa were
suggested [23]. The authors concluded that only proposed
resolutions of 70,000 or more FWHM led to reliable accurate
masses of elemental compositions differing in one CO, C2H4,
or N2 substructure.
A recent study attempted to determine the mass resolu-
tion and corresponding mass window width required to
obtain selectivity comparable to that of MS/MS [21]. This
benchmark was chosen because MS/MS is considered by
many mass spectrometrists as the gold standard that must be
met by any possible competing technique. The authors did
not focus on the question of whether the detection of a
particular exogenous analyte present at trace levels is
masked or impaired by endogenous compounds originating
from the matrix. They instead investigated the vacant m/z
space available in MS/MS and HRMS spectra of an ana-
lyzed blank honey matrix extract (honey is considered to be
a rather complex matrix). The idea behind this concept was
the assumption that monitoring a large number of such
randomly selected traces shows a number of traces contain-
ing one or more chromatographic peaks caused by endoge-
nous matrix compounds. Measuring the number and the
intensity of the detected peaks provided information regard-
ing the degree of detector selectivity obtained. A statistical
sampling procedure was developed, since it is not possible
to monitor all theoretically possible MS/MS transitions and
HRMS-resolved masses in a blank matrix. This procedure
consisted of the guided calculation of 100 so-called dummy
transitions and dummy exact masses. MS/MS transition
sampling restrictions were applied to permit only a precur-
sor mass range between 150 and 600m/z and a product ion
range between 70m/z and the mass of the precursor ion
minus 18 (corresponding to the neutral loss of water). Random
The current role of high-resolution mass spectrometry in food analysis 1237

RT: 0.00 - 15.60

2.46
2 46 NL:
NL
100 1.51E6
90

80
1.76 500 mDa m/z=
362.64628-
363.64628
5.44 MS
70 09_10_08_
Relattive Abu ndance

79
60 6.18

50
5.33
40 4.47 13.08
13 08
30 1.08
5 88
5.88
20 11 89
11.89
6.54
13.40
10 6.93 13.02
3 21 3
3.21 88
3.88 9 13.79
0.99 7 24
7.24 8.74 47
9.47 10 58 11.80
10.58 11 80 14 50
14.50
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (min)

RT: 0.00 - 15.60

2.46
2 46 NL:
100 1.51E6
m/z=

50 mDa
90
363.09628-
1.76
80 363.19628
MS
70 09_10_08_
Relattive Abu ndance

79
60
6.18
50

40

30

20

10 5.74
1.53 3.27 4.49 6.41 7.24 7.57 8.68 9.42 10.26 10.90 11.46 13.08 13.56 14.50
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (min)
RT: 0.00 - 15.60
6.18 NL:
100 8.21E5
m/z=
90

80
2 mDa 363.14428-
363.14828
MS
70 09_10_08_
09 10 08
ce
bundanc

79
60
Rellative Ab

50

40

30

20

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (min)

Fig. 1 Chromatogram of a bovine liver extract spiked with marbo- hal maximum (FWHM) (single-stage time-of-flight, TOF, instrument).
floxacin at 50 g/l. Shown is the exact mass of the [M+H]+ ion of The mass window was varied from 500 mDa to 2 mDa. Narrowing the
marbofloxacin at 363.14628 with a resolution of 10,000 full width at mass extraction window reduces the number of interfering peaks

sampling was permitted only within this defined transition was applied). Furthermore, it was taken into account that,
space. A corresponding mass range was defined for the ex- typically, differences between accurate and nominal masses
traction of ions to be determined by HRMS (no fragmentation are small and correlate with the mass of the ion. In addition,
1238 A. Kaufmann

RT: 0.00 - 15.60

3.72 NL:
100 9.95E4
90 m/z=
320.13847-
80 320.14247
MS
70 09_10_08_
ance

79
Abunda

60
6.40
50
Relative

40
R

30 5 00
5.00

20
5 95
5.95
10 10.45
8.07 9.92
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time ((min))

RT: 6.08 - 6.55

6 40
6.40 NL:
100 5.30E4
6.39 6.39
90 m/z=
320 13847
320.13847-
80 320.14247
6.40 MS
70 09 10 08
09_10_08_
undance

79
60
ative Abu

50

40
Rela

30 6.41

20

10

0
6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 6.55
Time (min)

Fig. 2 The same liver extract as shown in Fig. 1 was spiked with caused by endogenous matrix compounds (top). The amplified analyte
50 g/l norfloxacin. Even a narrow mass window (2 mDa) at the given peak (bottom) shows serious peak distortions
resolution (10,000 FWHM) was not sufficient to remove signals

the mass window width utilized was adjusted to correspond to
the four resolution settings investigated (50 ppm with 10,000
FWHM, 20 ppm with 25,000 FWHM, 10 ppm with 50,000
FWHM, and 5 ppm with 100,000 FWHM). The interrelation
of these two parameters was discussed above. As expected,
most extracted dummy traces were free of chromatographic
peaks. The dummy traces which contained chromatographic
peaks were integrated. In the next step, the SRM and HRMS
peak areas obtained were standardized. This was done by
determining an average peak area/concentration response of
some ten different antibiotics for the two MS techniques
investigated. Each SRM and accurate mass peak area was
standardized with this average peak area/concentration re-
sponse. This permitted a direct comparison between SRM-
and HRMS-based chromatographic signal intensities. Figure 4
compares the selectivity of the two techniques based on this
approach. The standardized concentrations of every detected
peak based on the monitored dummy signal intensities (y-axis)
are shown. The x-axis represents the chromatographic reten-
tion time at which the corresponding compound is eluted. The
four windows at the top refer to HRMS measurements
obtained with the four mass window resolution settings
The current role of high-resolution mass spectrometry in food analysis 1239

Norfloxacin
09_10_08_28 #473-476 RT: 6.37-6.41 AV: 4 NL: 1.75E4
T: FTMS {0,0} + p ESI Full ms [85.00-1000.00]
320.14050
100

90

80

Matrix
undan ce

70

60
Relativve Abu

50

40 320 16062
320.16062

30

20

10 319.51269 319.70224 319.92961

0
09_10_08_79
319.5 #4131319.6RT: 6.39 AV: 1 NL: 4.43E4
319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4
T: FTMS {0
{0,0}
0} + p ESI Full ms [85
[85.00-1000.00]
00 1000 00]
m/z
320.13855
100

90

80
dance

70

60
elative Abund

50

40
Re

30

20

10

0
319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4
m/z

Fig. 3 Spectra taken from the same separation as shown in Fig. 2. The separation of the analyte ion from an isobaric matrix compound. The
instrument utilized was a single-stage Orbitrap. The norfloxacin signal resolution of 10,000 FWHM does not resolve these two ions. Depend-
in liver extract was resolved at 100,000 FWHM (top) and 10,000 ing on the mass windows utilized, false-negative findings or inflated
FWHM (bottom). The higher-resolution setting permits the clear peak areas will be observed

described. The reduction of the number and concentration
of false-positive peaks in the blank honey extract inves-
tigated is clearly visible. The graph at the bottom repre-
sents the SRM performance. It can be concluded that an
HRMS resolution of 50,000 FWHM and a corresponding
mass window of 10 ppm provide selectivity as good as or
slightly higher than MS/MS. In the case of the honey
matrix investigated, QqQ selectivity seems to be poor for
late-eluted compounds. This is visible by the number of
rather intense peaks corresponding to compounds eluted in
the retention time region from 12 to 14 min (Fig. 4). It
has to be added that MS/MS provides the possibility to
achieve a higher selectivity by monitoring additional tran-
sitions. However, additional transitions seldom truly add
an orthogonal selectivity contribution [21]. Furthermore,
adding additional transitions consumes valuable dwell time
resources and significantly reduces the sensitivity for poor-
ly fragmenting analytes.
The latest generation of Orbitrap instrumentation pro-
vides resolutions well above 100,000 FWHM. Such instru-
ments provide a clearly higher selectivity than QqQ
instrumentation. The emergence of higher and higher re-
solving HRMS instruments is a significant advantage over
QqQ technology. It is a fact that in the recent past every
QqQ instrument generation showed an impressive increase
in detector sensitivity as compared with the previous gener-
ation. This provided not only lower limits of detection, but
also permitted the dilution of extracts in order to reduce
signal suppression effects. The other side of the coin is the
fact that detection selectivity remained virtually unchanged.
This resulted in SRM traces which show many peaks
corresponding to compounds being eluted before, after, or
even together with the targeted analyte [21]. It is unlikely
that QqQ technology will provide higher selectivity by
achieving improved resolution without significantly affect-
ing ion transmission. Some instrument manufacturers
1240 A. Kaufmann

FWHM = 10'000 FWHM = 25'000

200 200

180 180

160 160

140 140

120 120

conc
conc

100 100

80 80

60 60

40 40

20 20

0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
rt rt

FWHM = 50'000 FWHM = 100'000

200 200

180 180

160 160

140 140

120 120
conc

conc

100 100

80 80

60 60

40 40

20 20

0 0
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
rt rt
MS/MS
200

180

160

140

120
conc

100

80

60

40

20

0
-2 0 2 4 6 8 10 12 14 16
rt
 The current role of high-resolution mass spectrometry in food analysis
Fig. 4 Graphical comparison of liquid chromatography (LC)high-
resolution mass spectrometry (HRMS) versus LCtandem mass spec-
trometry (MS/MS) for a blank honey matrix. Data are given for the four
HRMS resolutions evaluated and the corresponding mass window
settings (see the text) as well as for the MS/MS transitions (bottom).
The x-axis represents the retention time, whereas the y-axis represents
the standardized concentrations of the observed dummy peaks (false-
positive signals caused by endogenous matrix compounds). Clearly
visible is the reduction of the number and intensity of dummy peaks
when the resolution is increased and the mass window width is corre-
spondingly adjusted. Furthermore, the MS/MS performance is approx-
imately equaled at 50,000 FWHM
realized the need for an additional degree of selectivity and
introduced ion-mobility-based interfaces in front of their
QqQ instruments. This clearly helps. However, the price to
be paid is a significant reduction in sensitivity and even
more important in flexibility. The relatively slow movement
of ions in the drift tube does not permit short SRM dwell
times. This makes it difficult to use such a technique for
multiresidue methods. Furthermore, such a device calls for
even more compound-specific tuning work and therefore
makes method development clearly less straightforward
than does full-scan HRMS-based detection.
Most of the currently available literature for HRMS
multiresidue screening [25, 26] is based on measure-
ments made with instruments capable of providing res-
olutions between 10,000 and 20,000 FWHM. Hence,
conclusions regarding selectivity and sensitivity derived
from such studies might no longer be relevant when
considering the latest HRMS instrument generation.
Currently, only a few articles have reported screening
approaches based on 50,000 or more FWHM HRMS
approaches. These include screening for marine toxins
[11, 12], water pollutants [18], pesticides [27], and
veterinary drugs [23, 24] and the combined detection
of veterinary drugs, mycotoxins, and pesticides [20].
It is safe to conclude that higher resolution leads to
superior results when detecting low concentrations of
analytes in complex matrices. However, depending on
the technology used, there is a price to be paid. TOF
instruments achieve higher resolution by employing very
long flight tubes. This might be a physical limitation
when it comes to installing such an instrument in a
laboratory. An alternative construction concept relying
on a folded flight path geometry reduces the duty cycle
and sensitivity. Orbitraps provide a user-defined resolu-
tion. There is no relevant sensitivity drop when choosing
a higher resolution. However, the resolution obtained is
proportional to the measurement time. The resulting con-
sequences are fewer data points per time unit. This is a
limitation when using UPLC equipment or when attempt-
ing polarity-switching experiments. Furthermore, higher
resolutions produce more data which have to be pro-
cessed by the instrument and later accessed from the
1241
hard disk when extracting mass traces from the raw data.
Hence, there is a need for sufficient computing power
and a highly developed data management algorithm.
Quantification
Sensitivity issues
Many mass spectrometrists believe that only quadrupole-
based tandem mass spectrometers (QqQ) are capable of
obtaining accurate trace level quantifications in biological
matrices. Few authors would dispute statements that tandem
quadrupole instruments can produce wider linear ranges,
higher sensitivity, and fewer matrix-dependent analyte
responses than traditional ion trap or older TOF instruments.
There have been statements such as Obviously, a Q-TOF
mass spectrometer instrument will most probably never take
the place of our trusted triple quadrupole or ion-trap instru-
ments [28]. In fact, only a few years ago, there was little
alternative technology available. The slow speed as well as
the high complexity and cost of sector-field or Fourier
transform ion cyclotron resonance instruments did not and
do not put these instruments in a position to compete against
MS/MS instruments. However, the dominance of MS/MS
was readdressed by authors who compared MS/MS with
some more modern HRMS techniques such as the new
TOF generation and the Orbitrap technology. TOF and
quadrupole TOF (Q-TOF) instruments based on time-to-
digital converter (TDC) technology with a resolution of
10,000 FWHM were reported to provide narrower linear
ranges and poorer sensitivity than QqQ instruments [27].
However, the authors were able to extend the dynamic range
by the use of a so-called dynamic range enhancement func-
tion. Inferior sensitivity and slightly poorer precision were
observed in the comparison of a TDC-based 10,000 FWHM
Q-TOF instrument with a QqQ system for a pesticide multi-
residue application [29]. Identical observations were made in a
similar comparison focusing on anabolic steroids [30]. Lower
sensitivities resulted for the 10,000 FWHM TOF instrument.
This was explained by both the signal-to-noise ratio (detector
sensitivity) and the significantly poorer selectivity of the
10,000 FWHM TOF instrument in comparison with MS/MS
[30]. Tandem quadrupole MS produced higher sensitivities
and wider dynamic ranges than an ion trap and 10,000 FWHM
Q-TOF instrument [6]. The authors of the articles mentioned
above acknowledged these limitations, but stressed the value
of Q-TOF MS because of its significantly higher qualitative
capabilities (confirmatory potential).
HRMS can be more sensitive than QqQ MS when cov-
ering a large number (e.g., 200 compounds) of analytes [31,
32]. This is due to the sensitivity reduction caused by the
shortening of the SRM dwell time. Only a few cases of
1242
HRMS sensitivities higher than QqQ MS sensitivities were
reported for non-multi-residue approaches [29, 33]. Better
signal to noise was reported [33] for the quantification of
avermectin and ivermectin in milk. These compounds form
a significantly higher abundance of [M+Na]+ than [M+H]+
analyte ions in the electrospray interface. The high affinity
for sodium compels the MS/MS user to attempt fragmenta-
tion of the very stable [M+Na] + ions, resulting in a low
abundance of product ions. On the other hand, the reported
HRMS method utilized the unfragmented [M+Na]+ exact
mass trace for quantification.
The latest HRMS instrument generation (TOF and Orbi-
trap) have significantly better sensitivity specifications than
the instruments used in the studies discussed above. This
was achieved by technological advances in detection tech-
nology, the introduction of new ion transition devices, and
the higher resolution power, which reduces isobaric inter-
ferences with matrix compounds. This generation of instru-
ments will permit the use of HRMS for applications with
significantly higher sensitivity requirements. The recently
introduced Q-Orbitrap provides the possibility to filter, ac-
cumulate, and store a limited mass range. This will signifi-
cantly increase sensitivity, at the price of a narrower mass
range
Still, single-residue applications which require maximum
sensitivity are preferably done with nonscanning QqQ
instruments (e.g., chloramphenicol and nitrofuran residues
in meat)
General quantification capabilities
Only a limited number of articles have evaluated the
general quantitative performance of MS/MS against the
latest generation of TOF or Orbitrap technology [10, 14,
15, 31, 33, 34]. Similar quantitative performance was
reported for a multimycotoxin study that compared an
LTQ Orbitrap against a QqQ instrument [15]. The com-
parison was complicated by the fact that the authors
used a micro-LC system as an inlet for the LTQ Orbi-
trap but connected a classic LC system to their MS/MS
instrument. In another study, an LTQ Orbitrap operated
at 100,000 FWHM was compared with a QqQ mass
spectrometer for the quantification of polyether toxins
in shellfish [10]. No relevant differences regarding the
limit of detection, intraday repeatability, and interday
repeatability were observed. A single-stage Orbitrap
(Exactive) operated at 50,000 FWHM was compared
with a QqQ MS instrument [31, 33] and with a TOF
instrument operating at 12,000 FWHM [34]. The vali-
dation procedure for some 40 different veterinary drugs
consisted of repeated spiking experiments covering a
number of analyte concentrations. A clearly inferior
performance (sensitivity, dynamic range, linearity,
A. Kaufmann
precision, and even accuracy) was observed for the
TOF-based measurements. The authors explained this
by the limitation caused by the characteristics of the
TDC detector and the insufficient TOF resolution as
well. Operating the Exactive instrument at the low res-
olution setting of 10,000 FWHM produced precision
accuracies and sensitivities similar to the TOF instru-
ment. This was interpreted as the proof that the ob-
served performance difference was caused by isobaric
interferences and is therefore directly related to the
instrument resolution and the corresponding mass win-
dow width. Hence, a higher-resolving non-TDC TOF
instrument was expected to provide quantification data
similar to those obtained with the Orbitrap. In another
study, a multiresidue pesticide method (230 compounds)
validated for MS/MS was directly migrated to an LC
Orbitrap platform [31]. Minimal investigations regarding
the compound independent interface settings and the
calculation and verification of compound-specific exact
masses were the only changes that were introduced. It
was the aim of that study to investigate the capability of
the current HRMS technology to accommodate methods
developed for MS/MS. The complete validation process
included three different matrices which were repeatedly
spiked at several concentrations. Despite the minimal
method adaptation efforts, very similar quantification
capabilities were observed regarding accuracy, precision,
and linearity. The MS/MS instrument showed a slightly
higher overall sensitivity. However, there were many
compounds which could be detected more sensitively
with the HRMS instrument. HRMS traces were clean-
er than the corresponding SRM traces owing to the
high selectivity provided by the resolution and mass
window setting chosen. The MS/MS measurements were
based on carefully time adjusted SRM windows. The
authors stressed that the instrument utilized did not
permit any further shortening of dwell times or prolon-
gation of measurement cycle times without significantly af-
fecting the sensitivity and precision the MS/MS-based
measurements. Such limitations do not exist for HRMS, in
which a theoretically unlimited number of analytes can be
monitored without any compromise of sensitivity.
Reliability and ruggedness of the technique
Modern HRMS instrumentation has become increasingly
rugged and user-friendly. Still, there are a number of
aspects which require some attention. Of uttermost impor-
tance is the accuracy and stability of the mass axis. Instru-
mental software has been greatly improved so that the
calibration of the mass axis is an easy and not very time
consuming task. Yet, there are significant differences
among the various instruments. TOF instrumentation
The current role of high-resolution mass spectrometry in food analysis
requires frequent recalibrations since temperature fluctua-
tions in the laboratory can cause the expansion and con-
traction of the flight tube, which directly affects the ion
flight time. Some instruments still require a permanent
internal mass calibration. This is mostly done by the con-
tinuous postcolumn infusion of a suitable calibration solu-
tion. Such a technique is not free of problems [35]. First, it
seems to be very difficult to introduce only the mass
calibration compound without any co-infused contami-
nants. Second, the mass calibrant signal can be lost when
injecting samples which cause intensive signal suppres-
sion. Such problems do not exist for systems which use a
baffle to introduce the calibrant solution in regular short
intervals. On the other hand, there is no sample data point
available when the instrument records the spectrum repre-
senting the mass calibrant. Narrow chromatographic peaks
corresponding to compounds which are eluted during such
a baffle switching lack a measurement data point. This
might not be visible because of the peak smoothing ap-
plied. Still, it affects the peak area measurements [35].
Some TOF instruments are sufficiently stable to permit a
mass axis calibration before or after a chromatographic
run. Nevertheless, the most stable mass axis seem to be
provided by the Orbitrap technology. The best mass accu-
racies are obtained by using an internal mass calibration.
This can be a baseline signal or a ubiquitous contaminant,
e.g., phthalate. In other words, it does not rely on the
postcolumn infusion of a calibrant solution. External cali-
bration produces only slightly poorer mass accuracies.
Practical experiences in our laboratory showed that cali-
bration frequencies of once a month are sufficient.
As mentioned above, older TDC-based TOF detectors
suffered [6, 28, 35] from mass shifts at high ion abundances.
This problem has been significantly improved with more
modern analog-to-digital converters. Still, mass shift at high
ion abundances is a particular concern for TOF measure-
ments. The interrelation between the measured mass devia-
tion and ion abundance is not a problematic issue for the
Orbitrap technology [36].
A critical feature of the current Orbitrap instrumentation
is the automatic gain control functionality. This feature is
intended to prevent an overfilling of the trap with ions
(charges). It is not the Orbitrap detection cell, but the col-
lecting device, the C-trap, which can be affected by a high
ion abundance. A prescan is performed to measure the
number of incoming ions (charges). This knowledge is used
to calculate the permitted accumulation time for the follow-
ing measurement scan. Hence, the C-trap will only accumu-
late ions for this calculated time period to prevent a
detrimental overfilling. The measured peak abundances of
the measurement scan are mathematically corrected for this
collection time reduction; therefore, quantification is not
affected. However, samples containing a lot of matrix or
1243
the existence of intense column bleed will cause the auto-
matic gain control to use short accumulation times. This
reduces not only the number of matrix ions, but also the
number of analyte ions. The consequences are that a low
analyte concentration can be easily detected in a pure stan-
dard but can no longer be reliably detected in a heavy matrix
sample.
There are indications that single-stage Orbitrap detection
(Exactive) is more strongly affected by matrix effects than
QqQ-based approaches. It was reported that the presence of a
high concentration of proteins (e.g., crude bovine liver
extracts) can cause the loss of low mass ions [37]. This was
tentatively explained by the strong electrostatic interaction
caused by the high charge density of such multiply charged
proteins as present in the C-trap. It is important to note that the
reported postinterface signal suppression affects only the in-
tensity of low mass ions but does not cause mass shifts of the
affected ions. The application where this problem was ob-
served was improved by a more extensive protein removal
step [36]. The modified method showed quantification
performance comparable to that of a QqQ-based method
[34]. Furthermore, newer Orbitrap instruments (e.g., Q
Exactive) provide low and high mass filters to reduce
the inflow of ions outside the user defined measurement
mass range.
Confirmation
As described in a European Commission decision (2002/657/
EC) [38], confirmation of a detected and quantified veterinary
drug residue is required. Although not compulsory, final proof
of the identity of a found peak has been adopted in many other
areas, such as pesticide analysis [25, 41]. Traditionally, con-
firmation is based on monitoring two MS/MS transitions. The
retention time of the suspected peak and the area ratio between
the two chromatographic peaks in the sample have to corre-
spond closely to the values observed when a pure standard is
injected. This approach has become the gold standard for
confirmatory techniques. Nevertheless, there were reports in-
dicating that this technique cannot completely rule out the
production of false-negative or false-positive findings. False-
negative findings were observed [42] for molecules that can
be protonated at two different sites in the molecule. The two
isobaric ions were shown to undergo completely different
fragmentation reactions. Signal suppression in the interface
was observed to significantly shift the ratio of the two isobaric
precursor ions and therefore distort the observed SRM ratio.
On the other hand, a false-positive finding caused by an
endogenous compound (neopellitorin A) was reported when
the pesticide sebuthylazine was analyzed in tarragon [49]. The
matrix compound met all MS/MS confirmation criteria, as
demanded by the European Commission decision. On the
1244
Fig. 5 UPLCTOF chromatogram of a suspected sebuthylazine-
containing tarragon extract (middle). The sebuthylazine signal is pres-
ent in the standard (top) and the spiked sample (bottom). The applica-
tion of a relatively wide mass window shows the presence of an intense
signal of an endogenous compound (neopellitorine A, C15H19NO) with
the same nominal mass as the analyte (sebutylazine, C9H16ClN5) and a
retention time of 5.22 min. The two compounds can be clearly distin-
guished by using a narrower mass window (not shown). The shown
chromatogram represents a carefully optimized separation using a sub
other hand, neopellitorin A was easily resolved from sebuthy-
lazine by HRMS. Furthermore, a chromatographic separation
could also be obtained by using a sub-2-m particulate sepa-
ration column. Figure 5 shows a sub-2-m separation of a
spiked tarragon sample. The chosen mass window of 0.05 Da
is wide enough that both isobaric compounds become visible.
A narrower mass window would show only the analyte with-
out the interfering matrix compound.
Product ion ratios without previous precursor selection
have also been used in single-stage GC- and LC-based
detection for confirmatory purposes. The quality of such
unit-mass-resolution GCMS spectra regarding confirma-
tion has been extensively discussed [41]. On the other hand,
the issue of the quality of non-precursor-selected LC
HRMS fragmentation spectra has not yet been thoroughly
investigated. There are reports that fragmentations (without
previous precursor selection) in the higher-energy collision-
al dissociation (HCD) cell of the Orbitrap are more repro-
ducible and accurate than those induced in the interface
A. Kaufmann
2-m particulate column method (the original, unmodified routine
method was not able to chromtographically resolve these two isobaric
compounds). Furthermore, the selected reaction monitoring ratio of the
analyte and the interfering compound is identical. Hence, even follow-
ing the EU confirmation procedure, a false-positive QqQ finding
resulted. On the other hand, even a medium-resolving HRMS instru-
ment (10,000 FWHM) was capable of mass-resolving the two isobaric
compounds
region [34]. However, the author also noted that HCD
fragmentations were affected to a minor degree by the
presence of a high abundance of ions with m/z values
differing from the value of the ion investigated.
HRMS criteria for a successful confirmation of suspected
residues in food have been discussed [38, 40, 41]. Unfortu-
nately, the European Commission decision [38] refers to the
required resolution criteria of 10,000 based on resolutions
defined at 10% of the mass peak height and not the currently
more commonly used 50% (FWHM). However, such a
resolution can be approximated to correspond to 20,000
FWHM [23]. It has been advocated [21, 23] that
HRMS- and MS/MS-based confirmation criteria should
be redefined in the light of modern HRMS technology.
This redefinition should include not only resolutions and
mass windows but also the diagnostic value of a tran-
sition (e.g., low confirmatory power of water loss) and
the chromatographic resolution provided by the separa-
tion system employed [21, 23].
The current role of high-resolution mass spectrometry in food analysis 1245

Fig. 6 13C2 mass peak of 1.99629

oxytetracycline (bottom) and
6000
five proposed (simulated) C22H26N6NaS2
elemental compositon spectra 4000
(above). These five elemental 2.00669
2000
compositons were the best hits
(based on the measured mass 0
1.99557
deviation) as proposed by the 4000
software. Ions containing sulfur 3000 2.00460
are clearly visible by an 2000
C14H29O11N4S
absolute mass difference of
1.996 Da from the 1000
monoisotopic peak. Such a 0
2.00595

Relative Abundance
display helps in reducing the
number of proposed elemental 3000
composition elucidation hits. 2000
C22H25O9N2
The surviving elemental
1000
compositions are only
C22H25O9N2 and C6H25O14N10. 0
2.00348
The first surviving elemental 3000
composition is the correct one,
as easily identified by the 2000 C6H25O14N10
intensity of the first carbon
1000
isotope signal (13C)
0
1.99560
10000
C14H30O2N8NaS3
5000

0
2.00641
6000
analyte
4000

2000

0
463.13 463.14 463.15 463.16 463.17 463.18
m/z

One of the most relevant differences between SRM and
HRMS is that exact-mass full-scan data contain much more
information than just the [M+H]+ or [M-H]- trace. This
includes the presence of other adducts, fragments, metabo-
lites, or degradation products. In other words, the informa-
tion content of an SRM trace is very finite. Users requiring
more confirmative information are compelled to define ad-
ditional SRM traces and monitor them in another dedicated
run. Hence, a posteriori hypothesis testing is not possible by
MS/MS but requires the redefinition of the MS experiment
followed by another sample injection.
Structure elucidation and data mining
Determination of elemental compositions and chemical
structures
Structure elucidation based on EI spectra and commercial
libraries is common in the field of GCMS [9, 41]. Techni-
cal aspects (i.e., requirement for nano-LC flow regimes as
well as poor ionization of intermediate and high mass com-
pounds) prevented the widespread acceptance of EI in LC
[43]. Commercial LC interfaces such as APCI and ESI are
so-called soft ionization techniques that produce little or no
diagnostic fragmentation. Hence, further structural investi-
gations require the use of fragmentation devices. There have
been extensive efforts to standardize ion trap and tandem
quadrupole collision chamber induced fragmentations [44].
However, these efforts were of limited success and have not
yet led to the availability of platform-independent commer-
cial MS/MS libraries. Hence, LCMS users cannot utilize
commercial EI spectra libraries. This must be considered a
relevant disadvantage of LCMS compared with GCMS.
On the other hand, the introduction of HRMS-
detection-based approaches provided food chemists with
new tools for structural elucidation. The combination of
exact mass data and measured relative isotopic abun-
dance ratios (RIA) permits the unequivocal elucidation
of elemental compositions of compounds ranging up to
300m/z [45]. Elemental compositions of heavier ions
have been determined with single-stage HRMS by
1246
additional measurements of the accurate masses of ob-
served product ions and interpreting the related neutral
losses [45]. Elemental compositions of ions up to 700m/z
could be elucidated [39] by the use of a single-stage
Orbitrap operated at a resolution of 100,000 FWHM.
Such resolutions permit the separation of 34S isotopes
from 13C2 and even 15N from 13C1. The relative height
of such signals can be used to estimate the number of
sulfur and nitrogen atoms within an elemental composi-
tion (see Fig. 6). Such orthogonal information permits the
inclusion of restrictions regarding the minimal and max-
imal number of atoms of each element (e.g., carbon,
nitrogen, chlorine) in order to reduce the number of
proposed elemental compositions. In contrast to the use
of commercial EI spectra libraries [46], the elucidation of
elemental compositions based on exact masses and RIA
of the unfragmented analyte ion is a comprehensive
Fig. 7 Signals obtained when recording a precursor scan with a
tandem quadrupole mass spectrometer. The first mass spectrometer
scanned, and the second quadrupole was set to m/z 156 to monitor
sulfonamides. Top: Standard solution containing several sulfonamides.
A. Kaufmann
approach. The real elemental composition of an investi-
gated peak will always be on the list of calculated
candidates, as long as the user does not enforce limita-
tions that are too stringent (e.g., mass accuracies or the
type and number of atoms permitted). EI libraries cannot
guarantee this since they are neither complete nor always
free of errors. This issue is relevant when metabolites or
degradation products are to be investigated. Few such
derived compounds are included in commercial libraries.
However, it has to be recognized that the information
content of an EI spectrum is superior to that of an
HRMS spectrum representing an unfragmented ion. In
addition, HRMS product ion spectra cannot be automat-
ically interpreted because of the lack of commercial
libraries. However, elemental compositions can be much
more easily assigned to HRMS-resolved product ions
than to unit-mass-resolved product ions. Knowing the
Bottom: Blank honey sample spiked with 50 g/kg sulfadimidine.
Sulfadimidine is eluted with a retention time of 5.5 min. The analyte
is visible together with a number of false-positive signals
The current role of high-resolution mass spectrometry in food analysis 1247

Fig. 8 Corresponds to Fig. 7.

However, the measurement was
made with a single-stage Orbi-
trap instrument operated at
50,000 FWHM. Fragmentation
energy was applied in the
higher-energy collisional disso-
ciation cell (no precursor selec-
tion). Shown is a narrow mass
trace corresponding to the
generic sulfonamide fragment.
Top: Standard mix. Bottom:
Blank honey sample spiked
with 50 g/kg sulfadimidine.
Sulfadimidine is eluted with a
retention time of 5.48 min. A
comparison with the
quadrupole-based precursor ion
scan (Fig. 7) shows a signifi-
cantly better signal-to-noise
ratio and no false-positive
signals

correct elemental composition of a product ion greatly
helps in the task of spectra interpretation.
A unique capability of HRMS is the possibility to search for
compounds containing particular atoms. Dedicated software
has been used to search for chlorine-containing unknowns
[41]. The approach utilized is based on the characteristic of
chlorine RIA, which shows a typical mass difference of
1.997 Da and an abundance ratio of 75.5/24.5. Such a search
is also possible when using unit-resolving MS. However, The
presence of unresolved matrix compounds will distort the RIA
of compounds present at low concentrations and consequently
leads to a large number of false-positive findings.
Comparison with unique tandem quadrupole scan modes
In contrast to QqQ, neutral loss or precursor scans are
not commonly available from single-stage HRMS
instruments. Even Q-TOF instruments do not permit
some typical QqQ scan-based experiments. However,
such scan information is available by using mathematical
instead of physical approaches. Neutral losses can be calculated
by testing spectra or more efficiently by investigating deconvo-
luted peaks for a particular exact mass neutral loss. There is,
however, a certain danger that the two ions found are not
precursor and product ion pairs, but are caused by two coeluted
compounds which just happen to show the accurate-mass-based
constant neutral loss mass difference investigated. On the other
hand, the neutral loss observed by a QqQ instrument might be
caused not by the expected precursor ion but by a chromato-
graphically coeluted isobaric structure.
Precursor ion scans are another typical MS/MS experiment
that aims at finding compounds containing the same diagnos-
tic substructure. Figure 7 shows a typical QqQ precursor scan
(product ion 156m/z) applied to detect a number of sulfona-
mides present in a pure standard solution (Fig. 7, top). This
QqQ scan mode is also capable of detecting the fortified
sulfadimidine (single compound) in a honey extract
(Fig. 7, bottom). However, the baseline is elevated, and a
number of false-positive signals [47] are caused by endog-
enous matrix compounds. The same sample was analyzed by
using a single-stage HRMS Orbitrap. Fragmentation was in-
duced in the HCD cell (without applying any precursor selec-
tion) and the instrument monitored the exact mass (10-ppm
mass window) of a fragment typical for sulfonamides (m/z
156.0114) at 50,000 FWHM. Only the analyte investigated
was observed in this mode and no false-positive matrix signals
were observed, as shown in Fig. 8 (bottom). The much clearer
results provided by monitoring narrow mass windows indicate
the power of HRMS accurate mass information over QqQ
unit-mass-based scan modes.
1248
Conclusion and future trends
In the last few years, we have seen a significant increase in
the number of studies reporting HRMS-based approaches
for food analysis applications. Increasingly, HRMS instru-
ments have been used in both quantitative and confirmative
work. However, the results of proficiency tests and interla-
boratory trials reveal a different picture. An overwhelming
number of participating laboratories still analyze pesticides,
veterinary drugs, or mycotoxins by using LCMS/MS or
even the older LCfluorescence- or LCUV-based technol-
ogy. Reported results derived from HRMS measurements
are still rare, which clearly reflects the fact that HRMS has
not yet been widely embraced by chemists working in
routine residue laboratories. This can certainly be explained
by the relatively recent availability of modern HRMS in-
strumentation and the reluctance of analytical chemists to
leave a time-proven technology such as MS/MS. It has to be
added that chemists who tried to solve their analytical prob-
lems with HRMS instead of MS/MS were sometimes dis-
appointed by the lack of speed and the limited user-
friendliness of the data processing software and some hard-
ware limitations [18, 20, 23, 31, 47].
This situation is likely to change. Most MS companies
have launched HRMS instruments designed for the routine
environment. The latest generation of HRMS instrumentation
is capable of providing higher resolution, superior sensitivity,
larger dynamic ranges, and improved speed. Furthermore,
downtime, cost, and time for maintenance as well as the
required user training have become much less relevant than
in the past. This will hopefully include the development of fast
and user-friendly dedicated HRMS data processing software.
Terms such as "Quan Qual and Quanfirmation have been ferent if the analyte is present in the sample rather than in the
coined, indicating that such instruments are not designed to
remain screening tools that require an additional MS/MS-
based quantification and confirmation step. Recently launched
HRMS screening software uses calculated exact masses and
not the experimentally determined retention time of analytes
as a qualifier for the initial identification of a compound.
Additional qualifiers to be utilized are the isotopic ratio of
the unfragmented analyte and the exact masses of typical
fragment ions. This approach works well for single-stage
HRMS, where fragmentation is obtained within or beyond
the interface. The application of a ramped collision energy
produces a sufficiently high yield of product ions, while
maintaining an acceptable ion count of the intact precursor
ion as well. Suitable software is able to indicate findings
which require an additional confirmation and quantification
by comparing the retention time and peak area for a final
confirmation and quantification. This will permit new
approaches for multiresidue analysis. Instead of producing
and injecting mixed standard solutions containing more than
100 pesticides, only the most often found compounds, or even
A. Kaufmann
only the internal standards, will be injected. The reference
solution will only be used for the actual quantification and
final confirmation of a positive finding. Such techniques could
theoretically also be used for unit-mass-resolution QqQ
approaches. However, the definition of narrow retention time
windows to accommodate the many SRM traces carries the
inherent risk that a potentially present compound will not be
detected if it is eluted outside the predefined retention time
window. The successful implementation of such a screening
technique which does not rely on reference compounds has
several advantages. The production and maintenance of mixed
standard solutions containing hundreds of different pesticides
is very challenging. It is not only that some reference com-
pounds are expensive or difficult to purchase. The most insta-
ble compound dictates the expiration date of a mixed standard
solution. Furthermore, screening approaches which do not
rely on the injection of reference compounds will not cause
the otherwise often observed carryover problems.
It is probably the major advantage of HRMS over QqQ-
based techniques that full-scan HRMS provides much more
comprehensive information. This does not only permit the
monitoring of metabolites or degradation products which
are not physically available for the analytes investigated.
HRMS is a great troubleshooting tool to detect and solve
MS problems. Poorly reproducible multiple reaction moni-
toring based peak areas or incomprehensible QqQ perfor-
mance can often be successfully explained and solved by
HRMS data. HRMS is ideally suited to identify coeluted
and therefore potentially suppressing matrix compounds. It
can also be utilized to detect analyte adducts with sodium,
acetonitrile, ammonia, etc. This might lead to the observa-
tion that the abundance ratios among these adducts is dif-
pure reference solution.
Structure elucidation by LCHRMS has two significant
advantages over more traditional GCMS studies. First, the
calculation of possible elemental composition is based on exact
masses and isotopic ratios (RIA). Exact masses and RIA of
compounds can be calculated exactly and are platform-
independent. As a result, this approach does not rely on exper-
imentally produced libraries. Hence, searches are exhaustive
and not affected by the completeness or even possible errors
within the library. Second, HRMS structure elucidation soft-
ware can be operated with little user training. This permits a
much larger number of analysts to venture into the field of
structure elucidation. More often than not, an observed inter-
esting finding or a suspected peak is not investigated further in
a busy routine laboratory. This has less to do with a lack of
scientific curiosity; it is rather the lack of time which prevents
further investigations. HRMS enables busy analytical chemists
to focus on such a finding and hopefully discover something
which might not have been have attempted without the avail-
ability of such an easy to use methodology.
The current role of high-resolution mass spectrometry in food analysis
It is likely that soon the main workload of routine trace
level quantification will be handled by HRMS, whereas MS/
MS will be used only for applications requiring ultimate
sensitivity or an orthogonal confirmatory information. Such
a development has been predicted for drug discovery bio-
analysis [48]. The authors of that article referred to this
ongoing change as a paradigm shift. The pharmaceutical
industry currently represents the major user of MS instru-
mentation. Therefore, it is likely that emerging HRMS hard-
ware and software designed for the pharmaceutical industry
will have a strong influence on how food residue chemists
approach future analytical problems.
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