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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), Introduction


which is used for residue analysis in food, has gained wider
acceptance in the last few years. This development is due to History of high-resolution mass spectrometry in food
the availability of more rugged, sensitive, and selective instru- analysis
mentation. The benefits provided by HRMS over classical
unit-mass-resolution tandem mass spectrometry are consider- High-resolution mass spectrometry (HRMS) has long been
able. These benefits include the collection of full-scan spectra, used in food analysis, but until recently it was associated
which provides greater insight into the composition of a with high instrumental complexity. Therefore, HRMS has
sample. Consequently, the analyst has the freedom to measure been limited to the most demanding applications, such as the
compounds without previous compound-specific tuning, the analysis of natural organic matter or dioxin-related com-
possibility of retrospective data analysis, and the capability of pounds. The appearance of modern HRMS instruments such
performing structural elucidations of unknown or suspected as time-of-flight (TOF) and Orbitrap instruments has funda-
compounds. HRMS strongly competes with classical tandem mentally changed this situation. Consequently, the capabil-
mass spectrometry in the field of quantitative multiresidue ities and limitations of current HRMS techniques in the field
methods (e.g., pesticides and veterinary drugs). It is one of of food analysis must be reevaluated.
the most promising tools when moving towards nontargeted Coupled with gas chromatography (GC), HRMS has been
approaches. Certain hardware and software issues still have to the core technology utilized for the analysis of halogenated
be addressed by the instrument manufacturers for it to dis- dioxin residues in food and the environment [1, 2]. It is inter-
lodge tandem mass spectrometry from its position as the esting to note that for some 30 years the same type of mass
standard trace analysis tool. spectrometer (sector-field instrument) has been utilized. This
choice of instrumentation was obvious during the early days of
Keywords High-resolution mass spectrometry . Food dioxin analysis. The complexity of matrices and the required
analysis . Oribitrap . Time-of-flight mass spectrometry . low detection limits (around 1 ppt) forced analysts to utilize this
Residue analysis capital-, operator-, and know-how-intensive mass spectrometry
(MS) technology. The high mass defect of ions containing
halogen atoms relative to endogenous nonhalogenated ions
Published in the special issue High-Resolution Mass Spectrometry with produced a very selective detection even in complex and chal-
guest editors Hans H. Maurer and David C. Muddiman.
lenging samples. The complexity of the sampling, extraction,
A. Kaufmann (*) cleanup, separation, and detection processes involved led to the
Official Food Control Authority,
definition and international acceptance of norms such as EPA
Fehrenstrasse 15,
8032 Zurich, Switzerland method 1613. Considering the technological progress in ana-
e-mail: anton.kaufmann@klzh.ch lytical chemistry, it is remarkable that the community of dioxin
analysts is still using the same type of instrument it accepted
A. Kaufmann
three decades earlier. On the other hand, analysts not involved
Kantonales Labor Zrich,
Fehrenstrasse 15, in dioxin analysis were not easily convinced of the suitability
8032 Zurich, Switzerland and ruggedness of HRMS in general and sector-field MS in
1234 A. Kaufmann

particular for their analytical problems. Until recently, the pesticide analysts [7, 8], attempts were made to introduce TOF
HRMS technique was probably considered too cumbersome spectrometry in GC [7, 8]. However, TOF spectrometry be-
for non-dioxin-related work. came more popular in the field of GC because of the fast scan
The analysis of persistent halogenated pesticide residues in speed rather than the mass resolution. Higher than unit mass
food and in the environment was initially performed with GC resolution GCMS detectors are yet to become commercially
and an electron capture detector. The widening range of com- successful. As a consequence, only a few articles have focused
pounds included in analysis led to the use of nitrogen- and on this otherwise promising technology [9]. This situation has
phosphorous-specific detectors and then to single-stage low- changed with the very recent commercial introduction of sev-
resolution MS. Electron ionization (EI) was and still is the eral new types of HRMS-based GCTOF instrumentation.
most commonly used type of GCMS ionization. Additional They will likely have a relevant impact in the field of GC
selectivity was obtained by chemical ionization techniques. MS. Generally, there is a much wider choice of commercially
The importance of monitoring very polar and thermolabile available HRMS detectors which can be readily coupled to LC
pesticides as well as derived degradation products led to the systems than to GC systems. This is clearly reflected by the
investigation of liquid chromatography (LC). Researchers number of published articles.
used single-stage ion trap or single-stage quadrupole instru-
ments or even non-MS-based detection [36]. Single-stage Current use of high-resolution mass spectrometry in different
LCMS was utilized for relatively simple matrices such as fields of food analysis
water, but seldom for interference-prone samples such as fruits
and vegetables. Currently, analysis of more and more analytes LCHRMS has become increasingly popular in the field of
has been transferred from the GC-based to the LC-based meth- pesticide analysis [7, 8] within the last few years. This
ods. It seems that only analytes that cannot be ionized by development was initiated by an ever-increasing number
electrospray ionization (ESI) or atmospheric pressure chemical of active compounds and derived degradation products to
ionization (APCI) are now analyzed by GC methods. On the be monitored within a single analytical method.
other hand, overlapping analytes which can be determined by The analysis of marine biotoxinssuch as paralytic shell-
GC and LC add a greater degree of dependability to the results. fish poisoning toxins, diarrheic shellfish poisoning toxins,
Because LCMS/MS was extensively accepted within a few domoic acid, tetrodotoxins, ciguatera, and azaspiracidshas
years, many analysts welcomed the switch from the GCMS to a unique history in that it relied until very recently on biolog-
the GCMS/MS method as well. The acceptance of the latter ical tests. The mice assay utilized is based on the survival rate
technology can be understood by the need for additional selec- of mice into which shell extracts have been injected. The high
tivity (caused by the lowering of detection limits and the number of false-positive and false-negative findings as well as
reluctance to employ an extensive sample cleanup) as well as the questionable ethics of such an assay resulted in pressure to
the familiarity with LCMS/MS. Users of the latter are familiar switch to alternative detection principles. However, this process
with selecting precursor and product ion masses. However, the was not straightforward since reference substances are not only
precursor ion mass in LCMS most often corresponds to the very expensive but also often consist of a number of isomeric
molecular mass of the analyte molecule plus a proton (positive analogues. Their limited availability as reference material and
ionization) or minus a proton (negative ionization). This guide- the assumption that toxicity varies significantly from analogue
line no longer exists for GCMS/MS, since the commonly to analogue made the development of LCMS/MS methods
utilized EI is a hard ionization technique causing extensive difficult. This is probably the reason why HRMS seems to be
fragmentation during the ionization process. This aspect might the preferred technique for the analysis of marine toxins
be less relevant if chemical or cold ionization techniques are [1012].
used. Still, more often than not, the user has to select one of the Many mycotoxins have to be detected at a very low level,
fragments produced as a precursor and must optimize the requiring a good cleanup and high-sensitivity detection. The
conditions for a suitable product ion as a second fragmentation. use of very specific but expensive and time-consuming immu-
The straightforwardness of such an approach is somewhat noassasy steps can be circumvented by employing LCMS/
questionable. There is a loss of absolute signal intensity (sensi- MS instead of classical LC-based detection (e.g., fluorescence
tivity) that is normally more than compensated by the higher or COBRA cell). HRMS is not yet widely used for the
selectivity provided by MS/MS (signal to noise improves). The analysis of mycotoxins. However, the limited availability of
process of fragmenting a relatively small molecule during many mycotoxin reference materials will make this technique
ionization and then fragmenting again the resulting EI ions in attractive soon [1315]. Furthermore, HRMS facilitates the
the collision chamber produces even smaller ions with limited monitoring of the so-called masked mycotoxins [16].
diagnostic quality. Hence, the overall selectivity is lower than Veterinary drug residues have long been analyzed by immu-
that derived from ESI-based LCMS/MS. Although until very noassay tests or by LCfluorescence detection. The relatively
recently GCHRMS was not used to any relevant extent by high molecular weight of these compounds and their high
The current role of high-resolution mass spectrometry in food analysis 1235

polarity required a derivatization step prior to their injection Screening techniques


into the gas chromatograph, which led to the early use of LC
MS technology. Ion trap instruments were known for their Advantages of high-resolution mass spectrometry
relatively low speed and their limited quantification capabilities for screening techniques
when utilized for trace level analysis of difficult matrices such
as liver and kidney. On the other hand, single-stage quadrupole- Screening is intended to produce a yesno answer, whether a
based LCMS instrumentation produced insufficient selectivity single or a set of different compounds is present in a large
and correspondingly poor sensitivity. As a consequence, LC number of samples. The focus is on speed and often also on
MS/MS was investigated earlier than in many other analytical cost-effectiveness. Although quantification capabilities might
fields. This process was accelerated by the extensive use of be desired, the focus is on a manageable number of false-
LCMS/MS in the pharmaceutical industry, which led to the positive findings at the relevant concentrations and the absence
availability of suitable hardware and software. HRMS was only of false-negative results. Screening techniques can be either
recently introduced by analysts to cope with the large number targeted or nontargeted. Targeted approaches focus on a pre-
of active compounds and derived metabolites that required defined list of compounds to be tested for. The ideal nontar-
detection and quantification. This can be partially explained geted approaches should be capable of detecting any unknown
by the compelling use of validation and confirmation protocols exogenous compound in a given sample. Given the complexity
(2002/657/EC) that were written with a strong focus on MS/ of such an approach, the vast majority of published screening
MS technology [5, 17]. approaches are still targeted techniques.
The analysis of organic contaminants originating from The high level of selectivity provided by MS (as compared
the environment has an extensive history of using GC as the with LCUV detection or GCflame ionization detection)
separation technique. The initial electron capture detectors permitted the monitoring of lower analyte concentrations in
were replaced by single-stage and then triple quadrupole complex matrices without producing an excessive number of
(QqQ) detectors. Probably more than any other analytical false-positive findings. The search for even higher selectivity
discipline, comprehensive GC (GCGC) has gained accep- led to MS/MS-based targeted multiresidue screening techni-
tance, which is due to the complexity of the matrix and the ques. Such approaches became feasible with modern MS/MS
attempted limits of detection. However, GCGC is currently instruments capable of monitoring selected reaction monitor-
not widely used as a routine technique. Again, LCHRMS ing (SRM) traces with very short dwell times. Still, analysts
and to a certain degree GCHRMS have been used by a have to strike a compromise between the number of transitions
number of authors [1, 2, 9]. There is increasing interest in to be monitored, the length of the dwell times, and the number
the field of water utility in using the latest generation of of data points across a chromatographic peak. The number of
single-stage LCHRMS technology [18] because of its suf- compounds to be monitored could be increased up to 1,000 by
ficient full-scan sensitivity. This permits monitoring of un- using high-end instruments and the application of retention-
expected contaminants (chemical spill) at relevant time-window-based SRM traces. It is obvious that this tech-
concentrations. nique requires knowledge of the exact retention time of every
Large, biologically active molecules such proteins with screened analyte. This becomes a serious limitation when the
allergenic potential are most often analyzed by bioassays number of compounds extends beyond 200. The user is then
such as the enzyme-linked immunosorbent assay. There is a forced to test the correctness of the expected retention time by
clear trend towards nonbioassays such as LCMS/MS and injecting standard solutions. Retention times often show nei-
LCHRMS. This development is clearly observable for the ther an absolute nor a consistent relative stability. Retention
detection of allergens in food [19]. times of acid or alkaline compounds may drift relative to those
The plethora of HRMS articles published in recent years of neutral compounds. Hence, SRM-based retention time
was motivated by several factors. The increasing number of windows not only must be set up carefully but also might
analytes covered by a single analytical method (multiresidue have to be readjusted during a long sample series. The com-
method) makes the use of a full-scan method an attractive bined difficulties associated with managing and monitoring a
option. In addition, the lack of commercial availability of large number of SRM traces motivated the shift towards full-
some reference substances (e.g., active compounds, degra- scan-based HRMS screening. Furthermore, a positive finding
dation products, and metabolites) requires instrumentation very often raises the question of additional information. Typ-
that does not need previous individual compound-specific ical examples are the tentative identification of a suspected
instrument tuning. Finally, HRMS is a powerful tool for drug that is known to be often co-administered with another
semitargeted or nontargeted screening while not only en- drug (e.g., trimethoprim, which is used in combination with
abling the analyst to detect suspected peaks, but also helping sulfonamides) or the detection of a pesticide that is known to
in identifying and confirming the underlying chemical produce a degradation product at a relevant concentration.
structure. Furthermore, there are occasions where it might be of interest
1236 A. Kaufmann

to know if a newly found residue was already present in algorithm utilized [22]. Applying too narrow a mass window
similar samples analyzed a year ago. Such information is around the exact mass of norfloxacin could even cause the
stored within the full-scan data on the hard disk. Access to complete disappearance of this signal when the measured
existing HRMS data files can quickly provide answers to such signal lies outside the defined mass range. Hence, there is a
questions and permit the formulation of a posteriori hypothe- risk of reporting false-negative findings if mass window widths
ses. This is a significant advantage over MS/MS-based narrower than the resolving capability of the instrument utilized
approaches, in which the analyst normally needs to have are defined [2024].
physical access to reference material before determining the Some studies have addressed the issue of the mass resolu-
correct MS/MS settings, which finally enables the analyst to tion required for trace analysis in food matrices [20, 21, 23].
build an acquisition method. Resolutions of 50,000 or more FWHM were suggested for
low levels of analytes in complex matrices such as animal feed
Selectivity issues [20]. The same authors concluded that a resolving power of
25,000 FWHM is sufficient for less complex matrices (e.g.,
HRMS permits selectivity for the monitoring of compounds honey). Their conclusion was based on experiments that in-
that share the same nominal mass but differ in their elemental volved analyzing matrix samples spiked at low levels with
composition and therefore have a different exact mass. Many various veterinary drugs, mycotoxins, and pesticides. The
reports in the literature described the use of a narrow mass absence of isobaric interferences was concluded when the
window to improve selectivity [20, 21]. Figure 1 shows the detected analytes showed mass deviations below 2 ppm at a
chromatogram of a bovine liver extract spiked with marboflox- concentration of some 10 ng/g [20]. On the basis of experi-
acin [21]. The detection relied on monitoring the [M+H]+ion by ments with synthetic hormones, screening resolutions of
HRMS (m 10,000 full width at half maximum, FWHM). 10,000 or more FWHM with mass windows of 50 mDa were
Different mass window widths were applied. A first look at suggested [23]. The authors concluded that only proposed
Fig. 1 might lead to the conclusion that the best results (selec- resolutions of 70,000 or more FWHM led to reliable accurate
tivity) appear to be obtained when the narrowest mass window masses of elemental compositions differing in one CO, C2H4,
width is used. Yet selectivity is not simply the product of the or N2 substructure.
narrowness of the defined mass window. Selectivity of detec- A recent study attempted to determine the mass resolu-
tion in complex matrices is only ensured when the HRMS tion and corresponding mass window width required to
instrument can physically resolve the analyte mass from that obtain selectivity comparable to that of MS/MS [21]. This
of other coeluted isobaric matrix compounds. Figure 1 should benchmark was chosen because MS/MS is considered by
therefore not be interpreted as demonstrating that selectivity many mass spectrometrists as the gold standard that must be
improves when reducing the mass extraction window width. met by any possible competing technique. The authors did
This will be more clearly illustrated by another compound not focus on the question of whether the detection of a
(norfloxacin), which was monitored in the same spiked extract particular exogenous analyte present at trace levels is
[21]. The observed accurate mass deviated slightly from the masked or impaired by endogenous compounds originating
calculated exact mass when an intermediate mass window and from the matrix. They instead investigated the vacant m/z
a resolution of 10,000 FWHM were used. Figure 2 shows the space available in MS/MS and HRMS spectra of an ana-
observed chromatogram of marbofloxacin in the liver extract lyzed blank honey matrix extract (honey is considered to be
where a 2-mDa mass window was applied. Even such a very a rather complex matrix). The idea behind this concept was
narrow mass window did not reduce the number of interfering the assumption that monitoring a large number of such
peaks as extensively (chromatogram at the top) as observed for randomly selected traces shows a number of traces contain-
norfloxacin in Fig. 1. Furthermore, there is a clear distortion of ing one or more chromatographic peaks caused by endoge-
the mass signal corresponding to marbofloxacin (amplified nous matrix compounds. Measuring the number and the
chromatogram at the bottom). The splitting of the peak was intensity of the detected peaks provided information regard-
caused by a single scan which recorded the mass peak of ing the degree of detector selectivity obtained. A statistical
marbofloxacin outside the defined mass window. Use of a sampling procedure was developed, since it is not possible
significantly higher resolution of 100,000 FWHM revealed to monitor all theoretically possible MS/MS transitions and
the presence of an isobaric matrix peak (Fig. 3). Close inspec- HRMS-resolved masses in a blank matrix. This procedure
tion of a deviating single scan, obtained at 10,000 FWHM consisted of the guided calculation of 100 so-called dummy
(Fig. 2), showed an accurate mass of 320.13855m/z, which transitions and dummy exact masses. MS/MS transition
deviated by 6 ppm from the theoretical value of 320.14047m/z. sampling restrictions were applied to permit only a precur-
This can be traced to the presence of a coeluted interfering sor mass range between 150 and 600m/z and a product ion
endogenous compound. The observed mass error is also de- range between 70m/z and the mass of the precursor ion
pendent on the relative mass peak intensity and the centroiding 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 investigated. Each SRM and accurate mass peak area was
the four resolution settings investigated (50 ppm with 10,000 standardized with this average peak area/concentration re-
FWHM, 20 ppm with 25,000 FWHM, 10 ppm with 50,000 sponse. This permitted a direct comparison between SRM-
FWHM, and 5 ppm with 100,000 FWHM). The interrelation and HRMS-based chromatographic signal intensities. Figure 4
of these two parameters was discussed above. As expected, compares the selectivity of the two techniques based on this
most extracted dummy traces were free of chromatographic approach. The standardized concentrations of every detected
peaks. The dummy traces which contained chromatographic peak based on the monitored dummy signal intensities (y-axis)
peaks were integrated. In the next step, the SRM and HRMS are shown. The x-axis represents the chromatographic reten-
peak areas obtained were standardized. This was done by tion time at which the corresponding compound is eluted. The
determining an average peak area/concentration response of four windows at the top refer to HRMS measurements
some ten different antibiotics for the two MS techniques 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 The latest generation of Orbitrap instrumentation pro-
of false-positive peaks in the blank honey extract inves- vides resolutions well above 100,000 FWHM. Such instru-
tigated is clearly visible. The graph at the bottom repre- ments provide a clearly higher selectivity than QqQ
sents the SRM performance. It can be concluded that an instrumentation. The emergence of higher and higher re-
HRMS resolution of 50,000 FWHM and a corresponding solving HRMS instruments is a significant advantage over
mass window of 10 ppm provide selectivity as good as or QqQ technology. It is a fact that in the recent past every
slightly higher than MS/MS. In the case of the honey QqQ instrument generation showed an impressive increase
matrix investigated, QqQ selectivity seems to be poor for in detector sensitivity as compared with the previous gener-
late-eluted compounds. This is visible by the number of ation. This provided not only lower limits of detection, but
rather intense peaks corresponding to compounds eluted in also permitted the dilution of extracts in order to reduce
the retention time region from 12 to 14 min (Fig. 4). It signal suppression effects. The other side of the coin is the
has to be added that MS/MS provides the possibility to fact that detection selectivity remained virtually unchanged.
achieve a higher selectivity by monitoring additional tran- This resulted in SRM traces which show many peaks
sitions. However, additional transitions seldom truly add corresponding to compounds being eluted before, after, or
an orthogonal selectivity contribution [21]. Furthermore, even together with the targeted analyte [21]. It is unlikely
adding additional transitions consumes valuable dwell time that QqQ technology will provide higher selectivity by
resources and significantly reduces the sensitivity for poor- achieving improved resolution without significantly affect-
ly fragmenting analytes. 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 1241

Fig. 4 Graphical comparison of liquid chromatography (LC)high- hard disk when extracting mass traces from the raw data.
resolution mass spectrometry (HRMS) versus LCtandem mass spec-
trometry (MS/MS) for a blank honey matrix. Data are given for the four
Hence, there is a need for sufficient computing power
HRMS resolutions evaluated and the corresponding mass window and a highly developed data management algorithm.
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
Quantification
visible is the reduction of the number and intensity of dummy peaks
when the resolution is increased and the mass window width is corre- Sensitivity issues
spondingly adjusted. Furthermore, the MS/MS performance is approx-
imately equaled at 50,000 FWHM
Many mass spectrometrists believe that only quadrupole-
based tandem mass spectrometers (QqQ) are capable of
realized the need for an additional degree of selectivity and obtaining accurate trace level quantifications in biological
introduced ion-mobility-based interfaces in front of their matrices. Few authors would dispute statements that tandem
QqQ instruments. This clearly helps. However, the price to quadrupole instruments can produce wider linear ranges,
be paid is a significant reduction in sensitivity and even higher sensitivity, and fewer matrix-dependent analyte
more important in flexibility. The relatively slow movement responses than traditional ion trap or older TOF instruments.
of ions in the drift tube does not permit short SRM dwell There have been statements such as Obviously, a Q-TOF
times. This makes it difficult to use such a technique for mass spectrometer instrument will most probably never take
multiresidue methods. Furthermore, such a device calls for the place of our trusted triple quadrupole or ion-trap instru-
even more compound-specific tuning work and therefore ments [28]. In fact, only a few years ago, there was little
makes method development clearly less straightforward alternative technology available. The slow speed as well as
than does full-scan HRMS-based detection. the high complexity and cost of sector-field or Fourier
Most of the currently available literature for HRMS transform ion cyclotron resonance instruments did not and
multiresidue screening [25, 26] is based on measure- do not put these instruments in a position to compete against
ments made with instruments capable of providing res- MS/MS instruments. However, the dominance of MS/MS
olutions between 10,000 and 20,000 FWHM. Hence, was readdressed by authors who compared MS/MS with
conclusions regarding selectivity and sensitivity derived some more modern HRMS techniques such as the new
from such studies might no longer be relevant when TOF generation and the Orbitrap technology. TOF and
considering the latest HRMS instrument generation. quadrupole TOF (Q-TOF) instruments based on time-to-
Currently, only a few articles have reported screening digital converter (TDC) technology with a resolution of
approaches based on 50,000 or more FWHM HRMS 10,000 FWHM were reported to provide narrower linear
approaches. These include screening for marine toxins ranges and poorer sensitivity than QqQ instruments [27].
[11, 12], water pollutants [18], pesticides [27], and However, the authors were able to extend the dynamic range
veterinary drugs [23, 24] and the combined detection by the use of a so-called dynamic range enhancement func-
of veterinary drugs, mycotoxins, and pesticides [20]. tion. Inferior sensitivity and slightly poorer precision were
It is safe to conclude that higher resolution leads to observed in the comparison of a TDC-based 10,000 FWHM
superior results when detecting low concentrations of Q-TOF instrument with a QqQ system for a pesticide multi-
analytes in complex matrices. However, depending on residue application [29]. Identical observations were made in a
the technology used, there is a price to be paid. TOF similar comparison focusing on anabolic steroids [30]. Lower
instruments achieve higher resolution by employing very sensitivities resulted for the 10,000 FWHM TOF instrument.
long flight tubes. This might be a physical limitation This was explained by both the signal-to-noise ratio (detector
when it comes to installing such an instrument in a sensitivity) and the significantly poorer selectivity of the
laboratory. An alternative construction concept relying 10,000 FWHM TOF instrument in comparison with MS/MS
on a folded flight path geometry reduces the duty cycle [30]. Tandem quadrupole MS produced higher sensitivities
and sensitivity. Orbitraps provide a user-defined resolu- and wider dynamic ranges than an ion trap and 10,000 FWHM
tion. There is no relevant sensitivity drop when choosing Q-TOF instrument [6]. The authors of the articles mentioned
a higher resolution. However, the resolution obtained is above acknowledged these limitations, but stressed the value
proportional to the measurement time. The resulting con- of Q-TOF MS because of its significantly higher qualitative
sequences are fewer data points per time unit. This is a capabilities (confirmatory potential).
limitation when using UPLC equipment or when attempt- HRMS can be more sensitive than QqQ MS when cov-
ing polarity-switching experiments. Furthermore, higher ering a large number (e.g., 200 compounds) of analytes [31,
resolutions produce more data which have to be pro- 32]. This is due to the sensitivity reduction caused by the
cessed by the instrument and later accessed from the shortening of the SRM dwell time. Only a few cases of
1242 A. Kaufmann

HRMS sensitivities higher than QqQ MS sensitivities were precision, and even accuracy) was observed for the
reported for non-multi-residue approaches [29, 33]. Better TOF-based measurements. The authors explained this
signal to noise was reported [33] for the quantification of by the limitation caused by the characteristics of the
avermectin and ivermectin in milk. These compounds form TDC detector and the insufficient TOF resolution as
a significantly higher abundance of [M+Na]+ than [M+H]+ well. Operating the Exactive instrument at the low res-
analyte ions in the electrospray interface. The high affinity olution setting of 10,000 FWHM produced precision
for sodium compels the MS/MS user to attempt fragmenta- accuracies and sensitivities similar to the TOF instru-
tion of the very stable [M+Na] + ions, resulting in a low ment. This was interpreted as the proof that the ob-
abundance of product ions. On the other hand, the reported served performance difference was caused by isobaric
HRMS method utilized the unfragmented [M+Na]+ exact interferences and is therefore directly related to the
mass trace for quantification. instrument resolution and the corresponding mass win-
The latest HRMS instrument generation (TOF and Orbi- dow width. Hence, a higher-resolving non-TDC TOF
trap) have significantly better sensitivity specifications than instrument was expected to provide quantification data
the instruments used in the studies discussed above. This similar to those obtained with the Orbitrap. In another
was achieved by technological advances in detection tech- study, a multiresidue pesticide method (230 compounds)
nology, the introduction of new ion transition devices, and validated for MS/MS was directly migrated to an LC
the higher resolution power, which reduces isobaric inter- Orbitrap platform [31]. Minimal investigations regarding
ferences with matrix compounds. This generation of instru- the compound independent interface settings and the
ments will permit the use of HRMS for applications with calculation and verification of compound-specific exact
significantly higher sensitivity requirements. The recently masses were the only changes that were introduced. It
introduced Q-Orbitrap provides the possibility to filter, ac- was the aim of that study to investigate the capability of
cumulate, and store a limited mass range. This will signifi- the current HRMS technology to accommodate methods
cantly increase sensitivity, at the price of a narrower mass developed for MS/MS. The complete validation process
range included three different matrices which were repeatedly
Still, single-residue applications which require maximum spiked at several concentrations. Despite the minimal
sensitivity are preferably done with nonscanning QqQ method adaptation efforts, very similar quantification
instruments (e.g., chloramphenicol and nitrofuran residues capabilities were observed regarding accuracy, precision,
in meat) and linearity. The MS/MS instrument showed a slightly
higher overall sensitivity. However, there were many
General quantification capabilities compounds which could be detected more sensitively
with the HRMS instrument. HRMS traces were clean-
Only a limited number of articles have evaluated the er than the corresponding SRM traces owing to the
general quantitative performance of MS/MS against the high selectivity provided by the resolution and mass
latest generation of TOF or Orbitrap technology [10, 14, window setting chosen. The MS/MS measurements were
15, 31, 33, 34]. Similar quantitative performance was based on carefully time adjusted SRM windows. The
reported for a multimycotoxin study that compared an authors stressed that the instrument utilized did not
LTQ Orbitrap against a QqQ instrument [15]. The com- permit any further shortening of dwell times or prolon-
parison was complicated by the fact that the authors gation of measurement cycle times without significantly af-
used a micro-LC system as an inlet for the LTQ Orbi- fecting the sensitivity and precision the MS/MS-based
trap but connected a classic LC system to their MS/MS measurements. Such limitations do not exist for HRMS, in
instrument. In another study, an LTQ Orbitrap operated which a theoretically unlimited number of analytes can be
at 100,000 FWHM was compared with a QqQ mass monitored without any compromise of sensitivity.
spectrometer for the quantification of polyether toxins
in shellfish [10]. No relevant differences regarding the Reliability and ruggedness of the technique
limit of detection, intraday repeatability, and interday
repeatability were observed. A single-stage Orbitrap Modern HRMS instrumentation has become increasingly
(Exactive) operated at 50,000 FWHM was compared rugged and user-friendly. Still, there are a number of
with a QqQ MS instrument [31, 33] and with a TOF aspects which require some attention. Of uttermost impor-
instrument operating at 12,000 FWHM [34]. The vali- tance is the accuracy and stability of the mass axis. Instru-
dation procedure for some 40 different veterinary drugs mental software has been greatly improved so that the
consisted of repeated spiking experiments covering a calibration of the mass axis is an easy and not very time
number of analyte concentrations. A clearly inferior consuming task. Yet, there are significant differences
performance (sensitivity, dynamic range, linearity, among the various instruments. TOF instrumentation
The current role of high-resolution mass spectrometry in food analysis 1243

requires frequent recalibrations since temperature fluctua- the existence of intense column bleed will cause the auto-
tions in the laboratory can cause the expansion and con- matic gain control to use short accumulation times. This
traction of the flight tube, which directly affects the ion reduces not only the number of matrix ions, but also the
flight time. Some instruments still require a permanent number of analyte ions. The consequences are that a low
internal mass calibration. This is mostly done by the con- analyte concentration can be easily detected in a pure stan-
tinuous postcolumn infusion of a suitable calibration solu- dard but can no longer be reliably detected in a heavy matrix
tion. Such a technique is not free of problems [35]. First, it sample.
seems to be very difficult to introduce only the mass There are indications that single-stage Orbitrap detection
calibration compound without any co-infused contami- (Exactive) is more strongly affected by matrix effects than
nants. Second, the mass calibrant signal can be lost when QqQ-based approaches. It was reported that the presence of a
injecting samples which cause intensive signal suppres- high concentration of proteins (e.g., crude bovine liver
sion. Such problems do not exist for systems which use a extracts) can cause the loss of low mass ions [37]. This was
baffle to introduce the calibrant solution in regular short tentatively explained by the strong electrostatic interaction
intervals. On the other hand, there is no sample data point caused by the high charge density of such multiply charged
available when the instrument records the spectrum repre- proteins as present in the C-trap. It is important to note that the
senting the mass calibrant. Narrow chromatographic peaks reported postinterface signal suppression affects only the in-
corresponding to compounds which are eluted during such tensity of low mass ions but does not cause mass shifts of the
a baffle switching lack a measurement data point. This affected ions. The application where this problem was ob-
might not be visible because of the peak smoothing ap- served was improved by a more extensive protein removal
plied. Still, it affects the peak area measurements [35]. step [36]. The modified method showed quantification
Some TOF instruments are sufficiently stable to permit a performance comparable to that of a QqQ-based method
mass axis calibration before or after a chromatographic [34]. Furthermore, newer Orbitrap instruments (e.g., Q
run. Nevertheless, the most stable mass axis seem to be Exactive) provide low and high mass filters to reduce
provided by the Orbitrap technology. The best mass accu- the inflow of ions outside the user defined measurement
racies are obtained by using an internal mass calibration. mass range.
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- Confirmation
bration produces only slightly poorer mass accuracies.
Practical experiences in our laboratory showed that cali- As described in a European Commission decision (2002/657/
bration frequencies of once a month are sufficient. EC) [38], confirmation of a detected and quantified veterinary
As mentioned above, older TDC-based TOF detectors drug residue is required. Although not compulsory, final proof
suffered [6, 28, 35] from mass shifts at high ion abundances. of the identity of a found peak has been adopted in many other
This problem has been significantly improved with more areas, such as pesticide analysis [25, 41]. Traditionally, con-
modern analog-to-digital converters. Still, mass shift at high firmation is based on monitoring two MS/MS transitions. The
ion abundances is a particular concern for TOF measure- retention time of the suspected peak and the area ratio between
ments. The interrelation between the measured mass devia- the two chromatographic peaks in the sample have to corre-
tion and ion abundance is not a problematic issue for the spond closely to the values observed when a pure standard is
Orbitrap technology [36]. injected. This approach has become the gold standard for
A critical feature of the current Orbitrap instrumentation confirmatory techniques. Nevertheless, there were reports in-
is the automatic gain control functionality. This feature is dicating that this technique cannot completely rule out the
intended to prevent an overfilling of the trap with ions production of false-negative or false-positive findings. False-
(charges). It is not the Orbitrap detection cell, but the col- negative findings were observed [42] for molecules that can
lecting device, the C-trap, which can be affected by a high be protonated at two different sites in the molecule. The two
ion abundance. A prescan is performed to measure the isobaric ions were shown to undergo completely different
number of incoming ions (charges). This knowledge is used fragmentation reactions. Signal suppression in the interface
to calculate the permitted accumulation time for the follow- was observed to significantly shift the ratio of the two isobaric
ing measurement scan. Hence, the C-trap will only accumu- precursor ions and therefore distort the observed SRM ratio.
late ions for this calculated time period to prevent a On the other hand, a false-positive finding caused by an
detrimental overfilling. The measured peak abundances of endogenous compound (neopellitorin A) was reported when
the measurement scan are mathematically corrected for this the pesticide sebuthylazine was analyzed in tarragon [49]. The
collection time reduction; therefore, quantification is not matrix compound met all MS/MS confirmation criteria, as
affected. However, samples containing a lot of matrix or demanded by the European Commission decision. On the
1244 A. Kaufmann

Fig. 5 UPLCTOF chromatogram of a suspected sebuthylazine- 2-m particulate column method (the original, unmodified routine
containing tarragon extract (middle). The sebuthylazine signal is pres- method was not able to chromtographically resolve these two isobaric
ent in the standard (top) and the spiked sample (bottom). The applica- compounds). Furthermore, the selected reaction monitoring ratio of the
tion of a relatively wide mass window shows the presence of an intense analyte and the interfering compound is identical. Hence, even follow-
signal of an endogenous compound (neopellitorine A, C15H19NO) with ing the EU confirmation procedure, a false-positive QqQ finding
the same nominal mass as the analyte (sebutylazine, C9H16ClN5) and a resulted. On the other hand, even a medium-resolving HRMS instru-
retention time of 5.22 min. The two compounds can be clearly distin- ment (10,000 FWHM) was capable of mass-resolving the two isobaric
guished by using a narrower mass window (not shown). The shown compounds
chromatogram represents a carefully optimized separation using a sub

other hand, neopellitorin A was easily resolved from sebuthy- region [34]. However, the author also noted that HCD
lazine by HRMS. Furthermore, a chromatographic separation fragmentations were affected to a minor degree by the
could also be obtained by using a sub-2-m particulate sepa- presence of a high abundance of ions with m/z values
ration column. Figure 5 shows a sub-2-m separation of a differing from the value of the ion investigated.
spiked tarragon sample. The chosen mass window of 0.05 Da HRMS criteria for a successful confirmation of suspected
is wide enough that both isobaric compounds become visible. residues in food have been discussed [38, 40, 41]. Unfortu-
A narrower mass window would show only the analyte with- nately, the European Commission decision [38] refers to the
out the interfering matrix compound. required resolution criteria of 10,000 based on resolutions
Product ion ratios without previous precursor selection defined at 10% of the mass peak height and not the currently
have also been used in single-stage GC- and LC-based more commonly used 50% (FWHM). However, such a
detection for confirmatory purposes. The quality of such resolution can be approximated to correspond to 20,000
unit-mass-resolution GCMS spectra regarding confirma- FWHM [23]. It has been advocated [21, 23] that
tion has been extensively discussed [41]. On the other hand, HRMS- and MS/MS-based confirmation criteria should
the issue of the quality of non-precursor-selected LC be redefined in the light of modern HRMS technology.
HRMS fragmentation spectra has not yet been thoroughly This redefinition should include not only resolutions and
investigated. There are reports that fragmentations (without mass windows but also the diagnostic value of a tran-
previous precursor selection) in the higher-energy collision- sition (e.g., low confirmatory power of water loss) and
al dissociation (HCD) cell of the Orbitrap are more repro- the chromatographic resolution provided by the separa-
ducible and accurate than those induced in the interface 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 well as poor ionization of intermediate and high mass com-
HRMS is that exact-mass full-scan data contain much more pounds) prevented the widespread acceptance of EI in LC
information than just the [M+H]+ or [M-H]- trace. This [43]. Commercial LC interfaces such as APCI and ESI are
includes the presence of other adducts, fragments, metabo- so-called soft ionization techniques that produce little or no
lites, or degradation products. In other words, the informa- diagnostic fragmentation. Hence, further structural investi-
tion content of an SRM trace is very finite. Users requiring gations require the use of fragmentation devices. There have
more confirmative information are compelled to define ad- been extensive efforts to standardize ion trap and tandem
ditional SRM traces and monitor them in another dedicated quadrupole collision chamber induced fragmentations [44].
run. Hence, a posteriori hypothesis testing is not possible by However, these efforts were of limited success and have not
MS/MS but requires the redefinition of the MS experiment yet led to the availability of platform-independent commer-
followed by another sample injection. 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.
Structure elucidation and data mining On the other hand, the introduction of HRMS-
detection-based approaches provided food chemists with
Determination of elemental compositions and chemical new tools for structural elucidation. The combination of
structures exact mass data and measured relative isotopic abun-
dance ratios (RIA) permits the unequivocal elucidation
Structure elucidation based on EI spectra and commercial of elemental compositions of compounds ranging up to
libraries is common in the field of GCMS [9, 41]. Techni- 300m/z [45]. Elemental compositions of heavier ions
cal aspects (i.e., requirement for nano-LC flow regimes as have been determined with single-stage HRMS by
1246 A. Kaufmann

additional measurements of the accurate masses of ob- approach. The real elemental composition of an investi-
served product ions and interpreting the related neutral gated peak will always be on the list of calculated
losses [45]. Elemental compositions of ions up to 700m/z candidates, as long as the user does not enforce limita-
could be elucidated [39] by the use of a single-stage tions that are too stringent (e.g., mass accuracies or the
Orbitrap operated at a resolution of 100,000 FWHM. type and number of atoms permitted). EI libraries cannot
Such resolutions permit the separation of 34S isotopes guarantee this since they are neither complete nor always
from 13C2 and even 15N from 13C1. The relative height free of errors. This issue is relevant when metabolites or
of such signals can be used to estimate the number of degradation products are to be investigated. Few such
sulfur and nitrogen atoms within an elemental composi- derived compounds are included in commercial libraries.
tion (see Fig. 6). Such orthogonal information permits the However, it has to be recognized that the information
inclusion of restrictions regarding the minimal and max- content of an EI spectrum is superior to that of an
imal number of atoms of each element (e.g., carbon, HRMS spectrum representing an unfragmented ion. In
nitrogen, chlorine) in order to reduce the number of addition, HRMS product ion spectra cannot be automat-
proposed elemental compositions. In contrast to the use ically interpreted because of the lack of commercial
of commercial EI spectra libraries [46], the elucidation of libraries. However, elemental compositions can be much
elemental compositions based on exact masses and RIA more easily assigned to HRMS-resolved product ions
of the unfragmented analyte ion is a comprehensive than to unit-mass-resolved product ions. Knowing the

Fig. 7 Signals obtained when recording a precursor scan with a Bottom: Blank honey sample spiked with 50 g/kg sulfadimidine.
tandem quadrupole mass spectrometer. The first mass spectrometer Sulfadimidine is eluted with a retention time of 5.5 min. The analyte
scanned, and the second quadrupole was set to m/z 156 to monitor is visible together with a number of false-positive signals
sulfonamides. Top: Standard solution containing several sulfonamides.
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 compounds which just happen to show the accurate-mass-based
helps in the task of spectra interpretation. constant neutral loss mass difference investigated. On the other
A unique capability of HRMS is the possibility to search for hand, the neutral loss observed by a QqQ instrument might be
compounds containing particular atoms. Dedicated software caused not by the expected precursor ion but by a chromato-
has been used to search for chlorine-containing unknowns graphically coeluted isobaric structure.
[41]. The approach utilized is based on the characteristic of Precursor ion scans are another typical MS/MS experiment
chlorine RIA, which shows a typical mass difference of that aims at finding compounds containing the same diagnos-
1.997 Da and an abundance ratio of 75.5/24.5. Such a search tic substructure. Figure 7 shows a typical QqQ precursor scan
is also possible when using unit-resolving MS. However, The (product ion 156m/z) applied to detect a number of sulfona-
presence of unresolved matrix compounds will distort the RIA mides present in a pure standard solution (Fig. 7, top). This
of compounds present at low concentrations and consequently QqQ scan mode is also capable of detecting the fortified
leads to a large number of false-positive findings. sulfadimidine (single compound) in a honey extract
(Fig. 7, bottom). However, the baseline is elevated, and a
Comparison with unique tandem quadrupole scan modes number of false-positive signals [47] are caused by endog-
enous matrix compounds. The same sample was analyzed by
In contrast to QqQ, neutral loss or precursor scans are using a single-stage HRMS Orbitrap. Fragmentation was in-
not commonly available from single-stage HRMS duced in the HCD cell (without applying any precursor selec-
instruments. Even Q-TOF instruments do not permit tion) and the instrument monitored the exact mass (10-ppm
some typical QqQ scan-based experiments. However, mass window) of a fragment typical for sulfonamides (m/z
such scan information is available by using mathematical 156.0114) at 50,000 FWHM. Only the analyte investigated
instead of physical approaches. Neutral losses can be calculated was observed in this mode and no false-positive matrix signals
by testing spectra or more efficiently by investigating deconvo- were observed, as shown in Fig. 8 (bottom). The much clearer
luted peaks for a particular exact mass neutral loss. There is, results provided by monitoring narrow mass windows indicate
however, a certain danger that the two ions found are not the power of HRMS accurate mass information over QqQ
precursor and product ion pairs, but are caused by two coeluted unit-mass-based scan modes.
1248 A. Kaufmann

Conclusion and future trends only the internal standards, will be injected. The reference
solution will only be used for the actual quantification and
In the last few years, we have seen a significant increase in final confirmation of a positive finding. Such techniques could
the number of studies reporting HRMS-based approaches theoretically also be used for unit-mass-resolution QqQ
for food analysis applications. Increasingly, HRMS instru- approaches. However, the definition of narrow retention time
ments have been used in both quantitative and confirmative windows to accommodate the many SRM traces carries the
work. However, the results of proficiency tests and interla- inherent risk that a potentially present compound will not be
boratory trials reveal a different picture. An overwhelming detected if it is eluted outside the predefined retention time
number of participating laboratories still analyze pesticides, window. The successful implementation of such a screening
veterinary drugs, or mycotoxins by using LCMS/MS or technique which does not rely on reference compounds has
even the older LCfluorescence- or LCUV-based technol- several advantages. The production and maintenance of mixed
ogy. Reported results derived from HRMS measurements standard solutions containing hundreds of different pesticides
are still rare, which clearly reflects the fact that HRMS has is very challenging. It is not only that some reference com-
not yet been widely embraced by chemists working in pounds are expensive or difficult to purchase. The most insta-
routine residue laboratories. This can certainly be explained ble compound dictates the expiration date of a mixed standard
by the relatively recent availability of modern HRMS in- solution. Furthermore, screening approaches which do not
strumentation and the reluctance of analytical chemists to rely on the injection of reference compounds will not cause
leave a time-proven technology such as MS/MS. It has to be the otherwise often observed carryover problems.
added that chemists who tried to solve their analytical prob- It is probably the major advantage of HRMS over QqQ-
lems with HRMS instead of MS/MS were sometimes dis- based techniques that full-scan HRMS provides much more
appointed by the lack of speed and the limited user- comprehensive information. This does not only permit the
friendliness of the data processing software and some hard- monitoring of metabolites or degradation products which
ware limitations [18, 20, 23, 31, 47]. are not physically available for the analytes investigated.
This situation is likely to change. Most MS companies HRMS is a great troubleshooting tool to detect and solve
have launched HRMS instruments designed for the routine MS problems. Poorly reproducible multiple reaction moni-
environment. The latest generation of HRMS instrumentation toring based peak areas or incomprehensible QqQ perfor-
is capable of providing higher resolution, superior sensitivity, mance can often be successfully explained and solved by
larger dynamic ranges, and improved speed. Furthermore, HRMS data. HRMS is ideally suited to identify coeluted
downtime, cost, and time for maintenance as well as the and therefore potentially suppressing matrix compounds. It
required user training have become much less relevant than can also be utilized to detect analyte adducts with sodium,
in the past. This will hopefully include the development of fast acetonitrile, ammonia, etc. This might lead to the observa-
and user-friendly dedicated HRMS data processing software. tion that the abundance ratios among these adducts is dif-
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 pure reference solution.
remain screening tools that require an additional MS/MS- Structure elucidation by LCHRMS has two significant
based quantification and confirmation step. Recently launched advantages over more traditional GCMS studies. First, the
HRMS screening software uses calculated exact masses and calculation of possible elemental composition is based on exact
not the experimentally determined retention time of analytes masses and isotopic ratios (RIA). Exact masses and RIA of
as a qualifier for the initial identification of a compound. compounds can be calculated exactly and are platform-
Additional qualifiers to be utilized are the isotopic ratio of independent. As a result, this approach does not rely on exper-
the unfragmented analyte and the exact masses of typical imentally produced libraries. Hence, searches are exhaustive
fragment ions. This approach works well for single-stage and not affected by the completeness or even possible errors
HRMS, where fragmentation is obtained within or beyond within the library. Second, HRMS structure elucidation soft-
the interface. The application of a ramped collision energy ware can be operated with little user training. This permits a
produces a sufficiently high yield of product ions, while much larger number of analysts to venture into the field of
maintaining an acceptable ion count of the intact precursor structure elucidation. More often than not, an observed inter-
ion as well. Suitable software is able to indicate findings esting finding or a suspected peak is not investigated further in
which require an additional confirmation and quantification a busy routine laboratory. This has less to do with a lack of
by comparing the retention time and peak area for a final scientific curiosity; it is rather the lack of time which prevents
confirmation and quantification. This will permit new further investigations. HRMS enables busy analytical chemists
approaches for multiresidue analysis. Instead of producing to focus on such a finding and hopefully discover something
and injecting mixed standard solutions containing more than which might not have been have attempted without the avail-
100 pesticides, only the most often found compounds, or even ability of such an easy to use methodology.
The current role of high-resolution mass spectrometry in food analysis 1249

It is likely that soon the main workload of routine trace 22. Kaufmann A, Butcher P (2006) Rapid Commun Mass Spectrom
20:35663572
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