Available online at www.sciencedirect.com
Journal of Chromatography A, 1175 (2007) 24–37
Multi-residue method for the analysis of 101 pesticides and their
degradates in food and water samples by liquid
chromatography/time-of-flight mass spectrometry
Imma Ferrer a,∗ , E. Michael Thurman b
a
Department of Analytical Chemistry, University of Almerı́a, Almerı́a, Spain
b University of Colorado, Boulder, CO, USA
Received 25 July 2007; received in revised form 24 September 2007; accepted 27 September 2007
Available online 17 October 2007
Abstract
A comprehensive multi-residue method for the chromatographic separation and accurate mass identification of 101 pesticides and their
degradation products using liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) is reported here. Several classes of compounds belonging to different chemical families (triazines, organophosphorous, carbamates, phenylureas, neonicotinoids, etc.) were carefully
chosen to cover a wide range of applications in the environmental field. Excellent chromatographic separation was achieved by the use
of narrow accurate mass windows (0.05 Da) in a 30 min interval. Accurate mass measurements were always below 2 ppm error for all the
pesticides studied. A table compiling the accurate masses for 101 compounds together with the accurate mass of several fragment ions is
included. At least the accurate mass for one main fragment ion for each pesticide was obtained to achieve the minimum of identification
points according to the 2002/657/EC European Decision, thus fulfilling the EU point system requirement for identification of contaminants
in samples. The method was validated with vegetable samples. Calibration curves were linear and covered two orders of magnitude (from
5 to 500 ␮g/L) for most of the compounds studied. Instrument detection limits (LODs) ranged from 0.04 to 150 ␮g/kg in green-pepper
samples. The methodology was successfully applied to the analysis of vegetable and water samples containing pesticides and their degradation products. This paper serves as a guide for those working in the analytical field of pesticides, as well as a powerful tool for finding
non-targets and unknowns in environmental samples that have not been previously included in any of the routine target multi-residue methods.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Liquid chromatography/mass spectrometry; Time-of-flight; Environmental samples; Pesticides
1. Introduction
The analysis of pesticides in food and water is a major
environmental concern and new instrumental techniques are
constantly being sought for better detection and monitoring.
One of the problems for multi-residue methods by conventional
LC/MS is the decision of which pesticides should be measured. With over 600 active ingredients currently in legal use
in Europe [1], one must choose analytes of interest for monitoring purposes. Recent reviews [2–4] on pesticides in food and
water have commented on the unique ability of accurate mass
∗
Corresponding author. Tel.: +34 950 014102.
E-mail address: [email protected] (I. Ferrer).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.09.092
to identify both target compounds and non-targets by liquid
chromatography/time-of-flight mass spectrometry (LC/TOFMS); thus, offering a possible solution to this conundrum.
Therefore, LC/TOF-MS is a relatively new and valuable technique for the control of pesticides to ensure food safety. In this
sense time-of-flight techniques can record an accurate full-scan
spectrum throughout the acquisition range and have resulted in
an excellent tool for the unequivocal target and non-target identification and confirmation of pesticide residues in vegetable and
fruits [5,6].
One of the weaknesses of LC/TOF-MS and liquid
chromatography/quadrupole time-of-flight mass spectrometry
(LC/Q-TOF-MS) has been the lack of quantitative results. However, recent breakthroughs in instrument design now make
LC/TOF-MS a quantitative tool [7] with mass accuracies that
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
are in the 1–2 ppm range, for several types of instruments when
used in environmental analyses [8–14]. These changes relate to
extending the linear dynamic range of the instrument by using
analog-to-digital converter (ADC) rather than time-to-digital
converter (TDC) [15]. Furthermore, innovations in chromatographic particle chemistry (from 5 to 3.5 or 1.8 ␮m packings, as
well as new bonding chemistries) have improved the separation
of pesticides [16].
In general, official routine laboratories analyze a certain
number of target compounds (ranging from 1 up to less
than 50 different compounds) [16,17] depending on the legal
requirements for positive identifications and the scope of the
methodology used in the respective labs. The literature has hundreds of papers reporting diverse LC–MS methodologies for
the analysis of all the different classes of pesticide compounds.
Several review papers have tried to compile all the existent information regarding mass spectrometric data (including fragment
ions) using different instrumentation (ion-trap, triple quad, TOF,
Q-TOF), but unfortunately, in every case, singular information
is obtained depending on the method of detection used [18].
For example when using tandem mass spectrometric techniques
the instrument parameters (especially the fragmentor voltage
and collision energy) play an important role on the number
of fragments and relative intensities obtained. For this reason,
many attempts to exploit MS–MS fragmentation mass libraries
have failed due to the differences in instrumentation and operating conditions. However, this is not the case of time-of-flight
techniques, since accurate mass measurements are specific and
universal for every target analyte and do not depend on the
instrumentation used. In this way, a number of publications
regarding the use of accurate mass databases of pesticides have
been reported recently [19,20]. Accurate mass determination
allows obtaining specific information for a given molecule plus
an additional confirmation if more fragments are present in the
spectra. For this reason, a study containing an extensive number
of compounds has been carried out in this work.
This paper describes a multi-residue method for 101 commonly used pesticides, including complete information on
accurate masses for the protonated molecules and fragment
ions, retention times on a C8 reversed-phase column, limits of
detection and calibration curves. We have evaluated the potential of LC/TOF-MS for the quantitative analyses of pesticides
in food and water samples at concentrations in the low ␮g/L
range. The proposed method for vegetable and fruit samples
consists of a sample treatment step using an extraction with
acetonitrile followed by quantitative analyses by LC/TOF-MS.
The sample treatment applied to water samples is based on
solid-phase extraction (SPE) using Sep-Pak C18 cartridges. The
method developed is sensitive for the detection of the 101 pesticides in food samples, which meets the current 0.01 mg/kg
standard of the EU 91/414/EC food directive. This method will
work well for accurate mass instruments since it is not instrument specific. Thus, it is highly useful for identification of at
least 101 pesticides in food and water matrices. Finally, the
proposed method has been successfully applied to real environmental samples including food commodities and surface water
samples.
25
2. Experimental
2.1. Chemicals and reagents
Pesticide analytical standards were purchased from both
Sigma (St. Louis, MO, USA) and Chem Service (West Chester,
PA, USA). Individual pesticide stock solutions (1000 ␮g/mL)
were prepared in pure acetonitrile and stored at −18 ◦ C. HPLC
grade acetonitrile and methanol were obtained from Merck
(Darmstadt, Germany). Formic acid was obtained from Fluka
(Buchs, Switzerland). A Milli-Q-Plus ultra-pure water system
from Millipore (Milford, MA, USA) was used throughout the
study to obtain the HPLC-grade water used during the analyses.
Anhydrous magnesium sulfate and sodium acetate were from
Sigma–Aldrich (Madrid, Spain). For the SPE procedure, Seppak
C18 cartridges (500 mg, 6 mL) obtained from Waters (Milford,
MA, USA) were used.
2.2. Sample preparation
2.2.1. Vegetable and fruit samples
The QuEchERS method (acronym for “quick, easy, cheap,
effective, rugged and safe) was used for the extraction of food
samples [21]. According to this method, a 15-g portion of food
sample previously homogenized was weighted in a 200 mL
PTFE centrifuge tube. Then, 15 mL of acetonitrile were added
and the tube was vigorously shaken for 1 min. After this time,
1.5 g of NaCl and 4 g of MgSO4 were added repeating then
the shaking process again for 1 min to prevent coagulation of
MgSO4 . The extract then was centrifuged (3700 rpm) for 1 min.
A 5 mL aliquot of the supernatant (acetonitrile phase) was then
taken with a pipette and transfer to a 15 mL graduated centrifuge
tube, containing 250 mg of PSA (propylamino SPE cartridge;
Supelco, Bellefonte, PA, USA) and 750 mg of MgSO4 , being
then energetically shaken for 20 s. After this, the extract was
centrifuged again (3700 rpm) for 1 min. Finally, an extract containing 1 g of sample per mL in 100% acetonitrile was obtained.
The extract was then evaporated near to dryness and reconstituted to initial mobile phase composition up to 1 mL. Prior to
analysis, the extract was filtered through a 0.45 ␮m PTFE filter
and transferred into a vial. Matrix extracts were used for validation of the method by appropriate spiking with the pesticide
mix. The scope of this work was simply to develop a method for
the screening, quantitation and confirmation of 101 pesticides
in vegetable and fruit matrices, so recovery of the compounds
from raw samples was not taken into account here. Vegetables
and fruit samples included green-peppers, tomatoes, cucumbers
and oranges.
2.2.2. Water samples
An off-line SPE was used for the pre-concentration of the
water samples. All the extraction experiments were performed
using an automated sample preparation with extraction column
system (ASPEC XL, Gilson, Villiers-le-Bel, France) fitted with
an external 306 LC pump for dispensing the water samples
through the SPE cartridges and with 817 switching valve for the
selection of each sample. Disposable cartridge columns packed
26
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
Table 1
LC/TOF-MS accurate masses for the protonated molecules and the main fragment ions for all the compounds studied (fragmentor voltage 190 V)
Compound
Retention time (min)
Elemental compositiona
Accurate mass [M + H]+
Frag ion 1
Acetamiprid
Acetochlor
Alachlor
Aldicarb
Aldicarb sulfone
Aldicarb sulfoxide
Atrazine
Azoxystrobin
Benalaxyl
Bendiocarb
Bensultap
Bromoxynil
Bromuconazole
Buprofezin
Butylate
Captan
Carbaryl
Carbendazim
Carbofuran
Cartap
Chlorfenvinphos
Chlorpyrifos methyl
Cyanazine
Cyproconazole
Cyromazine
DEET
Deethylatrazine
Deethylterbuthylazine
Deisopropylatrazine
Diazinon
Dichlorvos
Difeconazole
Difenoxuron
Diflubenzuron
Dimethenamide
Dimethoate
Dimethomorph
Diuron
Ethiofencarb
Fenamiphos
Fenuron
Flufenacet
Flufenoxuron
Fluoroacetamide
Fluroxypyr
Hexaflumuron
Hydroxyatrazine
Imazalil
Imazapyr
Imazaquin
Imidacloprid
Ioxynil
Iprodione
Irgarol 1051
Irgarol metabolite
Isoproturon
Lenacil
Lufenuron
Malathion
Mebendazole
Metalaxyl
Metamitron
Methidathion
Methiocarb
16.8
26.1
26.1
18.7
11.8
6.5
21.4
24.3
26.8
20.8
21.4
21.7
24.0 + 24.8
27.4
29.7
24.4
21.3
7
20.8
3.1
26.5
28.2
19.6
23.6
2.9
21.3
15.9
19.6
13
27.8
20
26.4 + 26.6
21.6
25.2
24.3
16.6
22.5 + 22.8
21.7
21.8
24.1
15.7
26.1
29.5
3.1
19.2
27.5
12.1
18.1
13.7
19
16
23
25.6
21.2
17
21.6
19.6
28.9
26
18.4
21.5
15.2
24.1
23.7
C10 H11 N4 Cl
C14 H20 NO2 Cl
C14 H20 NO2 Cl
C7 H14 N2 O2 S
C7 H14 N2 O4 S
C7 H14 N2 O3 S
C8 H14 N5 Cl
C22 H17 N3 O5
C20 H23 NO3
C11 H13 NO4
C17 H21 NO4 S4
C7 H3 NOBr2
C13 H12 N3 OCl2 Br
C16 H23 N3 OS
C11 H23 NOS
C9 H8 NO2 SCl3
C12 H11 NO2
C9 H9 N3 O2
C12 H15 NO3
C7 H15 N3 O2 S2
C12 H14 O4 PCl3
C7 H7 NO3 PSCl3
C9 H13 N6 Cl
C15 H18 N3 OCl
C6 H10 N6
C12 H17 NO
C6 H10 N5 Cl
C7 H12 N5 Cl
C5 H8 N5 Cl
C12 H21 N2 O3 PS
C4 H7 O4 PCl2
C19 H17 N3 O3 Cl2
C16 H18 N2 O3
C14 H9 N2 O2 F2 Cl
C12 H18 NO2 SCl
C5 H12 NO3 PS2
C21 H22 NO4 Cl
C9 H10 N2 OCl2
C11 H15 NO2 S
C13 H22 NO3 PS
C9 H12 N2 O
C14 H13 N3 O2 F4 S
C21 H11 N2 O3 F6 Cl
C2 H4 NOF
C7 H5 N2 O3 FCl2
C16 H8 N2 O3 F6 Cl2
C8 H15 N5 O
C14 H14 N2 OCl2
C13 H15 N3 O3
C17 H17 N3 O3
C9 H10 N5 O2 Cl
C7 H3 NOI2
C13 H13 N3 O3 Cl2
C11 H19 N5 S
C8 H15 N5 S
C12 H18 N2 O
C13 H18 N2 O2
C17 H8 N2 O3 F8 Cl2
C10 H19 O6 PS2
C16 H13 N3 O3
C15 H21 NO4
C10 H10 N4 O
C6 H12 N2 O4 PS3
C11 H15 NO2 S
223.0745
270.1255
270.1255
213.0668b
223.0747
207.0798
216.1010
404.1241
326.1751
224.0917
432.0426
275.8654
375.9614
306.1635
218.1573
299.9414
202.0863
192.0768
222.1125
126.0105
224.0837
238.0993
116.0528
148.0427
132.0478
174.0541
372.0979
294.1489
167.0703
290.0338
358.9768
321.9023
241.0963
292.1211
167.1040
192.1383
188.0697
202.0854
174.0541
305.1083
220.9532
406.0720
287.1390
311.0393
276.0820
230.0069
388.1310
233.0243
226.0896
304.1131
165.1022
364.0737
489.0435
78.0350
254.9734
460.9889
198.1349
297.0556
262.1186
312.1343
256.0596
371.8377
330.0407
254.1434
214.1121
207.1492
235.1441
510.9857
331.0433
296.1030
280.1543
203.0927
302.9691
226.0896
158.9763
201.1056
162.0947
263.9647
145.0648
160.0505
165.0910
150.0406
204.9373
124.9821
214.0854
125.0153
108.0556
119.0491
146.0228
146.0228
146.0228
169.0794
127.0155
337.0393
123.0441
158.0412
244.0557
198.9647
301.0626
72.0444
164.0706
276.0818
72.0444
194.0976
158.0412
208.9679
158.0412
156.0880
255.0086
234.1237
284.1394
209.0588
244.9879
198.0808
158.0495
165.1022
153.0659
158.0412
285.0015
264.0768
248.1281
175.0978
145.0066
169.0682
Frag ion 2
Frag ion 3
148.1121
162.1277
89.0419
166.0532
89.0419
146.0228
133.0886
208.1332
109.0284
148.0757
70.0651
86.0600
235.9693
123.0446
104.9827
155.0468
98.9842
70.0400
91.0542
153.1022
109.0049
251.0025
141.0146
168.0841
170.9698
124.9821
107.0491
152.0506
180.9730
158.9763
266.1288
175.0978
72.0444
127.0390
124.9821
220.1332
192.1383
85.0396
122.0726
121.0648
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
27
Table 1 (Continued )
Compound
Retention time (min)
Elemental compositiona
Accurate mass [M + H]+
Frag ion 1
Frag ion 2
Frag ion 3
Methiocarb sulfone
Methomyl
Metolachlor
Metolcarb
Metribuzin
Molinate
Monuron
Nicosulfuron
Nitenpyram
Oxadixyl
Parathion ethyl
Pendimethalin
Phosmet
Prochloraz
Profenofos
Promecarb
Prometon
Prometryn
Propachlor
Propanil
Propiconazole
Prosulfocarb
Simazine
Spinosad A
Spinosad D
Spiromesifen
Spiroxamine
Teflubenzuron
Terbuthylazine
Terbutryn
Thiabendazole
Thiacloprid
Thiocyclam
Thiosultap
Triclocarban
Triflumizole
Trifluralin
17.7
12.6
25.9
19.7
20.1
24.8
19.2
18.1
12.1
19.1
27.3
30.2
24.3
23
28.6
24.4
16.6
20.3
22.8
23.3
25.9 + 26.1
29
19.1
20.7
21.4
30.7
19.7
27.9
23.8
20.4
8.8
18.3
4.5
3.2
27.5
25.9
30.6
C11 H15 NO4 S
C5 H10 N2 O2 S
C15 H22 NO2 Cl
C9 H11 NO2
C8 H14 N4 OS
C9 H17 NOS
C9 H11 N2 OCl
C15 H18 N6 O6 S
C11 H15 N4 O2 Cl
C14 H18 N2 O4
C10 H14 NO5 PS
C13 H19 N3 O4
C11 H12 NO4 PS2
C15 H16 N3 O2 Cl3
C11 H15 O3 PSClBr
C12 H17 NO2
C10 H19 N5 O
C10 H19 N5 S
C11 H14 NOCl
C9 H9 NOCl2
C15 H17 N3 O2 Cl2
C14 H21 NOS
C7 H12 N5 Cl
C41 H65 NO10
C42 H67 NO10
C23 H30 O4
C18 H35 NO2
C14 H6 N2 O2 F4 Cl2
C9 H16 N5 Cl
C10 H19 N5 S
C10 H7 N3 S
C10 H9 N4 SCl
C5 H11 NS3
C5 H13 NO6 S4
C13 H9 N2 OCl3
C15 H15 N3 OF3 Cl
C13 H17 N3 O4 F3
258.0795
163.0536
284.1412
166.0863
215.0961
188.1104
199.0633
411.1081
271.0956
279.1339
292.0403
282.1448
318.0018
376.0381
372.9424
208.1332
226.1662
242.1434
212.0837
218.0134
342.0771
252.1417
202.0854
732.4681
746.4838
371.2217
298.2741
380.9815
230.1167
242.1434
202.0433
253.0309
182.0126
311.9698
314.9853
346.0929
336.1166
201.0580
106.0321
252.1150
109.0648
187.1012
126.0913
72.0444
213.0328
225.1027
219.1128
264.0090
212.0666
160.0393
308.0006
344.9111
151.1117
184.1193
200.0964
170.0367
161.9872
158.9763
128.1070
132.0323
544.3633
558.3789
255.1380
144.1383
158.0412
174.0541
186.0808
175.0324
126.0105
136.9548
232.0130
161.9872
278.0554
122.0726
88.0215
72.9981
94.0413
182.0560
196.0636
133.0886
235.9777
194.0560
99.0917
132.0808
265.9537
302.8642
109.0653
142.0723
158.0495
127.0183
91.0542
100.1121
146.0228
127.0183
In bold the base peak ion observed in the spectrum at 190 V.
a Elemental compositions correspond to the neutral molecule.
b Ion corresponding to the sodium adduct [M + Na]+ .
with 500 mg of Seppak C18 sorbent were used. The cartridges
were conditioned with 6 mL of methanol followed by 6 mL of
HPLC water at a flow rate of 1 mL/min. The water samples
(100 mL) were loaded at a flow rate of 10 mL/min. Elution of
the analytes from the cartridge was carried out with 3 mL of ethyl
acetate. The solvent was evaporated with a stream of nitrogen
to near dryness and re-dissolved in 0.3 mL of mobile phase for
LC/TOF-MS analysis.
2.3. LC/TOF-MS analyses
The separation of the selected herbicides was carried
out using an HPLC system (consisting of vacuum degasser,
autosampler and a binary pump) (Agilent Series 1100, Agilent Technologies, Santa Clara, CA, USA) equipped with a
reversed phase C8 analytical column of 150 mm × 4.6 mm and
5 ␮m particle size (Zorbax Eclipse XDB-C8). Column temperature was maintained at 25 ◦ C. The injected sample volume
was 50 ␮L. Mobile phases A and B were acetonitrile and water
with 0.1% formic acid, respectively. The optimized chromatographic method held the initial mobile phase composition (10%
A) constant for 5 min, followed by a linear gradient to 100%
A after 30 min. The flow-rate used was 0.6 mL/min. A 10 min
post-run time was used after each analysis. This HPLC system
was connected to a time-of-flight mass spectrometer Agilent
MSD TOF equipped with an electrospray interface operating
in positive ion mode, using the following operation parameters: capillary voltage, 4000 V; nebulizer pressure, 40 psig;
drying gas, 9 L/min; gas temperature, 300 ◦ C; fragmentor voltage, 190 V; skimmer voltage, 60 V; octopole d.c. 1, 37.5 V;
octopole RF, 250 V. LC/MS accurate mass spectra were recorded
across the range 50–1000 m/z. The data recorded was processed
with Applied Biosystems/MDS-SCIEX Analyst QS software
(Frankfurt, Germany) with accurate mass application-specific
additions from Agilent MSD TOF software. Accurate mass
measurements of each peak from the total ion chromatograms
were obtained by means of an automated calibrant delivery system using a dual-nebulizer ESI source that introduces the flow
28
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
from the outlet of the chromatograph together with a low flow
of a calibrating solution (calibrant solution A, Agilent Technologies), which contains the internal reference masses (purine
(C5 H4 N4 at m/z 121.0509 and HP-921 [hexakis-(1H,1H,3Htetrafluoro-pentoxy)phosphazene] (C18 H18 O6 N3 P3 F24 ) at m/z
922.0098. The instrument worked providing a typical resolution
of 9700 ± 500 (m/z 922).
3. Results and discussion
3.1. LC/TOF-MS separation and detection of 101 pesticides
The pesticides included in this study were selected among
different classes of compounds (triazines, organophosphates,
carbamates, phenylureas, etc.) and several chemical uses (insecticides, herbicides and fungicides). Most of these compounds are
currently analyzed by hundreds of laboratories performing target analysis of pesticides in both food and water samples, and
for this reason they were included in this study. Table 1 compiles the chemical formulae and exact accurate masses obtained
by TOF-MS, as well as the retention times for all the pesticides analyzed in this study. Of the 101 pesticides, 76 presented
an [M + H]+ peak as a base peak in the spectrum (base peak
ions are marked in bold in Table 1). Surprisingly, 25 pesticides
did not present the protonated molecule as a main base peak
in the spectrum in spite of the low fragmentor voltage used;
in all these cases the larger ion was a fragment ion. Only one
compound (aldicarb) presented a sodium adduct as a base peak
and in only one case (cartap) both the protonated molecule and
the sodium adduct were absent, only two fragments showed
up in the spectrum in this particular case. Some of the most
usual detected degradation products in environmental samples
were also included in this study (e.g. degradation products for
atrazine, aldicarb, etc.) for more complete and detailed information.
A linear gradient starting with 10% acetonitrile up to 100%
in 30 min was applied, which was first developed by our group
[8] and had proven to be successful for the separation of a
wide variety of pesticide compounds. Fig. 1 shows the total
ion chromatogram for the 101 pesticides analyzed. As it can
be observed in Table 1 from the retention times, the majority of
compounds elute in a 10 min time window comprised between
16 and 26 min, mainly due to the similarity in polarity among
the pesticides studied. Nevertheless, good chromatographic separation was obtained for all the compounds by using extracted
narrow mass windows of 0.05 Da.
3.2. Structural characterization of the analytes
3.2.1. Accurate mass of fragment ions
The fragmentor voltage role in LC–MS is critical to obtain
structural information of the target analytes, as well as a way
to get the best balance between sensitivity and fragmentation. For this reason, the fragmentor voltage was increased to
obtain additional information from characteristic fragments of
the compounds. Every compound was studied separately (single
Fig. 1. Total ion chromatogram (TIC) corresponding to the analysis of a mix of 101 pesticides (0.1 ␮g/mL) by LC/TOF-MS.
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
29
Fig. 2. (a) Extracted ion chromatogram (XIC) corresponding to the analysis of a blank green-pepper sample where the banned pesticide nitenpyram was detected.
(b) Spectrum of nitenpyram showing the characteristic isotopic chlorine signature.
Fig. 3. (a) Total ion chromatogram (TIC) corresponding to the analysis of a spiked tomato sample with the studied pesticides (0.05 mg/kg) by LC/TOF-MS. (b)
Extracted ion chromatograms (XICs) corresponding to some protonated molecules (mass window 0.05 Da).
30
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
compound injections were carried out at different fragmentor
voltages) in order to obtain specific information on the fragments obtained, and accurate mass was used to determine the
particular fragment for each compound. Among the 101 pesticides, 96 of them clearly showed at least one fragment ion at
a medium fragmentor voltage of 190 V (Table 1). About half
of the pesticides (49) presented at least two fragment ions, and
a smaller number of pesticides (12) showed as much as three
fragment ions. Some of these fragments are the base peak ions
in the corresponding spectra, so it is important to account for
them when carrying out quantitation in order to achieve maximum sensitivity. Most of these fragments have been reported
by other studies using tandem mass spectrometry techniques,
so all the results obtained here match in every case and they
demonstrate that time-of-flight without MS–MS can be used as
an identification tool using fragments from the in-source collision induced dissociation. In addition to fragmentation we obtain
accurate mass information for every specific fragment that is
highly useful for unequivocal identification. For example, in
some cases the exact formula and hence the accurate mass of the
protonated molecule was identical for four pairs of compounds:
alachlor/acetochlor, deethylterbuthylazine/simazine, ethiofencarb/methiocarb, and prometryn/terbutryn. In all these cases, the
specific information on their fragment ions, which are different,
is essential to tell both compounds apart and to make a correct
identification, especially if retention time is close. Thus, if necessary, the fragmentor voltage may be increased to get enhanced
sensitivity for the fragment ion for a positive confirmation of the
analyte.
3.2.2. Accurate mass of isotopes
Additional information can be obtained for those compounds
containing elemental isotopes, such as chlorine, bromine or sulfur. In these cases, the accurate masses for these isotopic signals
are obtained and offer an extra added identification point for
confirmatory purposes [13]. An example is shown in Fig. 2 for
the identification of nitenpyram, a non-authorized pesticide, in
a blank of green-pepper sample that was found to contain this
insecticide. As it can be seen in this figure, the chlorine isotopic signal is obtained and the accurate mass of the chlorine 37
isotopes can be measured with a very small error. It is important to note that only 30 out of the 101 pesticides studied did
not present an A + 2 isotopic signal, these were the compounds
that contained mainly C, H, O and N. For the rest of pesticides
(70%), the accurate mass value of the A + 2 ion is highly useful
for the correct identification of the analyte as shown in Fig. 2
and it should be used as a tool for identification.
In summary, the accurate mass analysis of the protonated
molecule together with that of additional characteristic fragment
ion(s) (including characteristic isotopic signals and retention
times) enables the unambiguous identification and confirmation
of the studied pesticides at low concentration levels. This fits
the requirements of the EU according to the identification point
system [22].
3.3. Analytical performance
To evaluate the usefulness of LC/TOF-MS for quantitative
analyses in vegetable matrices, the analytical performance of the
Fig. 4. Quantitation window showing some extracted ion chromatograms (XICs) corresponding to the base peak ion for 12 selected compounds (mass window
0.1 Da).
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
31
Table 2
Calibration data, correlation coefficients and instrument LODs for all the analytes studied in a green-pepper matrix sample
Compound
Calibration curve
R2
LODs (␮g/kg)
Acetamiprid
Acetochlor
Alachlor
Aldicarb
Aldicarb sulfone
Aldicarb sulfoxide
Atrazine
Azoxystrobin
Benalaxyl
Bendiocarb
Bensultap
Bromoxynil
Bromuconazole
Buprofezin
Butylate
Captan
Carbaryl
Carbendazim
Carbofuran
Cartap
Chlorfenvinphos
Chlorpyrifos methyl
Cyanazine
Cyproconazole
Cyromazine
DEET
Deethylatrazine
Deethylterbuthylazine
Deisopropylatrazine
Diazinon
Dichlorvos
Difeconazole
Difenoxuron
Diflubenzuron
Dimethenamide
Dimethoate
Dimethomorph
Diuron
Ethiofencarb
Fenamiphos
Fenuron
Flufenacet
Flufenoxuron
Fluoroacetamide
Fluoroxypyr
Hexaflumuron
Hydroxyatrazine
Imazalil
Imazapyr
Imazaquin
Imidacloprid
Ioxynil
Iprodione
Irgarol 1051
Irgarol metabolite
Isoproturon
Lenacil
Lufenuron
Malathion
Mebendazole
Metalaxyl
Metamitron
Methidathion
Methiocarb
y = 1.74 × 104 C − 4.43 × 103
0.999
0.998
0.998
0.999
0.988
0.987
0.999
0.999
0.996
0.994
0.985
0.989
0.996
0.999
0.997
0.992
0.999
0.998
0.998
0.988
0.992
0.997
0.999
0.996
0.998
0.998
0.999
0.997
0.994
0.996
0.994
0.995
0.998
0.996
0.998
0.999
0.998
0.998
0.999
0.997
0.997
0.998
0.999
0.986
0.988
0.993
0.992
0.999
0.998
0.997
0.998
0.992
0.987
0.998
0.997
0.999
0.996
0.995
0.998
0.998
0.999
0.997
0.995
0.997
3
2
3
5
3
4
0.5
0.1
0.04
5
281a
20
0.3
0.6
3
15
3
0.8
4
15
0.2
30
2
1
9
2
2
1.5
2
0.05
0.5
0.5
0.4
12
1
1.5
4
0.6
4
0.1
10
3
6
80
45
8
0.4
0.3
5
0.7
2
15
4
0.1
0.5
0.7
9
9
1.5
0.8
0.2
3
15
0.7
y = 2.11 × 104 C + 3.38 × 103
y = 1.6 × 104 C + 1.08 × 104
y = 8.78 × 103 C − 8.66 × 103
y = 7.19 × 103 C + 5.36 × 103
y = 1.8 × 104 C + 1.34 × 105
y = 1.74 × 105 C − 1.29 × 105
y = 3.47 × 104 C − 1.8 × 104
y = 1.81 × 105 C − 9.96 × 104
y = 1.30 × 104 C − 1.31 × 104
y = 9.82 × 102 C − 260
y = 3.42 × 102 C + 3.56 × 103
y = 1.35 × 104 C − 8.19 × 103
y = 5.06 × 105 C − 5.35 × 105
y = 7.24 × 103 C − 5.9 × 103
y = 7.80 × 102 C − 1.74 × 104
y = 5.39 × 104 C + 1.2 × 104
y = 5.91 × 104 C + 3.49 × 103
y = 4.39 × 104 C − 1 × 104
y = 2.62 × 103 C + 2.25 × 103
y = 2.86 × 104 C − 1.25 × 104
y = 5.03 × 102 C + 2.02 × 104
y = 1.22 × 104 C + 9.56 × 103
y = 8.06 × 104 C − 4.52 × 104
y = 1.29 × 104 C − 2.33 × 104
y = 3.74 × 104 C − 1.29 × 103
y = 3.84 × 104 C − 1.45 × 104
y = 2.85 × 104 C − 1.32 × 104
y = 2.74 × 104 C − 9.22 × 103
y = 6.95 × 105 C − 9.03 × 105
y = 6.67 × 103 C − 1.33 × 103
y = 2.68 × 104 C − 6.21 × 104
y = 1.46 × 105 C + 3.51 × 105
y = 1.03 × 103 C − 586
y = 2.84 × 104 C + 1.62 × 104
y = 1.03 × 104 C − 914
y = 5.04 × 104 C + 5.43 × 105
y = 1.6 × 104 C + 2.65 × 103
y = 7.61 × 104 C + 1.15 × 104
y = 1.53 × 105 C − 7.84 × 104
y = 2.9 × 104 C + 7.52 × 104
y = 9.97 × 103 C + 5.79 × 103
y = 1.17 × 103 C − 3.08 × 103
y = 1.85 × 103 C − 5.91 × 104
y = 3.93 × 102 C − 1.04 × 103
y = 7.69 × 102 C − 1.38 × 103
y = 9.74 × 104 C − 6.39 × 104
y = 9.66 × 104 C − 1.38 × 105
y = 7.44 × 104 C − 2.49 × 104
y = 1.36 × 105 C − 9.31 × 104
y = 5.84 × 103 C − 374
y = 1.05 × 103 C + 454
y = 1.55 × 103 C − 261
y = 2.89 × 105 C − 4.59 × 105
y = 9.48 × 104 C − 1.01 × 105
y = 1.63 × 105 C − 1.88 × 104
y = 2.42 × 104 C + 3.84 × 104
y = 7.74 × 102 C − 2.05 × 103
y = 8.82 × 103 C + 2.53 × 104
y = 6.59 × 104 C − 6.49 × 104
y = 8.47 × 104 C − 1.06 × 104
y = 3.6 × 104 C − 1.78 × 104
y = 3.78 × 103 C + 4.06 × 103
y = 2.28 × 104 C + 2.92 × 103
32
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
Table 2 (Continued )
Compound
Calibration curve
R2
LODs (␮g/kg)
Methiocarb sulfone
Methomyl
Metolachlor
Metolcarb
Metribuzin
Molinate
Monuron
Nicosulfuron
Nitenpyram
Oxadixyl
Parathion ethyl
Pendimethalin
Phosmet
Prochloraz
Profenofos
Promecarb
Prometon
Prometryn
Propachlor
Propanil
Propiconazole
Prosulfocarb
Simazine
Spinosad A
Spinosad D
Spiromesifen
Spiroxamine
Teflubenzuron
Terbuthylazine
Terbutryn
Thiabendazole
Thiacloprid
Thiocyclam
Thiosultap
Triclocarban
Triflumizole
Trifluralin
y = 4.01 × 103 C − 1.97 × 103
y = 2.13 × 104 C + 7.24 × 103
y = 7.19 × 104 C + 3.77 × 103
y = 1.24 × 104 C + 1.44 × 105
y = 1.1 × 105 C − 7.02 × 104
y = 8.04 × 103 C + 9.6
y = 2.4 × 104 C − 1.17 × 103
y = 8.0 × 104 C − 1.1 × 105
y = 2.05 × 104 C + 4.75 × 105
y = 3.28 × 104 C − 3.43 × 104
y = 5.79 × 102 C − 296
y = 4.11 × 103 C − 7.1 × 103
y = 9.93 × 103 C + 1.99 × 103
y = 3.86 × 104 C − 6.31 × 104
y = 5.61 × 103 C − 7.33 × 103
y = 3.11 × 104 C − 1.16 × 104
y = 2.07 × 105 C − 2.33 × 105
y = 3.11 × 105 C − 5.7 × 105
y = 3.28 × 104 C − 1.34 × 104
y = 6.52 × 103 C − 2.23 × 103
y = 3.45 × 104 C − 3.54 × 104
y = 2.04 × 104 C − 2.36 × 104
y = 4.9 × 104 C − 3.6 × 104
y = 4.74 × 104 C − 1.09 × 105
y = 5.48 × 103 C − 1.08 × 104
y = 2.69 × 102 C − 186
y = 2.47 × 104 C − 4.43 × 104
y = 4.01 × 102 C − 896
y = 1.9 × 105 C − 2.77 × 105
y = 3.11 × 105 C − 5.7 × 105
y = 3.17 × 104 C − 8.35 × 104
y = 1.73 × 104 C − 1.22 × 104
y = 7.55 × 103 C − 1.19 × 103
y = 6.45 × 102 C − 170
y = 3.05 × 103 C − 7.72 × 103
y = 9.33 × 103 C − 9.31 × 103
y = 2.02 × 102 C − 929
0.994
0.995
0.999
0.998
0.997
0.996
0.998
0.999
0.999
0.987
0.988
0.996
0.998
0.999
0.997
0.998
0.999
0.999
0.998
0.996
0.997
0.998
0.999
0.988
0.986
0.986
0.988
0.992
0.999
0.998
0.997
0.999
0.996
0.991
0.993
0.996
0.998
9
2
0.8
12
0.6
1.5
0.7
0.8
0.2
14
17
11
0.9
0.7
1
3
1
0.3
0.5
0.7
0.3
2
0.4
0.9
6
120
8
35
0.4
0.3
5
1.5
5
50a
12
3
85
a
Bensultap and thiosultap degraded in the standard solutions due to hydrolysis of the molecule.
proposed method was studied and validated in terms of linearity,
limits of detection and reproducibility of the technique for food
commodities.
3.3.1. Quantitation by LC/TOF-MS
Quantitation of the sample extracts was accomplished using
a calibration curve based on matrix-matched standards: blank
sample extracts from vegetable and fruits were evaporated until
near dryness under a nitrogen flow and then reconstituted with
the 101 pesticide mix standard solution at different concentrations ranging from 0.005 to 0.5 mg/kg in order to have a wide
range of concentrations. The use of matrix-matched standards
provides reliable quantitation capabilities for food pesticide
analysis [8]. Fig. 3 shows the total ion chromatogram of a
tomato-matched standard spiked at 0.05 mg/kg with the mixture
of the 101 pesticides as well as ion extracted chromatograms for
some selected pestides. Analytes can be easily distinguished
among the matrix by the use of narrow accurate mass windows as shown in this figure. Quantitation was performed by
measuring the peak area of the base peak ion of each analyte
(numbers in bold in Table 1). Fig. 4 shows an example of the
quantitation wizard for 12 compounds. As it can be seen in
this figure, the extracted ion chromatograms are automatically
obtained and data are compared to the retention time of each
analyte, based on a previous standard injection. If two or more
peaks are present in the chromatogram, the software assigns as
positive the closest peak to the correct retention time for the
analyte under study. For example, for aldicarb, two peaks are
obtained: one at 6.7 min and the other one at 18.8 min for the
extracted ion at m/z 89.0. The software assigns (in blue matching in the figure) aldicarb to the peak at 18.8 min based on a
previous standard injection. Aldicarb sulfoxide, a metabolite
of aldicarb, which presents obviously the same fragment ion
at m/z 89.0419, gets assigned at 6.7 min. Another interesting
case is the chromatographic coelution of acetochlor and alachlor
at 26.2 min, both having the same protonated molecule at m/z
270.1255 as mentioned before. Fig. 4 shows again how acetochlor and alachlor can be differenciated by their respective
fragment ions at m/z 148.1121 and 162.1277 (which are base
peak ions in their respective spectra in both cases). Interestingly, the peak next to acetochlor at 26.9 corresponds to one
of the fragments of benalaxyl (m/z = 148.0757) which elutes at
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
33
Table 3
Typical diagnostic ions and accurate mass of several pesticide families
Pesticide family
Compound
Phenylureas
Diuron
Fenuron
Isoproturon
Monuron
Organophosphates
Chlorpyrifos methyl
Dimethoate
Malathion
Triazines
Deethylatrazine
Deethylterbuthylazine
Deisopropylatrazine
Diagnostic ion
Accurate mass of diagnostic ion
72.0444
124.9821
146.0228
Atrazine
Terbuthylazine
146.0228 and 174.0541
Fluorobenzoylureas
Diflubenzuron
Flufenoxuron
Hexaflumuron
Lufenuron
Teflubenzuron
158.0412
Neonicotinoids
Acetamiprid
Thiacloprid
126.0105
Conazole fungicides
Bromuconazole
Propiconazole
Imazalil
158.9763
this retention time and happened to be extracted with the 148
ion.
3.3.2. Calibration curves
Linearity was studied in both solvent and matrix-matched
standard solutions of green pepper at five different concentration
levels. Quantitation was carried out using the peak area from the
extracted ion chromatograms (XIC) of the base peak ion (in bold
in Table 1) using a mass window of 0.05 Da. Table 2 shows the
calibration equations obtained for the 101 pesticides in greenpepper matrices and their correlation coefficients. As it can be
observed, the linearity of the analytical response within the studied range of two orders of magnitude is good, with correlation
coefficients equal or higher than 0.99 in all cases.
3.3.3. Limits of detection and reproducibilty
The instrument limits of detection (LODs) were estimated
from the injection of matrix-matched standard solutions with
low concentration levels giving a signal-to-noise ratio of 3. The
results are summarized in Table 2 as well. It should be pointed out
that the LODs were as low as 0.04 ␮g/kg in the case of benalaxyl.
The average values are about 3 ␮g/kg, which is enough to meet
the 10 ␮g/kg standard (Directive 91/414/EC) established for
pesticides in fruits and vegetables [23]. Only few compounds
showed higher LODs due to their low response under electrospray conditions (bromoxynil, captan, chlorpyrifos-methyl,
fluoroacetamide, fluoroxypyr, spiromesifen, teflubenzuron and
trifluralin). Bensultap and thiosultap were found to be degraded
in water solutions due to a possible hydrolysis of the standards
34
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
Fig. 5. Extracted ion chromatogram for m/z 72.0444 (from m/z 72.02 to 72.07, mass window 0.05 Da) corresponding to the analysis of an orange matrix-matched
standard spiked with the mixture of 101 pesticides. Peaks: (1) fenuron, (2) monuron, (3) isoproturon and (4) diuron.
made in water. For the rest of compounds, the signal-to-noise
ratios were good, thus illustrating the high sensitivity and
suitability of LC/TOF-MS for trace analysis of pesticides in
environmental matrices. The LODs for water samples were
similar to the ones obtained in food commodities (results not
shown here). The reproducibility, repeatability and accuracy of
the method were also evaluated on matrix-matched solutions at
two different concentration levels: 0.01 and 0.1 mg/kg. The RSD
(n = 5) values for intra-day analyses were in the range 0.9–4%
and the RSD for inter-day (n = 5) values were between 3.5
and 9%.
3.4. Potential application to non-target pesticides
From data compiled in Table 1 one can extrapolate some
useful information referent to fragment ions. Depending on the
family of pesticides (triazines, phenylureas, organophosphates,
etc.) a trend is observed for fragmentation ions present in their
Fig. 6. (a) Total ion chromatogram corresponding to the LC/TOF-MS analysis of an orange sample where imazalil was detected. (b) Extracted ion chromatogram of
imazalil at m/z 297 (inset: accurate mass spectrum).
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
35
Table 4
LC/TOF-MS accurate mass measurements for the protonated molecules and main fragment ions of positive findings in a surface water sample
Compound
Ion
Atrazine
[M + H]+
Elemental composition
m/z theoretical
m/z experimental
Error
mDa
ppm
Frag. Ion
C8 H14 ClN5
C5 H9 ClN5
216.1010
174.0541
216.1007
174.0538
−0.3
−0.3
−1.6
−1.7
DEET
[M + H]+
Frag. Ion
C12 H17 NO
C12 H17 NO
192.1383
119.0491
192.1382
119.0488
−0.1
−0.3
−0.5
−2.9
Deethylatrazine
[M + H]+
Frag. Ion
C6 H10 ClN5
C3 H5 ClN5
188.0697
146.0228
188.0695
146.0224
−0.3
−0.4
−1.3
−2.7
Deisopropylatrazine
[M + H]+
Frag. Ion
C5 H8 ClN5
C3 H5 ClN5
174.0541
146.0228
174.0537
146.0223
−0.4
−0.5
−2.3
−3.4
Diazinon
[M + H]+
Frag. Ion
C12 H21 N2 O3 PS
C8 H13 N2 S
305.1083
169.0794
305.1087
169.0796
0.4
0.2
1.2
1.2
Dimethenamide
[M + H]+
Frag. Ion
C12 H18 ClNO2 S
C11 H15 ClNOS
276.0820
244.0557
276.0819
244.0554
−0.05
−0.3
−0.2
−1.4
Diuron
[M + H]+
Frag. Ion
C9 H10 Cl2 N2 O
C3 H6 NO
233.0243
72.0444
233.0238
72.0448
−0.5
0.4
−2.1
5.7
Metolachlor
[M + H]+
Frag. Ion
C15 H22 ClNO2
C14 H19 ClNO
284.1412
252.1150
284.1414
252.1148
0.2
−0.2
0.8
−0.7
Prometon
[M + H]+
Frag. Ion
C10 H19 N5 O
C7 H14 N5 O
226.1662
184.1193
226.1661
184.1189
−0.1
−0.4
−0.6
−2.1
Simazine
[M + H]+
Frag. Ion
C7 H12 ClN5
C5 H9 N5 35 Cl
202.0854
132.0323
202.0851
132.0319
−0.3
−0.4
−1.5
−3.0
Fig. 7. (a) Total ion chromatogram corresponding to the LC/TOF-MS analysis of a surface water sample where DEET was detected. (b) Extracted ion chromatogram
of DEET (inset: accurate mass spectrum).
36
I. Ferrer, E.M. Thurman / J. Chromatogr. A 1175 (2007) 24–37
respective spectra. For example, almost all the organophosphate pesticides show the diagnostic ion at m/z 124.9821
corresponding to the elemental formula of C2 H6 O2 PS. Phenylureas diagnostic ion is primarily m/z 72.0444 corresponding
to the C3 H6 NO moiety. Other examples are shown in Table 3
for triazines, fluorobenzoylureas, neonicotinoids and conazole
fungicides. This information is highly useful for the identification of non-target compounds that have not been included in a
multi-residue method but that they may present a common fragment ion for identification. For example in Fig. 5 an extracted
chromatogram for the ion at m/z 72.0444 is shown corresponding to an orange matrix-matched standard spiked with the 101
mixture. All the phenylurea compounds listed in Table 3 are
thus clearly identified with this ion, showing the potential of
this tool for positive identification of compounds belonging to
the same family or degradates. If a new peak shows up in the
chromatogram then a new identification could be made by taking a look at the spectrum obtained for such peak and assigning
elemental formula information to the corresponding protonated
molecule. Moreover, if the mass of a fragment ion shows up
at a different retention time than the protonated molecule this
may indicate the presence of a possible degradation product that
either presents the same mass or contains that fragment ion [24].
3.5. Analysis of food and water samples
To evaluate the application of the proposed methodology,
it was applied to the analysis of real samples including food
commodities and surface water. All the samples were extracted
and analyzed as described in the experimental part and some
examples are described as follows.
3.5.1. Food samples
The methodology was applied to the analysis of several
fruits and vegetables (green-peppers, tomatoes, cucumbers and
oranges). About 82% of the samples analyzed (a total of 20)
showed at least two pesticides. Only 15% of the positive samples presented pesticides at concentrations higher than the MRL.
An example is shown in Fig. 6 where the identification and confirmation of imazalil is carried out in an orange sample. The
accurate mass of the chlorine isotope is used for additional information and it matches with the theoretical exact mass. Imazalil
has been reported by other authors [25] pointing out the importance of carrying out the identification of fungicides in citrus
fruits.
3.5.2. Surface water samples
A total of four surface water samples from Kansas (USA)
were analyzed with the methodology described in this paper and
the comprehensive screening for the 101 pesticides was carried
out. All the samples gave positive findings for several pesticides. As an example, Fig. 7 shows the total ion chromatogram
and the extracted ion chromatogram for DEET (meta-N,Ndiethyltoluamide) at 21.3 min; the corresponding spectrum is
also shown. As it can be observed in the spectrum, the accurate mass of the protonated molecule (m/z 192.1382) presents
an error of only −0.5 ppm as well as its main fragment ion at
m/z 119.0488 does. DEET is used as a repellent for mosquitoes
and other related insects and it is one of the banned insecticides
in some countries in Europe, although it is an authorized pesticide in the USA. Positive findings for other nine pesticides
were confirmed in the same surface water sample with excellent
mass accuracies (<3 ppm) (see Table 4). Triazine compounds
such as atrazine and simazine were identified in this sample
along with the main degradation products, deethylatrazine and
deisopropylatrazine. The other compounds detected were also
herbicides which are widely used in corn and soybean fields
in this geographic area, thus further illustrating the usefulness
and reliability of LC/TOF-MS for the analysis of pesticides in
environmental samples [26].
4. Conclusions
A study to evaluate the potential of LC/TOF-MS for identification and confirmation of pesticides in environmental samples
was carried out. The developed method allows the screening
of 101 pesticides in vegetables and water samples. The LODs
obtained with this method are in compliance with the regulations on food established by the EU. The applicability of the
method was demonstrated by analysis of real samples (food
and water) showing excellent selectivity and sensitivity, thus
making possible the unambiguous identification of the selected
pesticides.
Acknowledgements
This work was supported by the Ministerio de Ciencia
y Tecnologı́a (Spain, Contract: AGL2004-04838/ALI). I. F.
acknowledges the research contract (Contrato de retorno de
Investigadores) from the Consejerı́a de Educación y Ciencia de
la Junta de Andalucı́a, Spain. Dr. Mike Meyer from the US Geological Survey in Lawrence, Kansas (USA) is acknowledged for
surface water samples supply.
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