73 pestic and metabs in fruits and veg

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2004; 18: 2443–2450

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1645

A multi-residue screening method for the determination

of 73 pesticides and metabolites in fruit and vegetables

using high-performance liquid chromatography/tandem

mass spectrometry

Christel L. Hetherton1, Mark D. Sykes1*, Richard J. Fussell1 and David M. Goodall2

1Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK2Department of Chemistry, University of York, Heslington, York YO10 5DD, UK

Received 26 May 2004; Revised 16 August 2004; Accepted 17 August 2004

A multi-residue screening method was developed for the simultaneous analysis of 73 pesticides

and their metabolites using high-performance liquid chromatography coupled with electrospray

tandem mass spectrometry. These pesticides were determined under a single set of experimental

conditions involving a simple acetonitrile extraction without the requirement for a clean-up step.

Validation was achieved at 0.01 and 0.1 mg kg�1 levels in apple, lettuce and orange. Recoveries were

in the range 77–124% for the majority of pesticides. # Crown Copyright 2004. Reproduced with the

permission of Her Majesty’s Stationery Office. Published by John Wiley & Sons, Ltd.

The screening of food samples for pesticide residues provides

analytical chemists with a difficult challenge due to the

diverse physicochemical properties of pesticides. The

application of multi-residue methods based on both gas

chromatography/mass spectrometry (GC/MS) and liquid

chromatography/mass spectrometry (LC/MS) is necessary

for the comprehensive screening of more than 800 pesticides

currently listed in the Pesticide Manual.1 These pesticides are

applied to agricultural crops to control undesirable insects,

mites, fungi, weeds, nematodes and molluscs. Monitoring

programmes are necessary to ensure that pesticides are being

applied according to Good Agricultural Practice (GAP) and

that maximum residue levels (MRLs) are not exceeded. Tra-

ditionally, GC has been the main analytical technique. The

most widely used confirmatory technique for pesticide resi-

due analysis has been the mass selective detector (MSD) with

electron ionisation (EI).2 The introduction of GC/MS using

an ion trap detector led to the possibility of the simultaneous

screening of up to 200 pesticides and their metabolites.3,4

Most of the recent reviews on multi-residue pesticide analy-

sis are based on determinations by GC rather than LC.5,6

However, the requirement for LC/MS/MS is becoming

more important in monitoring programmes because the

majority of modern pesticides tend to be more amenable to

LC than GC. LC/MS/MS, with its enhanced selectivity,

promises to be the most useful technique complementary to

GC/MS analysis.

Historically, there has been a strong tendency for LC/MS/

MS methods to be developed for a single analyte or a

relatively small multi-residue set of analytes so that both the

LC and MS conditions can be fully optimised for maximum

sensitivity, dependent on the physicochemical properties of

the analytes. Recent improvements in LC/MS/MS instru-

mentation, in particular source design, sensitivity and

electronics, make it possible to increase the number and

diversity of pesticides that can be included in a single LC/

MS/MS analysis.

There are few references in the literature to LC/MS/MS

methods allowing the simultaneous analysis of more than 50

pesticides in fruit and vegetables. Those that do exist tend to

employ extraction methods requiring time-consuming clean-

up procedures that may result in the loss of some

pesticides.7,8 The method of Klein and Alder, based on

methanol extraction and solid-phase extraction (SPE),7 yields

low recoveries for a number of pesticides. Another method,

based on ethyl acetate extraction, necessitates a time-

consuming solvent-exchange step prior to LC/MS/MS

analysis.9 The principal objective of the present study was

to develop an LC/MS/MS method to determine a large

number of different pesticides using a simple acetonitrile

extraction scheme,10 preferably without the need for a clean-

up step. The pesticides to be investigated were selected based

on knowledge of compounds that give poor performance

with GC/MS (or require specific procedures prior to such

analysis, e.g., oxidation of thioether compounds to sulfones),

pesticide usage data,11 and findings presented in national

monitoring reports.12 The secondary objective was to

investigate the use of standard generic instrumental para-

meters in order to significantly reduce the time required to

build acquisition methods for simultaneous multi-residue

pesticide analysis.

# Crown copyright 2004. Reproduced with the permission of Her Majesty’s Stationery Office. Published by John Wiley & Sons, Ltd.

*Correspondence to: M. D. Sykes, Central Science Laboratory,Sand Hutton, York YO41 1LZ, UK.E-mail: [email protected]/grant sponsor: Department for Environment, Foodand Rural Affairs.

EXPERIMENTAL

Chemicals, reagents and materialsCertified reference pesticides were obtained from Qmx

LaboratoriesLtd. (SaffronWalden, UK),Sigma-AldrichChemi-

cal Co. (Poole, UK), Greyhound Chemicals (Birkenhead, UK),

ISK Biosciences (Brussels, Belgium), BASF Corporation

(Princeton, USA) or Bayer (Leverkusen, Germany). All LC/

MS/MS mobile phase components (water, methanol, ammo-

nium acetate, all HPLC fluorescence grade), acetonitrile and

acetone (both HPLC grade), and anhydrous sodium chloride

(Analytical Reagent grade), were obtained from Fisher

Scientific (Loughborough, UK). Anhydrous magnesium

sulfate was supplied by York Glassware (York, UK). Apples,

oranges and lettuce, labelled as organically produced, were

purchased from local retailers.

Preparation of working standard solutionsIndividual stock standard solutions (generally containing

1000 mg mL�1 in methanol or acetonitrile) were prepared

from the reference standard materials. A 10 mg mL�1 working

standard solution in methanol was prepared for each

pesticide by serial dilution of the stock standard solution. A

mixed working standard solution, containing 10mg mL�1 of

each analyte in methanol, was used for the multi-residue

experiments.

Sample preparationBlank samples were frozen at �208C for a minimum of 24 h

and then processed cryogenically, i.e., by milling the frozen

samples in the presence of dry ice. Subsamples (30–40 g) of

the composite were stored at �208C prior to analysis. Com-

minuted samples (10 g) were shaken vigorously for 1 min

with acetonitrile (10 mL). Anhydrous magnesium sulfate

(4 g) and sodium chloride (1 g) were added, and the samples

were then vortexed immediately to prevent formation of co-

agulated magnesium sulfate. After shaking for 30 s, the sam-

ples were centrifuged at 4300 g for 5 min. Aliquots (1 mL) of

the resulting raw extracts were transferred to micro-centri-

fuge tubes containing anhydrous magnesium sulfate

(150 mg). These were mixed and centrifuged at 5200 g for

1 min. An aliquot (500 mL) of the combined sample extract

was adjusted to volume (1 mL) with water, to give a solution

equivalent to a crop concentration of 0.5 g mL�1.

High-performance liquid chromatographyAn Agilent 1100 series (Agilent Technologies, Waldbronn,

Germany) HPLC binary pump and autosampler were used.

Chromatographic separation was achieved using a HyPUR-

ITY C18 analytical column 150� 2.1 mm (i.d.), 5mm particle

size (Thermo Hypersil-Keystone, Runcorn, UK) fitted with

a C18 Security Guard cartridge 4� 2 mm (i.d.) (Phenomenex,

Macclesfield, UK), with a mobile phase flow rate of

0.2 mL min�1. The mobile phase contained 10 mM aqueous

ammonium acetate (A) and unmodified methanol (B). Gradi-

ent elution was used with a starting composition of 10% B, ris-

ing linearly to 90% B over 20 min. The composition was held

at 90% B for a further 10 min before returning to the initial

conditions over 1 min. The column was re-equilibrated for

9 min at the initial mobile phase composition. The integral

switching valve on the mass spectrometer was used to divert

the first 2.5 min of eluent to waste.

Mass spectrometryAll mass spectrometry was performed using a Sciex API 2000

(Applied Biosystems, Ontario, Canada) triple quadrupole

instrument. The mass spectrometer was operated using the

TurboIonsprayTM (TIS) source in the positive mode. The ion-

isation source-specific parameters were: curtain gas (CUR), 50

arbitrary units (a.u.); ionspray voltage, 5000 V; temperature of

the turbo heater gas, 3508C; nebuliser gas (GS1), 40 a.u.; turbo

gas (GS2), 80 a.u. Nitrogen was used as the curtain gas, nebu-

liser gas and turbo gas. The exhaust gas and CUR regulators

were each set at 3.5 bar. The GS1/GS2 regulator was set at

6.5 bar. Unit mass resolution settings were used for Q1 and

Q3. Only the two most critical analyte-dependent parameters,

the declustering potential (DP) and the collision energy (CE),

were optimised for each compound. The remaining para-

meters, generic to all the compounds, were: focusing poten-

tial, 350 V; entrance potential, �10 V; collision cell entrance

potential, 15 V; collision cell exit potential, 5 V; collision-

induced dissociation (CID) gas, also nitrogen, 2 a.u. The

data were acquired using AnalystTM software, version 1.1.

RESULTS AND DISCUSSION

Ionisation optimisationSolutions used for infusion were at 5mg mL�1, prepared by

diluting the working standard solutions 1:1 (v/v) with water.

The CE and DP were optimised by constant infusion of each

pesticide at a rate of 5mL min�1. Negative mode ionisation

was not attempted because switching polarity requires a sta-

bilisation time delay of 700 ms; this would consume a signifi-

cant proportion of the total cycle time and would therefore

limit the total number of multiple reaction monitoring

(MRM) transitions possible in the acquisition programme.

One of the most recently developed pesticides, pyraclos-

trobin, (N-{2-[1-(4-chlorophenyl)-1H-pyrazol-3-yloxymethyl]-

phenyl(N-methoxy)carbamate),1 was chosen as an example

for the purposes of discussion. Pyraclostrobin, a new

fungicide of the strobilurin group, is a synthetic analogue of

strobilurin A, a naturally occurring antifungal metabolite of

the mushroom Strobillurus tenacellus.13 It is thermally labile

and therefore not amenable to analysis by GC/MS; its

structure is shown in Fig. 1(a). The mass spectrum

(Fig. 1(a)) shows that the dominant molecular species was

[MþH]þ at m/z 388, corresponding to the most abundant ion

containing the 35Cl isotope. Collision-induced dissociation

(Fig. 1(b)) produced dominant product ions at m/z 296, 194

and 164. The transition m/z 388! 194 corresponds to

cleavage of the ether bond. The product ion at m/z 296

corresponds to the loss of 92 Da, attributed to the loss of a

methanol molecule and a methyl formate molecule from

pyraclostrobin.

ChromatographyA generic LC method was developed that employs this

laboratory’s existing mobile phase components and column

used for multi-residue analyses of smaller pesticide suites.

Gradient elution conditions were altered to achieve optimum

# Crown copyright 2004. Reproduced with the permission of Her

Majesty’s Stationery Office. Published by John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 2443–2450

2444 C. L. Hetherton et al.

separation of the analytes within an acceptable run time (see

Experimental section). Retention time (tR) values are given in

Table 1.

Various solvent compositions of the final extract were

evaluated to ensure compatibility with the initial mobile

phase conditions. Figure 2 shows a comparison of the LC

peak shapes for imidacloprid (SRM transition m/z 256! 175)

obtained using acetonitrile/water 50:50 (v/v), methanol, and

acetonitrile. As illustrated in the chromatograms, fronting

occurs when neat acetonitrile is used as solvent. On the basis

of these results and our investigations into optimum injection

volume, a 10mL injection in acetonitrile/water 50:50 (v/v)

was selected to obtain acceptable peak shape and sensitivity.

MS/MSThe MS/MS transition that gave the highest MRM response

for each compound was used to build a screening method;

these transitions are given in Table 1. Figure 3 shows the chro-

matogram of a standard containing all 73 pesticides and

metabolites.

In order to minimise time, labour and cost, the acquisition

method should contain two transitions, one for screening

purposes and the second for confirmation of identity should

residues be found. However, it became apparent that the

software currently installed on the instrument allowed a

maximum of only 100 MRM transitions within any single

retention time window (period). It therefore became neces-

sary to investigate the use of two or more periods.

The dependence of peak intensity on dwell time had to be

considered in order to determine the maximum number of

MRM transitions possible in a single period. It is generally

accepted that the distribution of MRM functions into

windows based on analyte retention times permits the use

of longer dwell times for less intense peaks, thereby

increasing their signal-to-noise (S/N) values while maintain-

ing a short overall scan time. However, it has been

demonstrated that the use of very short dwell times results

in only a minor reduction in signal intensity;7 this was

confirmed in our experiments. On this basis, the use of two

periods was initially investigated, the first spanning 0–

22 min, the second 22–40 min. However, the software used

did not allow the use of overlapping time windows, and as a

consequence some of the peaks in the first period were cut off

in mid-acquisition where the greatest number of pesticides

elute. It was therefore decided to investigate the use of three

time periods. Period one covered 0–18 min, period two 18–

24 min and period three 24–40 min. The extracted ion

chromatogram (XIC) of each transition was checked to

ensure that none of the analytes fell outside the acquisition

time. The data for the first period were as predicted, but the

third period in particular showed some ‘ghost’ peaks, i.e.,

peaks were observed occurring at the same apparent

retention time for different (and unrelated) transitions. For

example, hexythiazox (m/z 353! 228), pyridate (m/z

379! 207), imazalil (m/z 297! 159), chlorpyrifos-methyl

(m/z 322! 125) and dimoxystrobin (m/z 327! 205) all

appeared to have exactly the same retention time of

24.65 min. A similar phenomenon was observed for other

transitions at different retention times. For example, difenco-

nazole (m/z 406! 251), tolylfluanid (m/z 347! 137), fenpyro-

ximate (m/z 422! 366) and deltamethrin (m/z 523! 281) all

appeared at 30.90 min although their anticipated retention

times were 25.7, 24.6, 28.3 and 28.6 min, respectively.

The unreliability of the method based on three periods

forced the decision to use only the strongest transition of each

analyte to build a screening method based on one period. This

had the disadvantage that, should any residues be found, it

would then be necessary to repeat analysis of the sample

using a second, dedicated acquisition method containing a

second transition for confirmation.

Since the validation of this method (detailed below), our

laboratory has received the upgraded software, Analyst

version 1.4. The original acquisition method, based on two

transitions per compound over three periods, was re-run

with the new software. No changes were made to the method

at all. The resulting data showed no sign at all of the ‘ghost

peak’ effect; the problem transitions described above were

specifically checked. Again, there was no sign of ‘ghost’ peaks

for those transitions in the third period. This suggests that

software revision, intentionally or otherwise, has cured this

problem. One of the deliberate revisions of the software by

the manufacturer was to increase above 100 the maximum

number of transitions that can be entered into a single period.

Unfortunately, for this work, the software upgrade did not

arrive in time to use it during validation.

Figure 1. (a) Mass spectrum of the pesticide pyraclostrobin.

The structure of the molecule is shown as an insert.

Spectrum recorded by infusion of a standard solution

(5mgmL�1 in water/methanol 1:1, v/v) to the ES ionisation

source in positive mode. (b) Product ion spectrum of the

[MþH]þ ion of pyraclostrobin (precursor ion m/z 388).

LC/MS/MS multi-residue method for pesticides in produce 2445



Table 1. Chromatographic and MS parameters for 73 pesticides and metabolites included in the screening method (a: second

ion not established for confirmation)

Compound tR/minScreeningtransitions DP/V CE/V

Confirmatorytransitions DP/V CE/V

Aldicarb 17.7 208! 89 10 23 208! 116 10 13Aldicarb sulfone 10.4 240! 148 15 19 223! 86 15 25Aldicarb sulfoxide 9.2 224! 132 15 15 207! 89 15 19Atrazine 21.0 216! 174 40 25 216! 96 40 35Azinphos-methyl 22.0 318! 160 30 10 318! 261 30 10Azoxystrobin 22.2 404! 372 40 20 404! 344 40 35Bendiocarb 19.4 224! 109 30 25 224! 167 30 15Carbaryl 20.1 202! 145 20 10 a a aCarbendazim 16.8 192! 160 45 28 192! 132 45 42Carbofuran 19.4 222! 123 40 30 222! 165 40 203-Hydroxy Carbofuran 15.5 238! 163 25 21 a a aChlorpyrifos 27.5 350! 198 47 24 350! 322 47 16Chlorpyrifos methyl 25.8 322! 125 30 26 322! 290 30 21Cyprodinil 22.3 226! 108 35 38 226! 93 35 30Deltamethrin 28.6 523! 281 50 23 521! 279 50 23Demeton-S-methyl 19.6 231! 89 10 25 231! 61 10 35Demeton-S-methyl sulfone 12.2 263! 125 20 35 263! 127 20 40Demeton-S-methyl sulfoxide 11.7 247! 169 12 20 247! 127 12 40Dichlorvos 19.3 221! 109 60 25 221! 127 60 25Diethofencarb 22.3 268! 226 50 15 268! 152 50 33Difenconazole 25.7 406! 251 100 35 406! 337 100 23Dimoxystrobin 24.5 327! 205 50 15 327! 116 50 30Disulfoton 25.6 275! 89 10 15 275! 61 10 45Disulfoton sulfone 21.0 307! 97 60 41 307! 125 60 20Disulfoton sulfoxide 20.8 291! 185 30 16 291! 97 30 45Dodine 28.4 228! 43 45 50 228! 57 45 40Ethiofencarb sulfoxide 14.2 242! 185 8 30 242! 164 8 15Fenamiphos 24.2 304! 217 50 30 304! 202 50 45Fenamiphos sulfone 19.9 336! 308 80 20 336! 266 80 26Fenamiphos sulfoxide 19.6 320! 233 60 30 320! 292 60 20Fenoxycarb 24.4 302! 88 40 30 302! 116 40 15Fenpropimorph 31.3 304! 147 15 40 304! 130 15 35Fenpyroximate 28.3 422! 366 60 22 422! 135 60 45Fensulfothion 21.3 309! 157 60 30 309! 253 60 25Flufenacet 23.7 364! 194 40 15 364! 152 40 27Fosthiazate 20.5 284! 104 20 30 284! 228 20 15Furathiocarb 26.4 383! 195 70 24 383! 252 70 15Furmecyclox 24.7 252! 170 25 19 252! 138 25 21Hexythiazox 27.3 353! 228 45 21 353! 168 45 35Imazalil 24.9 297! 159 70 30 297! 201 70 30Imidacloprid 14.5 256! 175 60 27 256! 209 60 21Isoprocarb 21.1 194! 137 20 15 194! 152 20 15Isoproturon 21.4 207! 165 70 20 207! 73 70 40Kresoxim-methyl 24.5 314! 206 30 15 314! 267 30 15Linuron 22.7 249! 160 51 27 249! 182 56 23Metalaxyl 21.3 280! 220 20 20 280! 248 20 15Methamidophos 4.0 142! 94 50 20 142! 125 50 20Methiocarb 22.8 226! 169 50 15 226! 121 50 25Methiocarb sulfone 16.2 258! 122 20 25 258! 107 60 51Methiocarb sulfoxide 15.0 242! 168 50 31 242! 185 50 37Methomyl 11.5 163! 88 8 16 163! 106 8 16Monocrotophos 13.1 224! 193 20 10 224! 98 20 18Nuarimol 22.5 315! 81 65 49 315! 252 65 31Omethoate 7.5 214! 183 20 15 214! 155 20 25Oxamyl 10.7 220! 72 12 20 220! 90 12 10Phorate 25.4 261! 75 20 16 261! 97 20 37Phorate sulfone 21.1 293! 97 50 40 293! 115 50 35Phorate sulfoxide 20.8 277! 97 30 45 277! 143 30 26Propamocarb (free base) 14.8 189! 102 35 23 189! 144 35 17Pymetrozine 12.7 218! 105 36 31 a a aPyraclostrobin 25.1 388! 194 50 18 388! 164 35 25Pyridate 30.7 379! 207 45 24 379! 351 45 15Spiroxamine 29.4 298! 144 80 30 298! 100 80 45

Continues




Method validationThe finalised LC/MS/MS method was validated for each

pesticide investigated by analysis of five replicate recoveries

at two spike levels (0.01 and 0.10 mg kg�1) together with ana-

lysis of one blank extract. In order to generate reliable and

accurate results, matrix-matched standards were used in

each calibration procedure. The method was validated in

three commodities, apple, orange and lettuce. The results

given in Table 2 show that the majority of mean recoveries

were in the range 79–119% at the 0.10 mg kg�1 spiking level,

with associated relative standard deviations (RSDs) <14%.

Exceptions were pymetrozine, which has a poor recovery

possibly due to instability at the pH of the extraction regime,

and furmecyclox which shows poor recovery results for let-

tuce. At the lower spiking level of 0.01 mg kg�1, most reco-

veries were in the range 77–124% with associated RSDs <20%.

Pesticides giving unsatisfactory recoveries were terbufos,

chlorpyrifos-methyl, deltamethrin, ethiofencarb sulfoxide,

methamidophos, oxamyl, pymetrozine, and thiabendazole.

Polar analytes with low tR values, such as aldicarb sulfoxide,

aldicarb sulfone, demeton-S-methyl sulfoxide, pymetrozine,

methamidophos, omethoate, monocrotophos and oxamyl,

suffered generally from broad peak shapes possibly due to

the injection of samples in 50% acetonitrile. However, most

are still considered acceptable for screening purposes. The

low recoveries for thiabendazole are difficult to explain, but

have been observed before.14

Testing of the methodIn order to provide an appropriate test for the screening

method developed, a European Union (EU) proficiency sam-

ple was supplied as a blind sample for analysis.15 The sample

was extracted using the acetonitrile procedure and analysed

(without clean-up) against matrix-matched standard calibra-

tions for the 73 pesticides.

The screening method successfully identified those resi-

dues present in the sample that could be recognised on the

basis of their inclusion in the present acquisition method. All

other pesticides were detected in a parallel GC/MS screen.

The results of the LC/MS/MS analysis are shown in Table 3,

together with the results previously obtained by this

laboratory using conventional methods, actual spike levels

and the assigned values. Omethoate, demeton-S-methyl and

methiocarb sulfoxide were found by LC/MS/MS, and their

presence confirmed by GC/MS analysis; in addition, the

levels found were close to those found by GC/MS analysis.

The LC/MS/MS screening analysis also identified methio-

carb sulfone and azinphos-methyl residues not reported by

the GC/MS method. In order to quantify and confirm the

residues, the sample was re-analysed using a separate

Table 1. Continued

Compound tR/minScreeningtransitions DP/V CE/V

Confirmatorytransitions DP/V CE/V

Terbufos 26.7 289! 103 30 15 289! 233 30 10Terbufos sulfone 22.3 321! 171 20 15 321! 265 20 10Terbufos sulfoxide 22.3 305! 187 50 20 305! 243 50 10Thiabendazole 18.4 202! 175 90 30 202! 131 90 30Thiamethoxam 12.4 292! 211 35 17 292! 181 35 31Thiodicarb 20.4 355! 88 50 30 355! 108 50 30Tolylfluanid 24.6 347! 137 45 35 347! 238 45 13Triadimefon 23.2 294! 197 30 21 294! 69 30 33Triadimenol 23.7 296! 70 25 35 298! 70 25 35Trifloxystrobin 25.6 409! 186 30 25 409! 206 40 20

Figure 2. SRM chromatogram (m/z 256! 175) of imidaclo-

prid standard dissolved in different solvents used to

demonstrate the effect of solvent composition on peak

shape. Injection volume 5 mL, analyte concentration

0.10mgmL�1: (a) acetonitrile/water, 50:50 (v/v); (b) 100%

methanol; and (c) 100% acetonitrile.




Figure 3. Combined MRM chromatogram of a matrix-matched standard at 0.05mgkg�1 prepared in orange.

Table 2. Recovery data (%) obtained for orange, apple and lettuce using the LC/MS/MS screening method. The validation

batches consisted of five replicate recoveries at each of two spike levels (0.01 and 0.10mg kg�1) alongside analysis of one blank

extract (nr: no result)

Spiking level 0.1 mg kg�1 Spiking level 0.01 mg kg�1

Orange Apple Lettuce Orange Apple Lettuce

Mean RSD Mean RSD Mean RSD Mean RSD Mean RSD Mean RSD

Aldicarb 114 4 96 2 102 10 104 9 94 23 100 8Aldicarb sulfone 100 8 92 11 98 8 140 12 nr nr nr nrAldicarb sulfoxide 87 5 97 3 79 5 91 17 92 5 104 15Atrazine 113 3 107 2 103 2 112 2 88 6 106 7Azinphos-methyl 108 3 97 5 100 3 111 19 77 14 99 6Azoxystrobin 110 6 105 4 104 4 110 6 99 6 108 5Bendiocarb 119 2 106 2 103 2 115 5 94 14 110 17Carbaryl 117 4 104 4 106 4 111 6 94 6 104 3Carbofuran 91 2 93 1 106 2 90 6 81 9 113 53-Hydroxy Carbofuran 115 3 98 4 98 3 118 5 111 12 103 7Carbendazim 103 3 94 3 93 4 93 6 102 10 97 3Chlorpyrifos 113 4 101 4 111 4 117 15 136 9 107 14Chlorpyrifos-methyl 111 8 98 9 105 12 124 21 106 25 123 23Cyprodinil 102 2 101 12 107 3 109 6 nr nr 109 8Diethofencarb 116 4 108 3 101 2 121 11 81 5 108 5Deltamethrin 108 3 107 9 104 18 nr nr 113 24 111 40Demeton-S-methyl 105 4 101 3 92 2 107 5 99 6 114 6Demeton-S-methyl sulfone 98 3 107 4 99 3 115 5 106 25 106 11Demeton-S-methyl sulfoxide 82 9 96 2 96 3 106 6 101 6 102 3Dichlorvos 110 11 98 7 111 5 130 32 104 8 92 18Difenconazole 106 5 96 7 102 7 116 7 78 10 117 13Dimoxystrobin 111 1 101 4 108 8 103 6 97 2 113 8Dodine 94 6 109 5 92 8 74 16 97 16 90 6

Continues




LC/MS/MS acquisition method including two transitions

per analyte for the residues found. The second, confirmatory,

transitions used were those shown in Table 2. The method

with two transitions per analyte confirmed the presence of

methiocarb sulfone, but not azinphos-methyl.

CONCLUSIONS

An LC/MS/MS screening method was successfully devel-

oped and employed to determine a large number of target

pesticides in apple, orange and lettuce. The potential of

LC/MS/MS for such multi-residue applications has been

shown to be important as a technique complementary to

GC analysis, as a result of its high sensitivity and selectivity.

The false positive result for the azinphos-methyl residue,

obtained for the blind sample by the screening method based

on one transition only, highlights the importance of using a

second transition for confirmation. Further modification of

the method is required to reduce the need for re-analysis of

samples containing residues. Pesticides such as carbendazim,

Table 2. Continued

Spiking level 0.1 mg kg�1 Spiking level 0.01 mg kg�1

Orange Apple Lettuce Orange Apple Lettuce

Mean RSD Mean RSD Mean RSD Mean RSD Mean RSD Mean RSD

Disulfoton 106 3 108 9 103 3 120 7 129 6 105 10Disulfoton sulfone 109 4 100 4 107 4 105 8 60 11 107 4Disulfoton sulfoxide 106 1 101 3 103 2 111 3 95 9 107 6Ethiofencarb sulfoxide 88 5 96 8 96 4 93 13 72 44 105 7Fenamiphos 118 4 99 4 104 3 113 9 115 6 107 6Fenamiphos sulfone 115 2 96 3 98 4 113 8 98 8 117 10Fenamiphos sulfoxide 98 3 101 4 99 5 113 8 92 17 101 11Fenoxycarb 114 2 106 9 101 5 121 4 92 11 108 3Fenpropimorph 102 3 97 2 105 9 113 6 119 9 108 11Fenpyroximate 109 3 99 4 109 7 99 8 115 6 100 17Fensulfothion 107 4 100 4 102 4 97 7 55 10 104 18Flufenacet 111 2 101 2 107 6 107 6 101 7 97 6Fosthiazate 117 2 102 3 109 4 109 8 93 3 109 8Furathiocarb 109 2 105 2 108 6 115 3 98 2 108 7Furmecyclox 113 2 102 2 8 2 110 6 99 4 87 35Hexythiazox 112 6 105 9 115 7 119 11 126 15 93 12Imazalil 103 4 102 3 100 3 93 10 100 15 113 10Imidacloprid 99 7 92 9 96 6 122 3 106 38 96 22Isoprocarb 119 2 106 4 102 3 122 7 87 11 107 5Isoproturon 111 3 101 4 95 4 136 22 115 13 98 16Kresoxim-methyl 113 7 97 5 100 1 120 13 92 22 90 4Linuron 104 4 101 5 104 8 118 9 124 7 103 4Metalaxyl 113 3 104 6 98 3 114 3 91 6 107 8Methamidophos 84 13 80 7 83 9 101 8 82 46 88 28Methiocarb 105 4 99 3 106 4 116 8 92 5 106 7Methiocarb sulfone 125 5 106 6 107 6 124 9 116 11 130 11Methiocarb sulfoxide 112 3 92 7 98 2 99 8 106 12 112 21Methomyl 104 2 110 9 97 4 110 8 118 15 103 7Monocrotophos 109 1 100 5 92 5 101 9 91 7 95 9Nuarimol 116 6 100 6 109 11 158 28 116 32 119 32Omethoate 83 13 92 9 82 6 74 22 98 10 100 3Oxamyl 100 3 99 11 104 6 109 4 54 24 103 8Phorate 106 7 105 12 102 7 115 12 129 13 109 15Phorate sulfone 106 2 113 5 106 2 112 9 78 12 135 21Phorate sulfoxide 131 4 104 2 105 3 119 7 88 5 113 4Propamocarb (free base) 79 7 84 4 79 2 75 7 91 3 86 3Pymetrozine 16 8 80 28 72 4 nr nr nr nr 71 4Pyraclostrobin 112 3 101 2 106 5 119 6 97 6 105 5Pyridate 105 8 96 3 105 7 138 9 122 7 123 12Spiroxamine 107 2 100 2 117 6 109 2 110 3 114 7Terbufos 115 6 93 11 96 14 nr nr 66 49 147 39Terbufos sulfone 120 7 115 4 110 6 113 11 66 13 112 10Terbufos sulfoxide 118 5 107 3 105 5 116 9 89 7 109 2Thiabendazole 84 5 87 3 100 3 74 15 65 6 93 12Thiamethoxam 101 4 89 6 89 2 105 4 93 20 94 7Thiodicarb 106 4 97 4 103 6 112 6 95 6 107 5Tolylfluanid 113 1 105 7 110 8 118 10 100 9 128 14Triadimefon 111 3 104 3 112 5 123 9 109 10 117 9Triadimenol 105 5 107 2 105 5 112 6 88 8 108 8Trifloxystrobin 112 2 103 3 105 5 111 3 84 5 106 5




thiabendazole, imazalil, aldicarb and its metabolites, which

are frequently found in food samples, should always be

represented by two ions in the method.

Within the limitations of the particular software version

used, the scope of the method could potentially be increased

to include a further 27 pesticides, enabling the simultaneous

analysis of 100 pesticides. With Analyst version 1.4, the scope

of the method could be further extended until limited by

other factors.

AcknowledgementsThe Department for Environment, Food and Rural Affairs

(Defra), is gratefully acknowledged for funding this work.

We thank BASF Corporation and Bayer for the provision of

standards.

REFERENCES

1. Tomlin CDS (ed). The e-Pesticide Manual, BCPC, (12th edn).version 2.2., 2002–2003.

2. Pihlstrom T. Development of Enhanced Analytical Method-ology in Pesticide Chemistry. 2003; Uppsala University;http://urn.kb.se/resolve?urn¼urn:nbn:se:uu:diva-3406.

3. Tuinstra L. Int. J. Environ. Anal. Chem. 1995; 58: 81.4. Lehotay S, Eller J. J. AOAC Int. 1995; 78: 821.5. Sherma J. J. AOAC Int. 2001; 84: 1303.6. Vander Hoff G, van Zoonen P. J. Chromatogr. A 1999; 843:

301.7. Klein J, Alder L. J. AOAC Int. 2003; 86: 1015.8. Kearney G, Alder L, Newton A, Klein J. Application Note

March 2003. http://www.waters.com.9. Jansson C, Pihlstrom T, Osterdahl B-G, Markides K. J.

Chromatogr. A 2003; 1023: 93.10. Anastassiades M, Lehotay SJ. J. AOAC Int. 2003; 86: 412.11. Thomas MR, Wardman OL. Review of Usage of Pesticides

in Agriculture & Horticulture Throughout Great Britain1986–1996, Pesticide Usage Survey Group Report. 1999; 150.

12. Pesticide Residues Committee Annual Reports, 2000–2002,Defra Publications, London. http://www.pesticides.gov.uk/committees/prc.htm.

13. Anke T. Fungal Biotechnology. Chapman & Hall: London,1997; 206–212.

14. Fussell RJ, Smith F, Patel K, Sykes MD. Central ScienceLaboratory Internal Report, FD 02/33, 2003.

15. European Commission’s Proficiency Test on Pesticide Resi-dues in Fruit and Vegetables, Proficiency Test 5, 2003,National Food Administration: Sweden.

Table 3. Results of analysis of EU proficiency sample analysed by the present LC/MS/MS screening method compared to

results obtained by GC/MS, showing actual spiking levels and assigned values. The azinphos-methyl residue found by the

screening method was not confirmed by the second ion (nr: no result)

Pesticide

Level / mg kg�1

LC/MS/MSscreening

LC/MS/MSconfirmation GC Spike value

Assignedvalue

Azinphos-methyl 0.328 nr nr nr nrDemeton-S-methyl sulfoxide 0.164 0.179 0.234 0.200 0.180Methiocarb sulfone 0.006 0.016 nr nr nrMethiocarb sulfoxide 0.538 0.566 0.477 0.500 0.504Omethoate 0.055 0.045 0.038 0.052 0.059




73 pestic and metabs in fruits and veg

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