Anal Bioanal Chem (2007) 389:643–651 DOI 10.1007/s00216-007-1253-8
ORIGINAL PAPER
Simplified multiresidue method for determination of pesticide residues in lettuce by gas chromatography with nitrogen–phosphorus detection José Fenoll & Pilar Hellín & Josefa López & Alberto González & Pilar Flores
Received: 29 January 2007 / Revised: 8 March 2007 / Accepted: 9 March 2007 / Published online: 29 March 2007 # Springer-Verlag 2007
Abstract A rapid and simple method has been developed for simultaneous determination of different classes of pesticide in different varieties of lettuce (Lactuca sativum). Lettuce samples were extracted by homogenization with acetone and partitioned into ethyl acetate–cyclohexane. Subsequent sample clean-up was not needed. Pesticide residues were determined by capillary gas chromatography with nitrogen–phosphorus detection (NPD). Confirmatory analysis of the pesticides was performed by capillary gas chromatography coupled with mass spectrometry in selected-ion-monitoring (SIM) mode. Recovery at two levels of fortification (ca. 0.05 and 0.20 mg kg−1) ranged from 63.9 to 118.6%, and relative standard deviations were below 9.5%. The proposed method was used to determine pesticide levels in different types of lettuce grown in soil from experimental fields. Keywords Multiresidue . Lettuce . Pesticides . Gas chromatography
Introduction Use of pesticides is common practice in modern agriculture to control pests and diseases that damage vegetables and fruit. Although these compounds bring unquestionable benefits in increasing agricultural production, the toxicity of pesticides makes it necessary to monitor the quality of fruit and vegetables to avoid possible risks to consumers J. Fenoll (*) : P. Hellín : J. López : A. González : P. Flores Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/ Mayor s/n, La Alberca, 30150 Murcia, Spain e-mail:
[email protected]
[1]. Multiresidue methods enabling analysis of different pesticide classes have been generally developed for determination of these compounds in several matrices. Classical methods for determination of several pesticides in fruit and vegetables are often expensive, laborious and slow. A large and diverse literature is available on the extraction and purification of pesticides from fruits and vegetables by methods which include liquid–liquid extraction (LLE) [2], solid-phase extraction (SPE) [3, 4], accelerated solvent extraction (ASE) [5], gel-permeation chromatography (GPC) [6, 7], microwave-assisted extraction (MAE) [8], matrix solid–phase dispersion (MSPD) [9] and supercritical-fluid extraction (SFE) [10]. Several chromatographic methods have been published for determination of different pesticides in foods. Pesticide residues have usually been analyzed by gas chromatography with different selective detectors, for example flame photometric (FPD) [11], pulsed flame photometric (PFPD) [12], nitrogen–phosphorus (NPD) [13], and electron-capture (ECD) detectors [14, 15] or, more recently, coupled to mass spectrometry (GC–MS) [16, 17]. For non-volatile and/or thermally unstable and/or polar pesticides and metabolites, liquid chromatography (LC) with diode-array detection (DAD) [18], fluorescence detection [19], mass spectrometry (MS) [20, 21], or tandem mass spectrometry (MS–MS) [22, 23] has been also used. Growing lettuce is one of the main cultivation activities in the Murcia region of Spain. It accounts for 2.28% of total agricultural land in the region, with a total production of 374,684 tons. Approximately 75% of the lettuce crop is exported, mainly to the EU [24]. For this reason it is necessary to develop an analytical method for simultaneous determination of pesticide residues in this vegetable. The principal objective of this work was to develop a rapid multiresidue method for analysis of 25 pesticides in
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Table 1 Retention time (RT, min), molecular weight (MW), target (T) and qualifier (Q1, Q2, and Q3) ions (m/z), and abundance ratios (%)for qualifier ion/target ion (Q1/T and Q2/T)a of the pesticides No.
Pesticide
RT
MW
T
Q1
Q2
Q3
Q1/T
Q2/T
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Dimethoate Propyzamide Pyrimethanil Pirimicarb Vinclozolin Metalaxyl Fenitrothion Malathion Chlorpyrifos ethyl Cyprodinil Pendimethalin Procimidone Fludioxonil Fluazifop-p-butyl Iprodione I Benalaxyl Tebuconazole Iprodione II 1-Cyhalothrin I 1-Cyhalothrin II Acrinathrin Cyfluthrin I Cyfluthrin II Cyfluthrin III Cyfluthrin IV Cypermethrin I Cypermethrin II Cypermethrin III Cypermethrin IV Fluvalinate-tau I Fluvalinate-tau II Difenoconazol I Difenoconazol II Deltamethrin Azoxystrobin
12.68 13.95 14.13 15.69 16.63 17.34 18.07 18.80 19.23 20.54 20.99 21.96 24.06 25.32 25.65 26.75 27.43 28.39 30.09 30.37 30.71 32.22 32.36 32.48 32.54 32.69 32.84 32.97 33.02 34.72 34.85 35.16 35.23 36.00 36.72
229.3 256.1 199.3 238.3 286.1 279.3 277.2 330.4 350.6 225.3 281.3 284.1 248.2 383.4 330.2 325.4 307.8 330.2 449.9 449.9 541.4 434.3 434.3 434.3 434.3 416.3 416.3 416.3 416.3 502.9 502.9 406.3 406.3 502.2 403.4
87 173 198 166 212 206 277 173 197 224 252 96 248 282 187 148 125 314 181 181 181 163 163 163 163 181 181 163 163 250 250 323 323 181 344
93 175 199 72 285 45 125 127 199 225 253 283 127 254 189 91 250 187 197 197 208 206 206 206 206 163 163 181 181 252 252 265 265 253 388
125 145 200 238 198 160 109 125 314 210 281 285 154 383 244 206 70 189 208 208 93 165 165 165 227 165 165 165 165 209 209 325 325 251 345
143 255 77 167 187 249 260 93 97 226 162 67 182 255 124 204 83 244 209 209 289 227 227 227 199 77 209 209 209 181 181 267 267 255 372
60.2 62.3 46.5 50.4 97.0 62.8 97.4 85.3 93.2 62.8 14.9 70.2 25.3 49.2 72.8 42.6 99.6 44.9 77.5 83.6 63.3 69.3 71.0 67.2 65.7 87.2 95.0 81.2 81.4 33.6 35.0 87.6 86.8 66.5 30.4
59.7 29.2 6.0 25.3 89.5 52.7 76.8 83.5 70.1 10.3 12.7 47.3 23.5 48.6 65.1 27.8 46.0 33.6 51.8 53.6 52.6 65.9 66.2 66.8 52.4 75.3 80.3 65.9 64.2 29.3 28.6 66.6 66.3 41.9 28.7
a
Q/T (%) ratios were obtained by dividing abundances of qualifier ions Q1 and Q2 by the abundance of the target ion (T) and multiplying by 100
five varieties of lettuce (baby leaf, romaine, red oak leaf, lollo biondo, and lollo rosso) commonly cultivated in Spain [25]. The method has advantages compared with other conventional methods because of the use of a small volume of organic solvent for sample extraction and because a chlorinated hydrocarbon solvent is not required. The different varieties of lettuce were extracted with acetone and the analytes were partitioned into ethyl acetate–cyclohexane, without use of a cleanup step. Residue levels in the lettuce were determined by gas chromatography (GC) with nitrogen–phosphorus detection (NPD).
Experimental Materials and standards Reference pesticide standards were purchased from Dr Ehrenstorfer (Augsburg, Germany); the purity ranged from 94 to 100%. Pesticide-residue grade acetone, acetonitrile, dichloromethane, ethyl acetate, cyclohexane, and n-hexane were obtained from Scharlau (Barcelona, Spain).Pesticide stock solutions (1000 μg mL−1) of individual pesticide standards were prepared by dissolving 0.025 g pesticide in 25 mL ethyl acetate–cyclohexane (1:1, v/v).
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a
645 54
1
35
pA
130
50
110
33
21
9
7
32
20
46
8
30 31
23 27 22 24 26 28 29 25
19
34
pA 90 42
4
30
70
3
50
32
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6
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13
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30 10
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35
pA
130
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110
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30 31
23 26 27 29 22 24 25 28
19
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8
pA 90
42
4
30
70 3
50
2
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10 11
6
32
34 17
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Time (min)
18
14 15 16
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150
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54 35
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23 26 27 22 2425 28 29
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42 4
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34 17
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Fig. 1 Chromatograms (NPD) obtained from: (a) Standard solution (0.25 mg kg−1). (b) Spiked lettuce (baby leaf) sample (0.25 mg kg−1). (c) Spiked lettuce (red oak leaf) sample (0.25 mg kg−1). Peak numbers are identified in Table 1
40
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a
150
54 pA
130 50
110 46
pA 90 42 30
70
32
34
36
Tim e (min)
50 30 10
15
20
25
30
35
40
Time (min)
b
150
54 pA
130 50
110 46
pA 90 42 30
70
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36
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54 pA
130 50
110 46
pA 90 42 30
70
32
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36
Tim e (min)
50 30 10
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20
25
30
35
40
Time (min)
Fig. 2 Chromatograms (NPD) obtained from: (a) A control lettuce (baby leaf) sample. (b) A control lettuce (romaine) sample. (c) A control lettuce (red oak leaf) sample. (d) A control lettuce (lollo biondo) sample. (e) A control lettuce (lollo rosso) sample
Anal Bioanal Chem (2007) 389:643–651
647
d
150
54 pA
130 50
110 46
pA 90 42 30
70
32
34
36
Tim e (min)
50 30 10
15
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25
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e
150
54 pA
130 50
110 46
pA 90 42 30
70
32
34
36
Tim e (min)
50 30 10
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Time (min)
Fig. 2 (continued)
A pesticide intermediate standard solution (10 μg mL−1) was prepared by transferring 1 mL from each pesticide solution into a 100 mL volumetric flask and diluting to volume with ethyl acetate–cyclohexane (1/1, v/v). Several standard solutions, with concentrations in the range 0.05– 2 μg mL−1, were injected to study the linearity of detector response and the detection limits of the pesticides. Chromatography and mass spectrometry GC–NPD analysis was performed with an Agilent (Waldbronn, Germany) model HP 6890 gas chromatograph equipped with a nitrogen–phosphorus detector and Agilent model 7683 automatic split–splitless injector. Compounds were separated on a 30 m×0.25 mm i.d. fused silica capillary column coated with a 0.25-μm film of HP-5MSI (Agilent). The injector and detector temperatures were 250 and 325 °C, respectively. Nitrogen was used as makeup gas at 25 mL
min−1, helium as carrier gas (constant pressure eluting, bromophos 20.08 min), and hydrogen and air as detector gases at 3 and 60 mL min−1, respectively. The column temperature was maintained at 70 °C for 2 min then programmed at 25° min−1 to 150 °C, then at 3° min−1 to 200 °C, and finally at 8° min−1 to 280 °C which was and held for 10 min. The total analysis time was 41.87 min. Samples (1 μL) were injected in splitless mode. The concentration of each compound was determined by NPD, by comparing the peak areas for the sample with those obtained for mixtures of pesticide standards of known concentration. An Agilent model HP 6890 gas chromatograph equipped with a model 5973N mass spectrometric detector was operated in electron-impact ionization mode with an ionizing electron energy of 70 eV. The spectrometer was scanned from m/z 500 to 50 at 3.21 scans s−1. The ionsource temperature was 230 °C and the quadrupole
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temperature 150 °C. The electron multiplier (EM) potential was maintained at 1300 V and a solvent delay of 4.5 min was used. Gas chromatography was performed under the same conditions as used for GC–NPD. Analysis was performed in selected-ion-monitoring (SIM) mode using primary and secondary ions. The pesticides, with their retention times, molecular mass, their target and qualifier ions, and their qualifier to target abundance ratios are listed in Table 1. The target and qualifier abundances were determined by injection of individual pesticide standards under the same chromatographic conditions using full-scan mode with the mass/ charge ratio ranging from m/z 45 to 500. Pesticides were confirmed by their retention times, identification of target and qualifier ions, and determination of qualifier-to-target ratios. The qualifier-to-target ion percentage was then determined by dividing the abundance of the selected qualifier ion (Q) by that of the target ion (T) and multiplying by 100. Retention times had to be within ±0.1 min of the expected time, and qualifier-to-target ratios had to be within a 10% range for positive confirmation.
Sample preparation Vegetable samples Pesticide-free lettuce (baby leaf, romaine, red oak leaf, lollo biondo, and lollo rosso) grown in a experimental field in Campo de Cartagena (Murcia, Spain) were used as blank samples for spiking and recovery studies. Real lettuce samples, treated with different pesticides, were collected from five experimental fields in the Murcia Region. Procedure A representative portion of the sample (10 g) was transferred to a 100-mL beaker and homogenized with 20 mL acetone for 2 min by means of an Polytron PT2000 homogenizer (Kinematica; Lucerne, Switzerland). After homogenization, 20 mL ethyl acetate–cyclohexane (1:1, v/v) was added and the sample was centrifuged for 10 min at 4,000 g with an Eppendorf (Hamburg, Germany) model 5810R centrifuge.
Table 2 Limits of detection (LOD, μg kg−1) and limits of quantification (LOQ, μg kg−1) of pesticides assayed by GC–NPD Pesticide
Variety of lettuce Baby leaf LOD
Dimethoate Propyzamide Pyrimethanil Pirimicarb Vinclozolin Metalaxyl Fenitrothion Malathion Chlorpyrifos ethyl Cyprodinil Pendimethalin Procimidone Fludioxonil Fluazifop-p-butyl Iprodione Benalaxyl Tebuconazole 1-Cyhalothrin Acrinathrin Cyfluthrin Cypermethrin Fluvalinate-tau Difenoconazol Deltamethrin Azoxystrobin
