AOAC Official Method 2007.01
Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate
Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry
First Action 2007
[Applicable for the following pesticides in grapes, lettuces, and oranges: atrazine, azoxystrobin, bifenthrin, carbaryl, chlorothalonil, chlorpyrifos, chlorpyrifos-methyl, l -cyhalothrin (incurred in lettuces), cyprodinil, o,p ¢-DDD, dichlorv os, endosulfan sulfate,ethion (incurred in oranges), imazalil, imidacloprid,kresoxim-methyl (incurred in grapes), linuron, methamidophos,methomyl, permethrins (incurred in lettuces) procymidone,pymetrozine, tebuconazole, thiabendazole (incurred in oranges),tolylfluanid (degraded in lettuces), and trifluralin. The were reprentative pesticide analytes chon in reprentative matrixes,and the method is expected to be applicable to many other similar pesticides and matrixes. Limits of quantitation were demonstrated to be <10 ng/g.]
See Tables 2007.01A–E for the results of the interlaboratory study supporting acceptance of the method.
A. Principle
The QuEChERS (quick, easy, cheap, effective, rugged, and safe)method us a single-step buffered acetonitrile (MeCN) extraction and salting out liquid–liquid partitioning from the water in the sample with MgSO 4. Dispersiv e-solid-pha extraction (dispersive-SPE) cleanup is done to remove organic acids, excess water, and other components with a combination of primary condary amine (PSA) sorbent and MgSO 4; then the extracts are analyzed by mass spectrometry (MS) techniques after a chromatographic analytical paration. Figure 2007.01 outlines the protocol in a box format. In brief, a well-chopped food sample along
with 1 mL of 1% acetic acid (HOAc) in MeCN and 0.5 g anhydrous MgSO 4/NaOAc (4/1, w/w) per g sample are added to a centrifuge tube or bottle, which is shaken and centrifuged. A portion of the MeCN extract (upper layer) is added to anhydrous MgSO 4/PSA sorbent (3/1, w/w; 200 mg per 1 mL extract), mixed, and centrifuged. This final extract is transferred to autosampler vials for analysis by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify and determine a wide range of pesticide residues. To achieve <10 ng/g detection limits in modern GC/MS, large volume injection (LVI) of 8 m L is typically needed, or the final extract can be concentrated and solvent exchanged to toluene (4 g/mL), in which ca 2
m L splitless injection is ud.
Both GC/MS and LC/MS/MS techniques are prone to matrix effects in pesticide residue analysis, albeit for different reasons [Erney, D.R., Gillespie, A.M., Gilvydis, D.M., & Poole, C.F. (1993)J. Chromatogr. 638, 57–63; Hajslova, J., & Zrostlikova, J. (2003) J.Chromatogr. A 1000, 181–197; Alder, L., Luderitz, S., Lindtner, K.,& Stan, H.J. (2004) J. Chromatogr. A 1058, 67–79]. To account for the effects, matrix-matched calibration was conducted (calibration standards in solvent solution may also be ud if matrix effects are shown not to occur). Due to the situation that some laboratories had LVI capability and others did not, the necessary amounts of matrix blank(s) and final extract volume was different for some laboratories than others. Depending on the water content of the matrix, a 15 g sample typically yields 11–14 mL of initial MeCN extract after centrifugation. In dispersive-SPE, roughly half of the extract is lost to the powders, thus about 6–7 mL of final extract can be expected for a 15 g sample. Two options were provided in the protocol to account for the different situations among the laboratories.
Table 2007.01A. Interlaboratory study results for incurred pesticides (and chlorpyrifos-methyl)Analyte
Matrix Avg. concn s r
a
RSD r b
, %S R c
, ng/g Rec., %RSD R d
, %
HorRat No. of labs
Outlier
labs e Chlorpyrifos-methyl
Grapes 165 14 8.535 8321 1.00116-C, 4-C Lettuces 178 20 11 30 89170.811011-SG
Oranges
174 25 14 36 87200.9812Kresoxim-methyl Grapes 9.2 1.921
f
3.2NA 35
f
1.0912Cyprodinil Grapes 112 NA
g
NA 18 NA 160.7313l -Cyhalothrin Lettuces 58 6.111 11 NA 200.80 911-C Permethrins Lettuces 112 9.88.741 NA 36
f
1.63 96-C, 1-C Imidacloprid Lettuces 12NA NA 1.6NA 140.4411Ethion Oranges 198 23 12 36 NA 180.891111-C Thiabendazole Oranges 53 3.87.27.6NA 140.5812Imazalil
Oranges
13
NA
NA
4.7
NA
35
f
1.15
8
7-SG
a s r = Standard deviation for repeatability (within laboratory).
b RSD r = Relative standard deviation for repeatability.
c s R = Standar
d deviation for reproducibility (among laboratories).d RSD R = Relativ
e standard deviation for reproducibility.e C = Cochran outlier; SG = single Grubbs outlier.
f RSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; and fewer than 8 laboratories in an asssment.g
NA = Not applicable.
Analyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine9.30.6 6.9 2.093210.6513
45 3.27.1 5.790130.4913
365 23 6.271 9119 1.0413
Azoxystrobin9.40.6 6.6 2.094210.6413
92 8.79.411 92120.51128-SG
182 17 9.226 91140.70128-SG Bifenthrin7.80.811 2.37830b0.89112-C, 10-C
86 5.9 6.914 86170.73126-C
923 71 7.7136 92150.9113
Carbaryl12 1.211 2.8104 27b0.85125-SG
50 6.413 11 100 220.8713
1003 70 7.0189 100 19 1.18125-C Chlorothalonil 6.30.914 2.163b33b0.97 810-C
网络游戏开发培训59 8.314 13 79230.9310
140 19 13 38 7027b 1.27b10
Chloropyrifos8.1 1.519b 3.08137b 1.1212
68 8.312 14 84200.8413
396 25 6.450 79120.681211-SG Cyprodinil c123 13 10 26 101 210.9513
240 20 8.363 9226b 1.32b13
581 42 7.3110 9519 1.0913
o,p¢-DDD8.9 1.416b 3.28936b 1.0912
42 3.17.37.084170.6512
445 32 7.147 89100.58116-C Dichlorvos7.2 1.014 1.372180.53118-SG
85 7.48.715 85180.77114-C
294 25 8.562 9821 1.1012
Endosulfan sulfate8.60.910 1.586170.52 7b10-C 115 14 12 21 77180.8111
415 56 14 111 8327b 1.47b11
Imazalil7.60.89.8 3.17641b 1.22b11
50 2.5 4.915 67b30b 1.19108-C
432 53 12 161 7837b 2.06b11
Imidacloprid8.80.88.9 3.08834b 1.0413
45 3.57.78.999200.7813
218 18 8.224 97110.56128-SG Linuron9.9 1.717b 2.99929b0.9011
99 7.47.415 99150.6712
971 65 6.7191 9720 1.23b12
Methamidophos10 2.929b 3.0101 30b0.95 95-SG
80 8.010 14 80180.7712
852 72 8.4119 85140.85118-SG Methomyl9.3 1.212 2.99332b0.9812
50 3.3 6.79.3100 190.7413
204 10 4.926 102 130.6313
Procymidone8.20.78.2 2.082240.74115-SG
64 6.09.416 8524 1.0113
hardly是什么意思428 16 3.870 86160.90129-C Pymetrozine 6.2 1.220b 1.662b27b0.7711
47 3.1 6.79.662b200.8111
341 20 5.859 68b170.9211
Tebuconazole9.2 1.112 1.292130.41123&4-DG
63 5.58.78.884140.5813
439 29 6.784 8819 1.0613
Tolylfluanid7.9 1.012 3.17939b 1.1913
34 4.313 13 67b37b 1.41b13
144 13 8.842 7229b 1.37b13
Trifluralin7.80.78.5 1.878230.681210-C
58 3.7 6.414 7725 1.0213arbitrary
379 19 5.148 76130.69106-C, 4-C, 11-SG a
C = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.
b
RSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an asssment.
c
Cyprodinil was incurred in the grapes and affected quantitation.
Analyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine9.9 1.515 1.899180.5611
70 7.9111593210.8812
930 50 5.4166 9318 1.11115-C Azoxystrobin10 0.8 7.6 1.8102 180.5612
47 2.4 5.2 6.593140.5512
rop531 32 6.188106 170.9412
Bifenthrin9.10.8 9.1 1.491160.4811
66 8.012 9.488140.5911
217 27 123387150.7711
Carbaryl9.4 1.112 2.094220.6712
92 6.1 6.7 9.092 9.80.43118-SG
589 38 6.4127 9822 1.24b12
Chlorothalonil 6.20.814 2.062b32b0.93 6b
28 10 37b147048b 1.77b 7b
exceeding
684 134 20b205 68b30b 1.77b 6b
Chloropyrifos9.0 2.124b 2.39026b0.79 912-SG, 10&11-DG
86 9.411208623 1.011111-SG
179 18 103090170.821111-SG Cyprodinil9.7 1.010 1.497140.441111-SG
44 2.7 6.1 8.989200.791111-SG
天道留学好不好848 61 7.2117 85140.84108-SG
o,p¢-DDD8.90.6 7.0 1.989210.66 8
81 4.8 5.91281150.63 911-C
214 19 8.72786130.6210
Dichlorvos 5.2 1.020b 2.452b45b 1.29b12
58 6.6111277200.8112
838 50 6.0224 8427 1.63b11
Endosulfan sulfate 5.6 3.359b 2.556b45b 1.28b 2b
38 9.625b157539b 1.48b 7b
769 330 43b312 7740b 2.44b 7b
Imazalil7.60.3 3.5 3.57639b 1.18 82-C
72 3.7 5.22457b33b 1.39b11
589 47 7.9229 59b39b 2.25b11
Imidacloprid c22 1.3 6.2 1.7100 7.90.28118-SG
84 6.2 7.4 8.197 9.60.4112
515 21 4.253101 100.58115-C Linuron8.6 1.112 1.586170.5311
46 2.2 4.9 7.491160.63102-C
234 14 5.82594100.5311
Methamidophos8.80.8 8.5 1.388150.46 86-C
66 4.5 6.91282180.7211
538 37 6.86384120.67 95-C, 8-SG Methomyl9.70.8 8.6 1.096100.3210
99 8.0 8.1 6.499 6.50.29102-SG
997 24 2.4168 100 17 1.0511
Procymidone10 0.6 6.2 2.2101 220.68 82-C
92 8.5 9.21592170.7311
casual967 118 12129 97130.8311
Pymetrozine 6.90.4 6.1 1.469b200.5910
33 1.6 4.7 4.667b140.51 911-C
127 8.5 6.71763b130.6110
Tebuconazole9.70.7 6.9 1.297130.40114-C
89 6.8 7.71189120.5212
948 42 4.4226 9524 1.48b114-C Tolylfluanid 3.7 1.130b 2.237b59b 1.59b 4b
9.3 3.740b 4.1 9.3b44b 1.37b 83-SG, 8-SG
142 22 158614b61b 2.84b 812-C, 3&8-DG Trifluralin10 1.413 1.7103 170.5411
42 4.511 9.084220.8311
ppf169 25 153084180.8411
a
C = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.
puzzlesb
RSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an asssment.
c
Imidacloprid was incurred in the lettuces unbeknownst to the SD.
Analyte Avg. C, ng/g s r, ng/g RSD r, %s R, ng/g Rec., %RSD R, %HorRat No. of labs Outlier labs Atrazine 8.9 1.011 1.989210.65112-C 908.29.11290130.5712
187 19 10 2793140.6912
Azoxystrobin 8.4 1.316b 1.884210.6511
65 5.28.0 8.186120.5212
853 35 4.18285 9.60.591111-C Bifenthrin 9.7 2.324b 2.397240.75109-SG
45 2.5 5.6 6.891150.59102-C
488 51 10 7698160.8712
Carbaryl 8.40.67.3 2.184250.7710
66 5.07.51488210.8811
172 8.8 5.13486200.9512
Chlorothalonil 4.80.816b 2.748b56b 1.57b 3b
7014 20b297042b 1.74b 6b
330 137 42b131 66b40b 2.09b 7b
Chloropyrifos11 1.614 5.0111 45b 1.58b 92-C
82 4.5 5.61282150.64109-C
953 97 10 284 9530b 1.85b1211-C Cyprodinil 8.70.910 2.087230.7212
56 4.58.0 9.075160.6512
199 12 6.23580180.8612
o,p¢-DDD 9.10.67.2 1.891200.60 99-C
74 5.1 6.9 9.899130.561010-C
967 81 8.41919720 1.22b11
Dichlorvos 9.30.88.1 1.093110.35 7b12-C
43 2.2 5.2 8.085190.73 812-SG
446 22 5.05489120.6810
Endosulfan sulfate12 5.444b 5.4124b43b 1.40b 4b3-SG 8319 23b198323 1.0110
240 35 15 618025 1.28b10
Imazalil c22 1.77.7 6.29628b0.98 87-C
58 4.37.41392220.91 9
186 9.7 5.2418722 1.0610
Imidacloprid10 1.110 2.8104 27b0.8611
93 6.57.01293130.5711
989 64 6.5124 99130.7811
Linuron 7.8 1.317b 2.77835b 1.0411
60 3.0 5.01386210.8611
387 26 6.64279110.59 9111-DG Methamidophos 9.2 1.112 1.592160.49 89-C
42 3.58.2 5.685130.52 84-C
211 12 5.53185150.73 94&9-DG Methomyl 8.50.88.9 2.88533b0.99 97-C
68 4.87.0 8.791130.5412
492 19 3.96098120.6912
Procymidone110.98.1 3.9108 36b 1.15 812-C
43 3.58.0 5.886140.531010-C
170 16 9.72585150.7111
Pymetrozine 7.5 1.318b 2.17528b0.8210
77 5.97.71077140.5710
789 38 4.8117 79150.89 912-C Tebuconazole 8.70.78.0 1.287140.4211
41 2.2 5.4 6.282150.5812
177 14 7.92888160.7612
Tolylfluanid 5.8 1.220b 1.458b240.69 911-SG 467.516b1461b31b 1.21b119-C
356 54 15 134 7138b 2.02b12
Trifluralin 8.60.4 4.5 2.48628b0.87 99-C 928.69.41192120.5412
915 60 6.5194 9221 1.31b116-C
a
C = Cochran outlier; SG = single Grubbs outlier; DG = double Grubbs outliers.
b
RSD r >15%; 120% < Rec. < 70%; RSD R >25%; HorRat >1.2; or fewer than 8 laboratories in an asssment.
c
Imazalil was incurred in the oranges unbeknownst to the SD.
In Option A, if the laboratory had LVI capability, then 1 or 2 mL extracts were taken for dispersive-SPE (the volume depended on the analyst preference and the type of centrifuge and tubes available in the laboratory). The final extract volume was 0.5 mL if 1 mL was taken for dispersive-SPE, and 1 mL if 2 mL underwent the cleanup step. In either ca, two 15 g blank samples were ud for the matrix blank (0-standard) and 6 matrix-matched calibration standards (5, 10, 50, 100, 250, and 1000 ng/g equiv alent concentrations). For dispersive-SPE of the matrix blanks, either 7 parate tubes using the same 1–2 mL extract volumes as the test samples could have been ud, or 1–2 dispersiv
e-SPE tube(s) with 7-fold greater extract volume(s).
In Option B, if LVI is not available for GC/MS, then »30 mL of matrix blank extract was needed after dispersive-SPE cleanup to prepare the matrix-matched calibration standards (or $60 mL initial extract). In this ca, 6 matrix blanks of 15 g each were extracted along with the test samples to provide enough blank extract volume, which were combined, and ven 8 mL aliquots were distributed to 7 dispersive-SPE tubes containing 0.4 g PSA + 1.2 g anhydrous MgSO4.
B. Apparatus and Conditions
Note: Tables 4 and 5 of the collaborative study [J. AOAC Int. 90, 485(2007)] list the analytical instrumentation and sources of sample preparation materials ud by each laboratory in the study. Further information appears in the full report. Since the time of the collaborativ e study, at least 3 v endors, United Chemical Technologies (Bristol, PA, USA), Restek (Bellefonte, PA, USA) and Supelco (Bellefonte, PA, USA) hav e introduced commercial dispersive-SPE products for QuEChERS and other applications. See Table 4 [J. AOAC Int. 90, 485(2007)] for sources of analytical instruments.
(a) Gas chromatograph/mass spectrometer.—An ion trap, quadrupole, time-of-flight (TOF), or other GC/MS instrument may be ud with electron impact (EI) ionization, an autosampler (AS), and comp
uterized instrument control/data collection. Either LVI of 8 m L for a 1 g/mL MeCN extract (e.g., 75°C ramped to 275°C at 200°C/min) or 2 m L splitless injection of 4 g/mL extracts in toluene a t250°C m a y b e u s e d.A3–5m,0.25m m i d, phenylmethyl-deactivated guard column must be ud as a retention gap in either ca. The analytical column is a 30 m, 0.25 mm id, 0.25 m m film thickness (5%phenyl)-methylpolysiloxane (low bleed) analytical column (DB-5ms or equiv alent). Set He head pressure on the column to be 10 psi or constant flow to be 1.0 mL/min with systems capable of electronic pressure/flow control. After an appropriate time for solv ent delay, u an appropriate ov en temperature program, for example, starting at 75°C for MeCN extracts or 100°C for toluene ramped to 150°C at 25°C/min, then to 280°C at 10°C/min, and hold for 10 min. All collaborators had much experience in pesticide residue analysis and were free to u their own analytical conditions provided that peak shapes were Gaussian, peak widths at half heights were <5 s, and signal-to-noi ratio (S/N) of the quantitative ion for the pesticides at 10 ng/g equivalent concentrations in the sample were >10. For qualitative purpos (which were not the focus of this study), at least 3 ions yielding relativ e abundances that reasonably match a contemporaneously analyzed reference standard are typically needed to make an analyte identification.
(b) Liquid chromatograph/tandem mass spectrometer.— A triple quadrupole, ion trap, or other LC/M
S/MS instrument may be ud provided it is capable of electrospray ionization (ESI) in the positive mode with computerized instrument control/data collection and has an AS. An injection volume (5–100 m L) will be determined for each instrument to achieve S/N > 10 for the quantitation ion for a 10 ng/g equivalent sample concentration. As in GC/MS, the collaborators had much experience in the analysis of pesticides and were free to u their own conditions. Suggested LC conditions, howev er, include a 15 cm long, 3.0 mm id, 3 m m particle size C18 column, flow rate of 0.3 mL/min, and gradient elution with an initial condition of 25% MeOH in 5 mM formic acid solution taken linearly in 15 min to 90% MeOH in 5 mM formic acid solution and held for 15 min. A short C18 guard column must be ud to protect the analytical column, and a bypass valve must be ud before the MS instrument to av oid introduction of the early and late eluting nonanalyte components into the detector. The MS/MS conditions were optimized in each laboratory using direct infusion into the ESI source to prov ide highest S/N for the quantitation ion of each LC-type analyte from a single MS/MS transition. A cond transition with reasonably matching relative abundance ratios vs a contemporaneously analyzed reference standard is typically needed for qualitative purpos.
(c) Centrifuge(s).—Capable of holding the 50 mL centrifuge tubes or bottles ud for extraction and 10–15 mL graduated centrifuge tubes or 2 mL mini-tubes ud in dispersiv e-SPE. Determine the rp
m ttings that yield a given relative centrifugal force (RCF), and ensure that maximum ratings of the centrifuge, tube/bottles, and rotors for the instrument are not exceeded.
(d) Balance(s).—Capable of accurately measuring weights from
0.05 to 100 g within ±0.01 g.
preem(e) Freezer.—Capable of continuous operation <–20°C.
(f) Furnace/oven.—Capable of 500°C operation.
Table 2007.01E. Averaged interlaboratory study results for the fortified and incurred pesticides a
Matrix Recovery, %RSD r, %RSD R, %HorRat No. of labs (n) Grapes86 ± 1110 ± 422 ± 80.90 ± 0.2912 ± 1 Lettuces87 ± 1210 ± 720 ± 90.83 ± 0.4510 ± 1 Oranges87 ± 1510 ± 620 ± 80.84 ± 0.3710 ± 2 Overall87 ± 1110 ± 621 ± 80.86 ± 0.3711 ± 2
Incurred NA b12 ± 422 ± 80.92 ± 0.3011 ± 2
a
Data from fewer than 7 laboratories in an asssment were excluded.
b
NA = Not applicable.
