Multi-Residue Screening Method of Acidic Pesticides in Agricultural Products by Liquid Chromatography/ Time of flight Mass Spectrometry

J. Pestic. Sci., 34(4), 000–000 (2009) DOI: 10.1584/jpestics.G09-29

Original Article

Multi-residue screening method of acidic pesticides in agricultural products by liquid chromatography/ time of flight mass spectrometry

Yumi AKIYAMA,* Tomofumi MATSUOKA and Takao MITSUHASHI

Hyogo Prefectural Institute of Public Health and Consumer Sciences, Arata-cho 2–1–29, Hyogo-ku, Kobe, Hyogo 652–0032, Japan (Received June 27, 2009; Accepted September 2, 2009)

A simple and rapid screening method of 95 acidic pesticides in agricultural products was developed. As acidic pesticides adsorb on the PSA mini-column with fatty acids which become interfering peaks during GC/MS analysis, they were not included in multi-residue analysis entailing PSA clean-up. In this study, the intermediate extracted solutions before PSA clean-up were analyzed by LC/TOF-MS. Accurate mass measurement of TOF- MS enabled the detection of molecular ions and fragment ions with high selectivity. Mean recoveries of 95 pesti- cides added to 6 agricultural products at 0.1 mg/g were 49–127% with RSD 20%. Limits of quantitation were 0.01–0.02 mg/g for 95 pesticides. The method was applied to 140 samples, and 2,4-D from lemon and orange, fluazifop from baby kidney bean, dichlorprop from apple were detected, respectively, at 0.02–0.03 mg/g below MRLs. The proposed method showed good sensitivity for 95 acidic pesticides and enabled rapid screening in combination with our multi-residue method targeted to 520 pesticides. © Pesticide Science Society of Japan Keywords: acidic pesticide, multi-residue, agricultural product, screening method, LC/TOF-MS.

GC/MS analysis,5) but it also retains pesticides containing Introduction acidic functions, such as carboxyl, phenol, sulfonyl, etc. So In Japan, the Positive List System was introduced for the reg- these pesticides were not included in our multi-residue analy- ulation of agricultural chemical residues in foods on May 29, sis. 2006. Maximum residue limits (MRLs) were set for 799 sub- In the Director Notice of the Department of Food Safety, stances, including feed additives and veterinary drugs, by the the Ministry of Health, Labour and Welfare of Japan showed addition of many provisional MRLs.1) Among them, MRLs 2 clean-up procedures for multi-residue analysis in agricul- for pesticides were 586, and increased to 600 until June tural products: one is with an aminopropyl/graphite carbon 2) 2009. As some MRLs are set for the sum of several pesti- black (NH2/GCB) mini-column for 338 pesticides analyzed cides and their metabolites, the number of compounds we by GC/MS and LC/MS; the other is with a silica-gel mini-col- have to aim to analyze as the target of regulatory monitoring umn for 58 acidic pesticides analyzed by LC/MS;6) however, is supposed to be more than 700. it is difficult for a few restricted inspectors to complete all We have been developing multi-residue analytical methods procedures in the routine analysis of many samples and a by gas chromatography/mass spectrometry (GC/MS) and liq- simple screening method would be preferable. Anastassiades uid chromatography/mass spectrometry (LC/MS), and more suggested analyzing phenoxyalcanoic acids without clean- than 500 pesticides are simultaneously extracted from foods up by liquid chromatography/tandem mass spectrometry and purified with octadecylsilyl (ODS) and primary second- (LC/MS/MS) in negative ion mode in his multi-residue ary amine (PSA) mini-columns.3,4) PSA is effective to remove method, named QuEChERS.7) LC/MS/MS gives high sensitiv- fatty acids which generate large interfering peaks during ity in the multiple reaction monitoring mode and enables sam- ple dilution. On the other hand, liquid chromatography/ time- *To whom correspondence should be addressed. of-flight mass spectrometry (LC/TOF-MS) gives accurate E-mail: [email protected] mass information and enables the detection of an unlimited Published online XXXXX number of compounds with high selectivity in full-scan acqui- ©Pesticide Science Society of Japan sition mode.8) 2Y. Akiyama et al. Journal of Pesticide Science

In this study, we investigated the use of intermediate ex- Aldrich); guard column, Inertsil ODS3 (10 mm, 3.0 mm, tracted solutions before PSA clean-up of our multi-residue 3 mm) (GL Sciences, Japan); mobile phase, CH3CN–10 mM → method for the analysis of acidic pesticides in agricultural CH3COONH4 [(15 : 85) (95 : 5)]/16 min (95 : 5) 9 min; products by LC/TOF-MS. Dispersive solid-phase extraction flow rate, 0.5 ml/min; column temp., 40°C; sample cooler, (D-SPE) with GCB is a simple and rapid clean-up procedure 15°C. According to the injector program, 4 ml of the sample to adsorb the pigments,7–9) especially chlorophyll, which extract was mixed with 16 ml water, and then 20 ml was in- could be removed by PSA clean-up5); however, GCB also ad- jected in each run. The MS parameters were as follows: capil- sorbs pesticides with a planar structure.10) We evaluated the lary voltage, 4000 V (positive), 3500 V (negative); nebulizer effect of GCB clean-up on LC/TOF-MS analysis and the re- gas, 50 psi; drying gas, 10 L/min (350°C); fragmentor voltage covery of pesticides. As a result, it was clarified that 95 pesti- (FV), 100 V and 250 V. LC/TOF-MS accurate mass spectra cides retained on PSA were detected quantitatively without were recorded across the range of m/z 50–1050 (1 cycle/sec). remarkable interfering peaks only by solvent exchange. Internal mass calibration was performed automatically using a Screening these pesticides would be performed in a series of dual-nebulizer ESI with an automated calibrant delivery sys- multi-residue analyses of other pesticides. tem. Purine (C5H4N4, m/z 121.050873 (positive), 119.036320 (negative)) and HP-0921 (hexakis-(1H,1H,3H-tetrafluoropen- Materials and Methods toxy)-phosphazene, C18H18O6N3P3F24, m/z 922.009798 (posi- 1. Chemicals tive), 1033.988109 (negative, trifluoroacetate adduct)) were Acetone, acetonitrile, and anhydrous sodium sulfate (Wako used for internal reference masses. Data were processed with Pure Chemical Ind., Japan), n-hexane and sodium chloride Agilent Mass Hunter software (version B02.00). (Kanto Chemical, Japan) were of pesticide analysis grade. Acetonitrile (Wako) used for LC/TOF-MS was of liquid chro- 3. Sample preparation matography grade, and others were of analytical grade. As an All agricultural products were collected at local markets in internal standard (IS) solution, triphenylphosphate (TPP) Hyogo prefecture. We confirmed that the samples used as (Wako) and 1-ethyl-3-phenylurea (EPU) (Frinton Laborato- blanks or fortifications were pesticide-free with this proposed ries, USA) were mixed at each 5 and 10 mg/ml with acetone- method. About 500 g sample was chopped in a MK-K58 food n-hexane (1 : 4). processor (Panasonic, Japan) for more than 1 min to obtain ODS: Isolute C18 (endcapped), 1 g (Biotage, Sweden) was thoroughly mixed homogenates. conditioned with 10ml acetonitrile and 10ml water; GCB: Su- To 25 g portions of chopped samples (20 ml water was pelclean ENVI-Carb 120/400 (Sigma-Aldrich, USA) was added in case of low-moisture content samples, and 3–5 g used without conditioning. sodium acetate was added to citrus fruits for neutralization), Pesticides and their metabolites were obtained from Wako, 0.25 ml IS solution was added except for the blank sample, Kanto, Sigma-Aldrich, Hayashi Pure Chemical Ind. (Japan) and 1 ml fortification standard solution was added to give a and Dr. Ehrenstorfer (Germany). Individual stock standard so- final concentration of 0.1 mg/g for the recovery test. After lutions (250 mg/ml) were prepared with acetone. Mixed stan- standing for 30 min, the sample was extracted with 60 ml ace- dard solutions each containing 20–30 compounds at 10 mg/ml tonitrile by a HF93 homogenizer (SMT, Japan) for 3 min and were prepared from stock solutions with acetone. Fortification then filtered. The filtrates were cleaned up through an ODS standard solution containing 95 compounds at 2.5 mg/ml was (1 g) mini-column. Acetonitrile was separated by salting-out prepared from mixed solutions with acetone. Solvent standard with 10 ml of 2 M phosphate buffer/saturated brine solution solutions for LC/TOF-MS analysis were freshly prepared by (pH 7) and 6 g sodium chloride, and then 36 ml acetonitrile evaporating 0.1–1.0 ml of fortification standard and 0.25 ml IS layer was collected. After evaporation to dryness, the residue solution under a gentle stream of nitrogen, and dissolving was adjusted to 3 ml with acetone-n-hexane (1 : 1). A 2 ml with 2.5 ml acetonitrile. Matrix-matched standard solutions aliquot was purified with PSA (200 mg) mini-column for were prepared by evaporating 0.4 ml blank sample extracts multi-residue analysis of 520 pesticides by GC/MS and and dissolving with 0.2 ml each concentration of solvent stan- LC/MS.3,4) In this study, the residual 0.4 ml was evaporated to dard solutions. dryness and dissolved with 0.2 ml acetonitrile for LC/TOF- MS analysis. A 0.2 ml aliquot of the final acetonitrile solution 2. LC/TOF-MS analysis corresponds to 2 g sample matrix. Fig. 1 summarizes the pro- The Agilent 1200 series LC system (consisting of a binary cedure. pump, vacuum degasser, column oven, and autosampler) was connected to a time-of-flight mass spectrometer Agilent 6210 4. Clean-up with GCB MSD TOF (Agilent Technologies, USA) equipped with an To investigate the effect of GCB, clean-up by D-SPE was per- electrospray interface (ESI) operating in both the positive formed before solvent exchange; that is, 5 mg GCB was and negative ion mode. The LC conditions were as follows: added to the residual acetone-n-hexane (1 : 1) solution (ca. column, Ascentis C18 (100 mm, 3.0 mm, 3 mm) (Sigma- 1 ml). The mixture was shaken for 30 sec, mixed on a TM-105 Vol. 34, No. 4, 000–000 (2009) Screening method of acidic pesticides by LC/TOF-MS 3

others. The details, including molecular formulas, are pre- sented in Supplemental Table 1. Some pesticides were not fragmented even with FV 250 V. The relative mass error for each compound was kept within 5 ppm with a real-time refer- ence mass correction system. Target compounds were auto- matically found according to the database of RT and molecu- lar formula. In addition, target ion monitored at FV 100 V and qualifier ion at FV 250 V were extracted with a range m/z 0.01 at RT 1 min. The ratio of the peak area detected on each chromatogram and mass spectra of these peaks, includ- ing the isotopic pattern, made it easy to confirm the positive analytes. The peak area of the target ion was used for quanti- tation and the limits of detection (LODs) presented in Table 1 were equal to the peak height of 150 counts. In positive ion mode, both EPU and TPP used for IS were detected. The ratio of these 2 peaks was effective to estimate the matrix effect. As EPU with short RT easily suppressed its ionization, TPP usually played the role of IS.

2. Sample preparation In our procedure shown in Fig. 1, acetonitrile content for both Fig. 1. Sample preparation method for multi-residue analysis extraction and ODS clean-up is maintained between 70 and 75% according to the California Department of Food and Vortex mixer (Thermonics, Japan) for 30 sec, and then cen- Agriculture (CDFA) method.11) Non-polar co-extractives were trifuged for 10 min at 3000 rpm. A 0.4 ml supernatant sample effectively removed by ODS clean-up. Pigments were consid- was evaporated to dryness and dissolved with 0.2 ml acetoni- erably removed by this step, but remained in pre-PSA solu- trile. tions. With the addition of 5 mg GCB, as described in 4. Clean-up with GCB, chlorophyll was removed from pre-PSA Results and Discussion solutions, although some carotinoides remained for green and 1. LC/TOF-MS analysis yellow vegetables, as recommended by Anastassiades.7) The LC conditions were same as those previously set3,4) for To investigate the matrix effect on LC/TOF-MS analysis, multi-residue analysis by single quadrupole LC/MS. A col- matrix-matched standard solutions were prepared as described umn of 3.0 mm diameter was chosen to load high amounts of in 1. Chemicals. Suppression of ionization was observed for sample solution. For the final sample solution, composition compounds with short RT in positive mode such as pyridate close to the initial mobile phase is preferable. Otherwise metabolite, flumetsulam, cyromazine, etc. Peak areas of those peaks of early-eluted polar compounds become broad or split- compounds were decreased to almost half by the coexistence shape; however, water-rich solvent such as 20% acetonitrile in of matrix, for which there was no remarkable difference be- water (v/v) could not dissolve sample matrix well so low- tween with/without GCB clean-up. The difference was also polar compounds with a long retention time (RT) gradually not observed on the chromatograms. precipitated with matrix while waiting for the order of injec- Some structurally planar pesticides retained on PSA were tion. To solve this problem, we prepared the final sample solu- included in the target of this study, and they had good affinity tion with acetonitrile and diluted with water just before auto- to the planar structure of GCB. The recovery of these pesti- matic injection by adopting an injector program. After injec- cides was reduced by GCB clean-up, as shown in Table 2. tion, the loop was on-line and could be cleaned up by mobile From the above results, we decided not to adopt GCB clean- phase. To achieve high sensitivity, each sample was injected 2 up and applied pre-PSA solutions to LC/TOF-MS analysis times in positive and negative ion mode operated separately. only by solvent exchange. During one analysis, MS data were acquired for both FVs set at 100 V to detect mainly protonated or deprotonated mole- 3. Recovery test cules and at 250 V for fragment ions. As TOF-MS is highly The recovery tests were performed for 6 agricultural products vacuumed, high FV (250 V) was required compared with (brown rice, spinach, lemon, lettuce, sweet pepper and Japan- quadrupole MS (200 V). ese pear) at a level of 0.1 mg/g. The data are summarized in Table 1 shows the qualitative parameters of the studied pes- Table 1. The test for each product was conducted on a differ- ticides. Among 95 pesticides, 43 were in the sulfonyl group, ent day and details for each are shown in Supplemental Table 29 in the carboxyl group, 11 in the hydroxy group, and 12 2. 4Y. Akiyama et al. Journal of Pesticide Science

Table 1. LC/TOF-MS qualitative parameters and recovery data for pesticides

c) RT a) Monitor ion (m/z) LODb) Mean recovery (%) Mean Pesticide c) (min) Target Qualifier (ng) Solvent st.d) Matrix st.e) RSD (%)

Positive ionization mode Asulam 1.14 248.0700 253.0253 0.04 35.0 49.2 16.3 Imazethapyr 1.35 290.1499 290.1499 0.01 42.5 58.4 12.5 Cyromazine 1.37 167.1040 167.1040 0.01 49.8 85.7 12.6 Pyridate met. 2.04 207.0320 207.0320 0.02 51.7 94.4 8.6 Benzobicyclon met. 2.47 372.0667 355.0401 0.17 81.4 98.7 10.7 Nicosulfuron 2.72 411.1081 182.0560 0.01 70.3 72.1 8.9 Diflufenzopyr 2.75 162.0662 162.0662 0.15 84.7 91.7 8.1 Imazaquin 2.90 312.1343 312.1343 0.01 55.5 72.3 7.0 Flumetsulam 2.98 326.0518 129.0385 0.06 82.2 105.7 5.7 Thifensulfuron-methyl 3.03 388.0380 167.0564 0.09 106.4 86.3 5.6 Metsulfuron-methyl 3.34 382.0816 167.0564 0.07 130.1 93.7 4.3 Tepraloxydim met. 3.48 358.1416 266.1387 0.15 78.9 89.2 7.5 Chlorsulfuron 3.96 358.0371 141.0771 0.06 135.9 89.2 3.8 Rimsulfuron 4.18 432.0642 182.0560 0.08 148.9 88.3 5.3 Azimsulfuron 4.32 425.1099 182.0560 0.05 87.8 84.6 4.4 Foramsulfuron 4.57 453.1187 182.0560 0.10 102.9 86.0 6.5 Trinexapac-ethyl 4.58 253.1071 179.0703 0.06 87.0 97.8 9.9 Clofencet 4.72 279.0531 261.0425 0.03 40.9 53.3 8.8 Pyrasulfotole 4.95 363.0621 250.9984 0.05 94.9 66.7 10.8 Mesotrione 4.98 293.0478 315.0298 0.29 65.0 73.0 7.7 Sulfosulfuron 5.07 471.0751 261.0288 0.06 102.7 92.5 4.6 Cinosulfuron 5.10 414.1078 183.0513 0.02 125.6 125.8 5.1 Flucarbazone 5.10 397.0424 130.0611 0.29 105.6 87.8 7.4 Florasulam 5.24 360.0373 129.0385 0.04 111.8 96.5 6.7 Imazosulfuron 5.25 413.0429 156.0768 0.01 85.6 105.8 8.6 Propoxycarbazone 5.28 399.0969 116.0455 0.12 133.5 87.1 6.9 Flazasulfuron 5.33 408.0584 182.0560 0.01 93.4 89.2 9.2 Clethodim sulfone 5.51 392.1293 300.1264 0.02 120.1 106.1 6.6 Triasulfuron 5.55 402.0633 141.0771 0.02 136.2 97.5 4.4 Naptalam 5.63 292.0968 144.0808 0.06 80.9 72.1 7.6 Ethametsulfuron-methyl 5.82 411.1081 196.0829 0.01 90.1 84.8 4.6 Iodosulfuron-methyl 5.86 507.9782 167.0564 0.03 114.5 85.6 4.6 Pyrithiobac 5.86 327.0201 309.0095 0.02 77.1 84.3 5.8 Pyrazosulfuron-ethyl 5.92 415.1030 182.0560 0.02 91.3 85.8 5.0 Tribenuron-methyl 6.08 396.0972 155.0927 0.01 94.3 82.1 5.3 Mesosulfuron-methyl 6.28 504.0853 504.0853 0.02 139.8 98.0 3.6 IS (Ethylphenylurea) 6.28 165.1022 94.0651 0.01 Halosulfuron-methyl 6.32 435.0484 182.0560 0.02 91.5 100.9 9.3 Benzylaminopurine 6.34 226.1087 91.0542 0.01 51.7 70.2 10.8 Bispyribac 6.76 431.1197 413.1092 0.01 114.0 95.0 5.0 Trifloxysulfuron 6.82 438.0690 182.0560 0.01 111.2 95.6 6.5 Vol. 34, No. 4, 000–000 (2009) Screening method of acidic pesticides by LC/TOF-MS 5

Table 1. (Continued)

c) RT a) Monitor ion (m/z) LODb) Mean recovery (%) Mean Pesticide c) (min) Target Qualifier (ng) Solvent st.d) Matrix st.e) RSD (%)

Warfarin 6.85 309.1121 251.0703 0.01 84.6 92.7 7.5 Chlorimuron-ethyl 7.00 415.0474 186.0065 0.02 108.6 94.3 6.8 Metosulam 7.01 418.0138 174.9950 0.01 113.3 94.5 6.1 Penoxsulam 7.03 484.0709 484.0709 0.01 108.8 94.3 5.0 Ethoxysulfuron 7.07 399.0969 261.0288 0.01 85.7 114.4 3.5 Cloransulam-methyl 7.15 430.0383 398.0121 0.02 122.0 106.1 8.3 Diclosulam 7.28 405.9938 160.9794 0.03 113.4 94.5 7.5 Imazamox-methyl 7.56 320.1605 320.1605 0.14 77.3 92.2 7.5 Bensulfuron-methyl 8.52 411.0969 182.0560 0.01 99.2 87.1 4.4 Triflusulfuron-methyl 8.64 493.1112 264.0703 0.01 117.8 96.1 4.9 Sulfentrazone 8.89 404.0157 386.9891 0.03 102.7 93.5 4.8 Cyclosulfamuron 9.87 422.1129 261.0288 0.01 106.0 87.0 5.1 Diclomezine 11.07 255.0086 255.0086 0.03 84.5 87.9 9.7 Fenhexamid 12.17 302.0709 302.0709 0.02 80.2 94.9 6.0 Brodifacoum 12.28 523.0903 523.0903 0.02 85.5 91.4 9.8 Pinoxaden 13.09 401.2435 317.1860 0.01 52.5 66.0 8.5 IS (Triphenylphosphate) 14.30 327.0781 327.0781 0.01 Negative ionization mode Cyflumetofen met. 1.36 189.0169 145.0271 0.23 58.5 55.2 7.7 4-CPA 3.95 185.0011 126.9956 0.34 53.0 67.8 17.1 Bentazone 3.95 239.0496 239.0496 0.04 101.0 100.4 9.2 Cloprop 4.35 199.0167 126.9956 0.10 84.7 78.1 10.0 Spiromesifen met. 4.83 271.1340 271.1340 0.01 87.6 77.8 5.6 Bromoxynil 5.11 273.8509 275.8488 0.02 107.8 94.4 10.3 DNOC 5.32 197.0204 197.0204 0.01 88.2 78.1 10.3 MCPA 5.46 199.0167 141.0113 0.06 80.1 66.2 10.7 2,4-D 5.54 218.9621 160.9566 0.11 74.7 74.3 9.2 Mecoprop 5.89 213.0324 141.0113 0.09 90.4 79.6 8.1 Dichlorprop 6.02 232.9778 160.9566 0.07 90.4 75.9 8.1 IS (Ethylphenylurea) 6.28 223.1088 163.0877 0.01 Ioxynil 6.30 369.8231 126.9050 0.02 97.4 99.6 6.8 2,4,5-T 6.49 252.9232 194.9177 0.06 84.4 81.5 12.4 DADK 6.52 168.0779 168.0779 0.17 110.2 93.0 8.6 Clomeprop acid 6.78 246.9934 174.9723 0.09 106.0 79.4 8.9 Pindone 6.78 229.0870 229.0870 0.24 82.3 58.2 9.8 Clodinafop acid 6.80 310.0288 238.0077 0.17 101.2 106.4 7.8 Fenoprop 6.85 266.9388 194.9177 0.04 95.4 90.8 9.8 DA 6.89 198.0707 198.0707 0.02 107.1 99.0 7.1 Prosulfuron 6.99 418.0802 139.0625 0.05 110.8 107.8 5.4 Fluazifop 7.05 326.0646 254.0434 0.08 106.2 90.8 6.5 Quizalfop 7.43 343.0491 271.0280 0.11 86.2 88.7 9.6 2,4-DB 7.59 160.9566 160.9566 0.10 108.3 88.9 7.0 6Y. Akiyama et al. Journal of Pesticide Science

Table 1. (Continued)

c) RT a) Monitor ion (m/z) LODb) Mean recovery (%) Mean Pesticide c) (min) Target Qualifier (ng) Solvent st.d) Matrix st.e) RSD (%)

MCPB 7.69 227.0480 141.0113 0.17 105.3 95.7 8.9 Fenoxaprop 7.82 332.0331 260.0120 0.12 107.3 86.7 11.2 Thidiazuron 7.98 219.0346 70.9835 0.01 95.2 76.5 10.8 Haloxyfop 8.17 360.0256 288.0045 0.09 91.9 96.6 11.1 Dinoseb 8.30 239.0673 239.0673 0.01 95.5 79.1 5.9 Fenoxaprop met. 8.30 167.9858 132.0091 0.03 106.2 93.9 10.0 Primisulfuron-methyl 8.40 467.0290 176.0277 0.04 184.4 126.8 6.5 Acifluorfen 8.42 359.9892 315.9994 0.06 99.2 96.7 8.0 Dinoterb 8.70 239.0673 239.0673 0.01 97.8 90.8 8.5 Tecloftalam 9.01 399.8430 401.8400 0.06 85.3 77.6 13.6 Fomesafen 9.07 436.9827 436.9827 0.02 130.9 120.4 6.0 Pentachlorophenol 9.10 262.8397 264.8368 0.04 88.6 76.2 9.0 Forchlorfenuron 9.74 246.0440 127.0068 0.01 66.8 65.6 12.7 Flusulfamide 11.15 412.9383 412.9383 0.01 106.8 109.0 5.8 Dinocap 11.78 295.1299 295.1299 0.05 153.1 97.4 9.4 Fluazinam 14.57 462.9441 462.9441 0.01 104.2 94.2 3.5

a) Retention time. b) Limit of detection defined by peak filter set at 150 counts of peak height, 0.04 ng is equivalent to 0.001 mg/g in foods. c) The mean value of the recovery tests for 6 agricultural products fortified at 0.1 mg/g (brown rice (n3), spinach (n3), lemon (n3), let- tuce (n5), sweet pepper (n5), and Japanese pear (n5)). d) Recovery data calculated for solvent standard. e) Recovery data calculated for matrix-matched standard. met.: metabolite. IS: Internal standard.

The mean recoveries of 95 pesticides ranged 35–184% for tion of the water phase by salting-out at acidic pH for acidic solvent standard, asulam in positive mode showed low recov- pesticides and at pH 7.0 for others,6) we performed salting-out ery and primisulfuron-methyl and dinocap in negative mode at pH 7.0 for all pesticides. It was not the best condition for showed high recovery, which was improved in the range of acidic pesticides, and more polar compounds, such as en- 49–127% by calculating the matrix-matched standard, and 82 dothal, imazapyr, imazapic, etc., were not included in the tar- pesticides showed satisfactory values between 70 and 120%. get of this method. For the multi-residue method, Lee11) rec- RSDs were below 10% for 78 pesticides and below 20% ommended salting-out at pH 7.0, but currently salting out at for all. For individual samples, imazethapyr, nicosulfuron, weak-acidic pH has been adopted by Anastassiades7) and Oki- mesotrione, and naptalam showed poor recoveries in lemon. hashi.12) If salting-out at pH 5–5.5 was adopted after verifying Although the official multi-residue method adopts separa- the applicability for the target pesticides in our multi-residue method, recovery of acidic pesticides would be improved. This method is considered useful for the screening method, Table 2. Effect of GCB clean-up on recovery of pesticides even though it is difficult to prepare matrix-matched standard with planar structure added to spinach solutions for all kinds of samples in routine analysis. At the with GCB without GCB limits of quantitation (LOQs,) defined by the signal to noise Pesticide ratio (S/N10), 0.01 mg/g was available for most pesticides, (0.1 mg/g added) Recovery RSD Recovery RSD except for 0.02 mg/g for mesotrione, flucarbazone, 4-CPA, (%)a) (%) (%)a) (%) pindone and cyflumetofen metabolite.

Cyromazine 20.0 21.7 45.6 12.6 4. Monitoring results Benzylaminopurine 28.1 34.3 63.1 7.9 We applied this method to routine analysis of FY 2008. Diclomezine 56.1 22.0 109.6 12.0 Among 140 agricultural products (80 domestic and 60 im- Thidiazuron 56.1 19.4 97.7 10.2 ported), 3 pesticides were detected from 4 samples (Table 3). Fig. 2 shows, as an example, the extracted ion chromatograms a) Recoveries were calculated for solvent standard (n3). and mass spectra for fluazifop detected from baby kidney Vol. 34, No. 4, 000–000 (2009) Screening method of acidic pesticides by LC/TOF-MS 7

Table 3. Targeted pesticide residues found in agricultural products during FY2008

No. No. Producing Residue MRL Samplea) Pesticide analyzed detected district (mg/g) (mg/g)b)

Lemon 5 1 America 2,4-D 0.03 2 Orange 5 1 South Africa 2,4-D 0.02 2 Baby kidney bean (frozen) 5 1 China Fluazifop 0.02 0.1 Apple 1 1 Japan (Hyogo) Dichlorprop 0.02 3

a) Positive samples among 39 kinds of 80 domestic samples and 22 kinds of 60 imported samples. b) Maximum Residue Limit under the Japanese Food Sanitation Law. bean extracts. The exact coincidence between the sample and procedure covers more than 600 pesticides and is applicable the standard was observed on m/z to three decimal places. to most agricultural products. Conventional LC/MS/MS will During 1-year analysis, a decline of the sensitivity and resolu- provide more sensitive analysis for acidic pesticides and en- tion caused by damage to the column and instrument was not able sample dilution, but information is limited for target pes- observed. ticides. On the other hand, the number of pesticides found and Residues shown in Table 3 were low compared with MRLs. confirmed by LC/TOF-MS can be increased unlimitedly, even For acidic pesticides, MRLs are often set for the sum of sev- after analysis. The analytical data of LC/TOF-MS were used eral compounds. In 2,4-D, total analysis of 2,4-D, ester deriva- not only for acidic pesticides but also for the confirmation of tives, amine salts, etc. is required by hydrolysis and butyl es- pesticides usually determined after PSA clean-up. terification to specify the violation of MRLs. So the purpose of the multi-residue analysis is rapid screening rather than References precise determination. If the total residue of 2,4-D analyzed 1) Notification of Ministry of Health, Labour and Welfare of Japan, by this method and ester derivatives analyzed by GC/MS in No. 499, Nov. 29, (2005). our multi-residue method is high, reexamination by the offi- 2) Notification of Ministry of Health, Labour and Welfare of Japan, 6) cial method, notified individually would be performed. No. 325, Jun. 4, (2009). The proposed method showed good sensitivity for 95 acidic 3) Y. Akiyama, N. Yoshioka and T. Matsuoka: “Pesticide Chem- pesticides and allowed for rapid screening in combination istry” ed. by H. Ohkawa, H. Miyagawa and P. W. Lee, Willey- with our multi-residue method. The time and labor were con- VCH, Weinheim, pp. 395–399, (2007). siderably reduced by comparing with the official multi- 4) Y. Akiyama: Agriculture of this Month (Kongetsu no Nougyou), residue method using a silica-gel mini-column. The whole 51, 50–55 (2007) (in Japanese).

Fig. 2. Extracted ion chromatograms and mass spectra for fluazifop detected from baby kidney bean extracts. 8Y. Akiyama et al. Journal of Pesticide Science

5) Y. Akiyama, M. Yano, T. Mitsuhashi, N. Takeda and M. Tsuji: J. 9) L. Li, W. Li, D. Qin, S. Jiang and F. Liu: J. AOAC Int. 92, 538– Food Hyg. Soc. Jpn. 87, 351–362 (1996) (in Japanese). 547 (2009). 6) Director Notice of Department of Food Safety, Ministry of 10) M. Anastassiades, S. J. Lehotay, D. Stajnbaher and F. J. Schenck: Health, Labour and Welfare of Japan, Syoku-An No.0124001, J. AOAC Int. 86, 412–431 (2003). Jan. 24, 2005 (Final amendments were made on Feb. 18, 2008). 11) S. M. Lee, M. L. Papayhakis, H.-M. C. Feng, G. F. Hunter and J. 7) M. Anastassiades: QuEChERS, http://www.quechers.com/docs/ E. Carr: Fresenius J.Anal.Chem. 339, 376–383 (1991). quechers_en_oct2005.pdf (Fellbach, Germany). 12) M. Okihashi, Y. Kitagawa, K. Akutsu, H. Obana and Y. Tanaka: 8) M. Mezcua, O. Malato, J. F. Garcia-Reyes, A. Molina-Diaz and J. Pestic. Sci. 30, 368–377 (2005). A. R. Fernandes-Alba: Anal. Chem. 81, 913–929 (2009). Vol. 34, No. 4, 000–000 (2009) Screening method of acidic pesticides by LC/TOF-MS 9

Supplemental Table 1. LC/TOF-MS qualitative parameters for pesticides

Molecular Monoisotopic RT Monitor ion (m/z) Pesticide formula m.w. (min) Target Formula Qualifier Formula

Positive ionization mode

Asulam C8H10N2O4S 230.0361 1.14 248.0700 C8H14N3O4S 253.0253 C8H10N2O4SNa

Imazethapyr C15H19N3O3 289.1426 1.35 290.1499 C15H20N3O3 290.1499 C15H20N3O3

Cyromazine C6H10N6 166.0967 1.37 167.1040 C6H11N6 167.1040 C6H11N6

Pyridate met. C10H7N2OCl 206.0247 2.04 207.0320 C10H8N2OCl 207.0320 C10H8N2OCl

Benzobicyclon met. C16H15O5SCl 354.0329 2.47 372.0667 C16H19NO5SCl 355.0401 C16H16O5SCl

Nicosulfuron C15H18N6O6S 410.1009 2.72 411.1081 C15H19N6O6S 182.0560 C7H8N3O3

Diflufenzopyr C15H12N4O3F2 334.0877 2.75 162.0662 C8H8N3O 162.0662 C8H8N3O

Imazaquin C17H17N3O3 311.1270 2.90 312.1343 C17H18N3O3 312.1343 C17H18N3O3

Flumetsulam C12H9N5O2SF2 325.0445 2.98 326.0518 C12H10N5O2SF2 129.0385 C6H5NF2

Thifensulfuron-methyl C12H13N5O6S2 387.0307 3.03 388.0380 C12H14N5O6S2 167.0564 C6H7N4O2

Metsulfuron-methyl C14H15N5O6S 381.0743 3.34 382.0816 C14H16N5O6S 167.0564 C6H7N4O2

Tepraloxydim met. C17H24NO5Cl 357.1343 3.48 358.1416 C17H25NO5Cl 266.1387 C14H20NO4

Chlorsulfuron C12H12N5O4SCl 357.0299 3.96 358.0371 C12H13N5O4SCl 141.0771 C5H9N4O

Rimsulfuron C14H17N5O7S2 431.0569 4.18 432.0642 C14H18N5O7S2 182.0560 C7H8N3O3

Azimsulfuron C13H16N10O5S 424.1026 4.32 425.1099 C13H17N10O5S 182.0560 C7H8N3O3

Foramsulfuron C17H20N6O7S 452.1114 4.57 453.1187 C17H21N6O7S 182.0560 C7H8N3O3

Trinexapac-ethyl C13H16O5 252.0998 4.58 253.1071 C13H17O5 179.0703 C10H11O3

Clofencet C13H11N2O3Cl 278.0458 4.72 279.0531 C13H12N2O3Cl 261.0425 C13H10N2O2Cl

Pyrasulfotole C14H13N2O4SF3 362.0548 4.95 363.0621 C14H14N2O4SF3 250.9984 C9H6O3SF3

Mesotrione C14H13NO7S 339.0413 4.98 293.0478 C14H13O5S 315.0298 C14H12O5SNa

Sulfosulfuron C16H18N6O7S2 470.0678 5.07 471.0751 C16H19N6O7S2 261.0288 C7H9N4O5S

Cinosulfuron C15H19N5O7S 413.1005 5.10 414.1078 C15H20N5O7S 183.0513 C6H7N4O3

Flucarbazone C12H11N4O6SF3 396.0351 5.10 397.0424 C12H12N4O6SF3 130.0611 C4H8N3O2

Florasulam C12H8N5O3SF3 359.0300 5.24 360.0373 C12H9N5O3SF3 129.0385 C6H5NF2

Imazosulfuron C14H13N6O5SCl 412.0357 5.25 413.0429 C14H14N6O5SCl 156.0768 C6H10N3O2

Propoxycarbazone C15H18N4O7S 398.0896 5.28 399.0969 C15H19N4O7S 116.0455 C3H6N3O2

Flazasulfuron C13H12N5O5SF3 407.0511 5.33 408.0584 C13H13N5O5SF3 182.0560 C7H8N3O3

Clethodim sulfone C17H26NO5SCl 391.1220 5.51 392.1293 C17H27NO5SCl 300.1264 C14H22NO4S

Triasulfuron C14H16N5O5SCl 401.0561 5.55 402.0633 C14H17N5O5SCl 141.0771 C5H9N4O

Naptalam C18H13NO3 291.0895 5.63 292.0968 C18H14NO3 144.0808 C10H10N

Ethametsulfuron-methyl C15H18N6O6S 410.1009 5.82 411.1081 C15H19N6O6S 196.0829 C7H10N5O2

Iodosulfuron-methyl C14H14N5O6SI 506.9726 5.86 507.9782 C14H15N5O6SI 167.0564 C6H7N4O2

Pyrithiobac C13H11N2O4SCl 326.0128 5.86 327.0201 C13H12N2O4SCl 309.0095 C13H10N2O3SCl

Pyrazosulfuron-ethyl C14H18N6O7S 414.0958 5.92 415.1030 C14H19N6O7S 182.0560 C7H8N3O3

Tribenuron-methyl C15H17N5O6S 395.0900 6.08 396.0972 C15H18N5O6S 155.0927 C6H11N4O

Mesosulfuron-methyl C17H21N5O9S2 503.0781 6.28 504.0853 C17H22N5O9S2 504.0853 C17H22N5O9S2

IS (Ethylphenylurea) C9H12N2O 164.0950 6.28 165.1022 C9H13N2O 94.0651 C6H8N

Halosulfuron-methyl C13H15N6O7SCl 434.0411 6.32 435.0484 C13H16N6O7SCl 182.0560 C7H8N3O3

Benzylaminopurine C12H11N5 225.1014 6.34 226.1087 C12H12N5 91.0542 C7H7

Bispyribac C19H18N4O8 430.1125 6.76 431.1197 C19H19N4O8 413.1092 C19H17N4O7

Trifloxysulfuron C14H14N5O6SF3 437.0617 6.82 438.0690 C14H15N5O6SF3 182.0560 C7H8N3O3 10 Y. Akiyama et al. Journal of Pesticide Science

Supplemental Table 1. (Continued)

Molecular Monoisotopic RT Monitor ion (m/z) Pesticide formula m.w. (min) Target Formula Qualifier Formula

Warfarin C19H16O4 308.1049 6.85 309.1121 C19H17O4 251.0703 C16H11O3

Chlorimuron-ethyl C15H15N4O6SCl 414.0401 7.00 415.0474 C15H16N4O6SCl 186.0065 C6H5N3O2Cl

Metosulam C14H13N5O4SCl2 417.0065 7.01 418.0138 C14H14N5O4SCl2 174.9950 C7H7NCl2

Penoxsulam C16H14N5O5SF5 483.0636 7.03 484.0709 C16H15N5O5SF5 484.0709 C16H16N5O5SF5

Ethoxysulfuron C15H18N4O7S 398.0896 7.07 399.0969 C15H19N4O7S 261.0288 C7H9N4O5S

Cloransulam-methyl C15H13N5O5SClF 429.0310 7.15 430.0383 C15H14N5O5SClF 398.0121 C14H10N5O4SClF

Diclosulam C13H10N5O3SCl2F 404.9865 7.28 405.9938 C13H11N5O3SCl2F 160.9794 C6H5NCl2

Imazamox-methyl C16H21N3O4 319.1532 7.56 320.1605 C16H22N3O4 320.1605 C16H22N3O4

Bensulfuron-methyl C16H18N4O7S 410.0896 8.52 411.0969 C16H19N4O7S 182.0560 C7H8N3O3

Triflusulfuron-methyl C17H19N6O6SF3 492.1039 8.64 493.1112 C17H20N6O6SF3 264.0703 C8H9N5O2F3

Sulfentrazone C11H10N4O3SCl2F2 385.9819 8.89 404.0157 C11H14N5O3SCl2F2 386.9891 C11H11N4O3SCl2F2

Cyclosulfamuron C17H19N5O6S 421.1056 9.87 422.1129 C17H20N5O6S 261.0288 C7H9N4O5S

Diclomezine C11H8N2OCl2 254.0014 11.07 255.0086 C11H9N2OCl2 255.0086 C11H9N2OCl2

Fenhexamid C14H17NO2Cl2 301.0636 12.17 302.0709 C14H18NO2Cl2 302.0709 C14H18NO2Cl2

Brodifacoum C31H23O3Br 522.0831 12.28 523.0903 C31H24O3Br 523.0903 C31H24O3Br

Pinoxaden C23H32N2O4 400.2362 13.09 401.2435 C23H33N2O4 317.1860 C18H25N2O3

IS (Triphenylphosphate) C18H15O4P 326.0708 14.30 327.0781 C18H16O4P 327.0781 C18H16O4P Negative ionization mode

Cyflumetofen met. C8H5O2F3 190.0242 1.36 189.0169 C8H4O2F3 145.0271 C7H4F3

4-CPA C8H7O3Cl 186.0084 3.95 185.0011 C8H6O3Cl 126.9956 C6H4OCl

Bentazone C10H12N2O3S 240.0569 3.95 239.0496 C10H11N2O3S 239.0496 C10H11N2O3S

Cloprop C9H9O3Cl 200.0240 4.35 199.0167 C9H8O3Cl 126.9956 C6H4OCl

Spiromesifen met. C17H20O3 272.1412 4.83 271.1340 C17H19O3 271.1340 C17H19O3 81 Bromoxynil C7H3NOBr2 274.8581 5.11 273.8509 C7H2NOBr2 275.8488 C7H2NOBr( Br)

DNOC C7H6N2O5 198.0277 5.32 197.0204 C7H5N2O5 197.0204 C7H5N2O5

MCPA C9H9O3Cl 200.0240 5.46 199.0167 C9H8O3Cl 141.0113 C7H6OCl

2,4-D C8H6O3Cl2 219.9694 5.54 218.9621 C8H5O3Cl2 160.9566 C6H3OCl2

Mecoprop C10H11O3Cl 214.0397 5.89 213.0324 C10H10O3Cl 141.0113 C7H6OCl

Dichlorprop C9H12N2O 233.9850 6.02 232.9778 C11H15N2O3 160.9566 C9H11N2O

IS (Ethylphenylurea) C9H8O3Cl2 164.0950 6.28 223.1088 C9H7O3Cl2 163.0877 C6H3OCl2

Ioxynil C7H3NOI2 370.8304 6.30 369.8231 C7H2NOI2 126.9050 I

2,4,5-T C8H5O3Cl3 253.9304 6.49 252.9232 C8H4O3Cl3 194.9177 C6H2OCl3

DADK C7H11N3O2 169.0851 6.52 168.0779 C7H10N3O2 168.0779 C7H10N3O2

Clomeprop acid C10H10O3Cl2 248.0007 6.78 246.9934 C10H9O3Cl2 174.9723 C7H5OCl2

Pindone C14H14O3 230.0943 6.78 229.0870 C14H13O3 229.0870 C14H13O3

Clodinafop acid C14H11NO4ClF 311.0361 6.80 310.0288 C14H10NO4ClF 238.0077 C11H6NO2ClF

Fenoprop C9H7O3Cl3 267.9461 6.85 266.9388 C9H6O3Cl3 194.9177 C6H2OCl3

DA C8H13N3OS 199.0779 6.89 198.0707 C8H12N3OS 198.0707 C8H12N3OS

Prosulfuron C15H16N5O4SF3 419.0875 6.99 418.0802 C15H15N5O4SF3 139.0625 C5H7N4O

Fluazifop C15H12NO4F3 327.0718 7.05 326.0646 C15H11NO4F3 254.0434 C12H7NO2F3

Quizalfop C17H13N2O4Cl 344.0564 7.43 343.0491 C17H12N2O4Cl 271.0280 C14H8N2O2Cl

2,4-DB C10H10O3Cl2 248.0007 7.59 160.9566 C6H3OCl2 160.9566 C6H3OCl2 Vol. 34, No. 4, 000–000 (2009) Screening method of acidic pesticides by LC/TOF-MS 11

Supplemental Table 1. (Continued)

Molecular Monoisotopic RT Monitor ion (m/z) Pesticide formula m.w. (min) Target Formula Qualifier Formula

MCPB C11H13O3Cl 228.0553 7.69 227.0480 C11H12O3Cl 141.0113 C7H6OCl

Fenoxaprop C16H12NO5Cl 333.0404 7.82 332.0331 C16H11NO5Cl 260.0120 C13H7NO3Cl

Thidiazuron C9H8N4OS 220.0419 7.98 219.0346 C9H7N4OS 70.9835 C2HNS

Haloxyfop C15H11NO4ClF3 361.0329 8.17 360.0256 C15H10NO4ClF3 288.0045 C12H6NO2ClF3

Dinoseb C10H12N2O5 240.0746 8.30 239.0673 C10H11N2O5 239.0673 C10H11N2O5

Fenoxaprop met. C7H4NO2Cl 168.9931 8.30 167.9858 C7H3NO2Cl 132.0091 C7H2NO2

Primisulfuron-methyl C15H12N4O7SF4 468.0363 8.40 467.0290 C15H11N4O7SF4 176.0277 C5H4N3O2F2

Acifluorfen C14H7NO5ClF3 360.9965 8.42 359.9892 C14H6NO5ClF3 315.9994 C13H6NO3ClF3

Dinoterb C10H12N2O5 240.0746 8.70 239.0673 C10H11N2O5 239.0673 C10H11N2O5 37 Tecloftalam C14H5NO3Cl6 444.8401 9.01 399.8430 C13H4NOCl6 401.8400 C13H4NOCl5( Cl)

Fomesafen C15H10N2O6SClF3 437.9900 9.07 436.9827 C15H9N2O6SClF3 436.9827 C15H9N2O6SClF3 37 Pentachlorophenol C6HOCl5 263.8470 9.10 262.8397 C6OCl5 264.8368 C6OCl4( Cl)

Forchlorfenuron C12H10N3OCl 247.0512 9.74 246.0440 C12H9N3OCl 127.0068 C5H4N2Cl

Flusulfamide C13H7N2O4SCl2F3 413.9456 11.15 412.9383 C13H6N2O4SCl2F3 412.9383 C13H6N2O4SCl2F3

Dinocap C18H24N2O6 364.1634 11.78 295.1299 C14H19N2O5 295.1299 C14H19N2O5

Fluazinam C13H4N4O4Cl2F6 463.9514 14.57 462.9441 C13H3N4O4Cl2F6 462.9441 C13H3N4O4Cl2F6 12 Y. Akiyama et al. Journal of Pesticide Science 5) (%) RSD n 5) Japanese pear ( (%) RSD n (%) RSD 5) pepper ( Sweet n (%) RSD 3) Lettuce ( n Recovery of pesticides added to agricultural products Recovery (%) RSD 3) Lemon ( n Supplemental Table 2. (%) 3) Spinach ( RSD n Brown rice ( Brown st. st. st. st. st. st. st. st. st. st. st. st. Recovery (%)Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Solvent sticide g/g added)

m Pe (0.1 sitive ionization mode sitive rinexapac-ethyl 82.5 114.5 4.5 94.2 103.2 14.6 77.9 84.3 11.3 76.7 90.7 9.0 105.4 113.5 7.9 85.5 80.5 11.8 80.5 7.9 85.5 113.5 105.4 9.0 90.7 11.3 76.7 84.3 14.6 77.9 103.2 rinexapac-ethyl4.5 94.2 114.5 82.5 epraloxydim met. epraloxydim 100.6 1.5 97.4 6.4 116.2 75.2 52.0 2.0 71.3 122.8 6.6 12.1 16.3 64.2 78.4 79.5 83.8 67.3 oramsulfuron126.5 9.7 74.1 3.2 85.2 187.9 32.9 0.7 77.3 11.3 94.5 7.6 97.1 106.4 81.4 6.8 97.0 72.9 Benzobicyclon met. 95.1 81.6 8.5 77.9 98.0 7.7 79.2 61.4 7.1 90.1 153.7 6.0 58.6 72.3 24.8 87.5 125.1 9.9 125.1 24.8 87.5 72.3 6.0 58.6 153.7 7.1 90.1 61.4 7.7 79.2 Asulam98.0 Imazethapyr 8.5 77.9 81.6 Cyromazinemet.95.1 met. Pyridate 62.1 64.3 0.8 Benzobicyclon 66.5 75.8 1.7 29.7 31.3 50.1 Nicosulfuron 14.8 49.0 8.6 6.6 49.9 15.2 68.1 72.5 3.0 8.3 28.2 89.3 14.1 11.3 12.6 15.6 20.1 23.0 54.8 45.6 90.7 16.3 Diflufenzopyr 51.4 79.4 4.2 10.1 37.1 80.4 45.3 64.5 75.8 77.1 5.5 11.5 59.6 53.9 9.3 65.4 86.7 10.1 Imazaquin 51.3 82.7 5.7 19.9 34.1 98.8 51.4 42.0 15.3 32.8 82.1 109.7 5.6 146.9 7.2 100.9 Flumetsulam 58.7 105.0 60.9 2.3 13.1 105.6 2.9 101.6 117.9 10.0 113.0 80.1 113.5 1.4 48.3 22.5 Thifensulfuron-methyl 68.3 79.1 5.7 56.1 97.4 8.3 14.1 16.1 86.6 94.4 13.1 28.9 49.4 Metsulfuron-methyl 5.8 111.1 53.7 76.9 121.9 89.8 70.3 1.2 54.0 88.3 9.9 111.9 70.0 0.0 60.9 68.5 10.7 5.4 T 10.0 102.6 0.7 95.4 2.1 102.1 0.6 92.7 3.1 101.3 83.5 113.2 133.5 85.7 90.1 0.8 81.1 6.7 10.0 199.8 103.5 72.4 112.7 7.2 139.2 83.2 63.6 93.4 97.3 94.2 85.3 4.5 131.7 5.8 8.2 90.8 52.7 13.0 Chlorsulfuron 40.5 71.1 119.1 86.2 95.5 93.6 4.8 5.6 8.4 89.1 95.6 128.4 60.1 66.2 5.2 Rimsulfuron 43.3 65.8 8.5 Azimsulfuron 64.9 69.1 7.1 16.3 131.9 61.8 5.7 F 222.8 92.1 3.9 76.9 144.8 97.7 1.7 T 139.6 94.9 150.4 84.8 5.4 56.5 4.7 4.4 222.2 93.1 4.7 74.8 91.4 1.7 88.6 94.9 113.0 1.4 118.7 69.9 Clofencet 2.0 42.3 3.5 78.6 4.3 47.9 139.9 97.2 5.8 88.1 94.0 5.3 12.6 81.6 0.5 69.2 105.8 107.2 7.8 Pyrasulfotole84.6 6.1 68.4 92.2 100.1 7.1 84.7 8.9 105.9 129.2 75.6 194.1 92.8 6.0 7.7 90.3 Mesotrione27.7 132.5 12.5 Sulfosulfuron11.2 36.4 143.9 11.3 68.5 112.7 75.4 Cinosulfuron 100.5 73.2 33.4 51.0 9.2 7.7 1.1 76.3 14.1 96.5 12.0 71.4 51.6 45.4 60.0 8.5 Flucarbazone84.4 75.5 30.9 37.6 1.7 82.4 Florasulam8.6 85.1 87.5 3.7 80.6 110.0 4.9 93.3 2.4 131.4 5.2 100.6 95.2 9.9 42.5 46.8 63.5 7.1 108.1 3.9 180.0 3.5 105.7 85.5 5.7 94.8 5.5 139.9 126.0 152.8 1.2 103.0 127.1 115.2 136.0 21.9 116.3 6.3 128.3 99.6 2.5 Imazosulfuron 47.2 66.8 96.3 105.7 45.6 57.7 2.1 71.3 9.3 11.4 101.7 1.5 105.4 9.5 85.6 5.7 Propoxycarbazone96.5 116.0 85.9 6.0 10.1 103.6 80.0 125.5 140.4 44.7 81.3 91.7 1.6 62.8 106.2 3.0 104.5 2.6 13.0 11.9 79.9 4.4 97.2 5.5 86.8 147.9 103.0 104.8 79.0 112.5 107.9 6.8 111.7 7.7 135.4 88.6 76.5 0.9 12.4 98.9 5.9 135.7 102.9 87.1 7.4 91.3 190.6 172.7 121.9 40.5 65.2 76.9 Po Vol. 34, No. 4, 000–000 (2009) Screening method of acidic pesticides by LC/TOF-MS 13 5) (%) RSD n 5) Japanese pear ( (%) RSD n (%) RSD 5) pepper ( Sweet n (Continued) (%) RSD 3) Lettuce ( n Supplemental Table 2. (%) RSD 3) Lemon ( n (%) 3) Spinach ( RSD n Brown rice ( Brown st. st. st. st. st. st. st. st. st. st. st. st. Recovery (%)Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Solvent sticide g/g added)

m Pe (0.1 arfarin 86.6 97.9 3.3 82.1 93.4 2.3 81.8 96.4 15.9 64.3 73.5 3.8 96.0 103.7 12.9 96.9 91.5 7.0 91.5 12.9 96.9 103.7 3.8 96.0 73.5 arfarin15.9 64.3 96.4 2.3 81.8 93.4 3.3 82.1 97.9 86.6 riasulfuron98.5 3.1 117.2 4.7 134.8 98.6 3.0 92.1 4.3 158.1 138.3 88.2 3.1 137.1 113.8 90.2 8.2 135.0 ribenuron-methyl 103.1 73.5 0.8 86.4 75.2 3.5 103.5 81.3 6.1 91.8 74.8 4.5 12.5 85.8 94.9 95.5 92.6 4.4 rifloxysulfuron91.2 3.7 101.7 110.0 4.5 16.8 117.4 8.8 97.5 2.2 84.7 2.9 85.9 119.1 120.1 72.8 106.5 133.8 riflusulfuron-methyl102.2 4.5 125.7 2.3 92.0 4.0 142.9 85.0 5.8 113.9 133.7 115.0 90.9 6.3 99.2 81.0 6.5 102.1 enoxsulam86.3 4.6 103.0 7.3 81.6 102.1 0.9 92.1 6.3 134.4 97.1 5.6 108.4 127.2 84.9 5.1 79.5 121.9 Clethodim sulfone 111.6 105.8 2.8 130.4 103.4 2.7 120.0 121.0 13.6 128.1 92.1 6.0 115.2 115.9 10.8 115.0 98.3 3.9 98.3 10.8 115.0 115.9 6.0 115.2 92.1 13.6 128.1 121.0 2.7 120.0 sulfone103.4 Flazasulfuron2.8 130.4 105.8 Clethodim 111.6 T 12.0 Naptalam 67.6 73.3 67.8 99.5 2.2 125.2 77.3 Ethametsulfuron-methyl 2.1 111.2 94.6 8.4 75.7 61.5 3.7 12.4 86.8 1.3 18.0 113.5 102.2 Iodosulfuron-methyl 72.5 76.9 98.0 90.8 116.0 51.2 3.3 3.3 64.5 93.4 7.2 114.2 89.0 3.1 100.6 6.2 101.9 82.8 121.3 4.9 92.4 6.4 145.9 Pyrithiobac 139.0 94.6 86.2 3.6 100.5 77.0 6.6 Pyrazosulfuron-ethyl 89.4 98.3 7.1 69.1 80.2 6.7 80.8 89.3 99.5 3.5 5.1 T 112.9 35.1 20.7 9.7 90.1 5.7 52.2 2.6 89.4 79.4 94.7 4.7 105.4 71.5 6.8 85.4 90.8 81.7 Mesosulfuron-methyl51.2 8.6 7.1 53.0 95.7 5.5 94.1 82.4 4.4 79.0 94.0 98.5 93.3 8.2 98.0 2.0 5.2 105.0 2.6 104.6 3.5 85.5 131.0 84.0 5.5 102.5 3.2 98.4 2.1 86.7 3.6 Halosulfuron-methyl95.5 7.9 71.7 192.8 137.7 113.8 103.3 5.9 136.6 127.0 10.3 90.8 96.2 74.4 63.9 13.0 Benzylaminopurine103.7 5.3 127.2 89.2 3.6 72.7 6.4 21.9 70.3 110.5 119.2 106.9 127.2 85.3 5.4 35.3 Bispyribac 106.6 19.0 55.9 90.9 T 63.1 65.9 7.9 36.1 53.7 5.3 W Chlorimuron-ethyl7.9 10.1 86.5 4.4 74.3 91.6 105.5 3.2 88.2 Metosulam79.2 8.8 115.9 4.2 103.9 91.9 3.5 90.8 6.0 99.1 13.9 102.1 117.3 125.3 6.7 82.9 146.6 125.3 90.0 2.1 94.6 86.2 P 3.3 122.1 98.4 99.6 2.5 96.9 7.3 65.7 13.3 131.9 10.3 Ethoxysulfuron 87.5 85.2 95.6 117.4 80.5 10.6 7.2 106.0 103.1 8.3 104.3 114.1 108.6 12.0 96.5 5.3 130.0 Cloransulam-methyl165.6 96.0 88.6 91.9 3.4 3.7 160.0 Diclosulam88.1 124.1 3.3 1.9 63.5 95.6 98.4 86.2 2.2 128.9 5.7 10.0 79.8 90.3 1.4 74.3 112.7 107.0 89.2 10.3 Imazamox-methyl 103.1 141.6 96.9 6.2 104.2 114.1 86.5 4.7 132.7 9.0 73.2 Bensulfuron-methyl88.4 T 81.8 127.3 89.9 5.8 87.9 5.5 117.3 87.7 4.0 113.7 0.3 128.8 54.2 3.1 112.0 85.7 5.5 Sulfentrazone 86.7 122.2 101.6 4.7 98.6 94.0 7.7 97.7 79.5 7.1 88.9 87.0 87.6 Cyclosulfamuron 8.3 12.5 132.8 94.7 106.1 83.3 6.4 Diclomezine104.5 1.7 110.6 3.1 123.0 106.7 4.7 84.9 7.1 82.6 99.9 47.7 0.6 99.9 4.7 66.2 102.1 101.5 5.7 123.3 92.6 8.3 92.0 5.3 93.8 123.5 85.4 2.6 81.7 5.7 125.8 97.4 103.6 75.9 9.7 111.1 92.5 85.1 6.0 12.0 109.6 93.2 11.0 51.5 85.5 14 Y. Akiyama et al. Journal of Pesticide Science 5) (%) RSD n 5) Japanese pear ( (%) RSD n (%) RSD 5) pepper ( Sweet n (Continued) (%) RSD 3) Lettuce ( n Supplemental Table 2. (%) RSD 3) Lemon ( n (%) 3) Spinach ( RSD n Brown rice ( Brown st. st. st. st. st. st. st. st. st. st. st. st. 92.9 101.6 1.3 100.8 89.0 22.1 118.0 104.1 2.4 133.3 106.3 4.9 103.4 111.3 8.9 106.3 4.9 104.1 2.4 103.4 22.1 81.6 3.3 133.3 94.1 101.6 1.3 118.0 89.0 100.8 92.9 Recovery (%)Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Solvent sticide g/g added)

m Pe (0.1 ADK 110.8 93.4 5.8 11.4 105.3 83.4 122.9 97.4 0.8 103.7 8.1 18.1 99.0 123.5 93.0 99.5 87.1 7.4 enoprop 111.4 100.1 17.4 91.6 61.4 6.7 79.4 53.2 6.9 120.9 135.7 7.2 78.3 122.0 11.1 90.8 72.1 9.5 72.1 11.1 90.8 122.0 7.2 78.3 135.7 120.9 6.9 enoprop53.2 6.7 79.4 61.4 17.4 91.6 100.1 111.4 enhexamid 68.8 115.8 4.1 78.4 100.1 3.2 88.7 101.0 11.7 90.6 89.6 2.9 69.8 85.0 7.9 84.9 78.2 5.9 78.2 7.9 84.9 85.0 2.9 69.8 89.6 11.7 90.6 101.0 enhexamid3.2 88.7 100.1 4.1 78.4 115.8 68.8 Quizalfop 92.9 88.7 6.3 85.4 116.2 11.1 68.7 56.5 16.5 117.9 109.4 6.0 69.7 90.5 11.2 82.6 70.8 6.4 70.8 11.2 82.6 90.5 6.0 69.7 Prosulfuron109.4 Fluazifop117.9 16.5 Quizalfop56.5 10.2 2,4-DB11.1 68.7 110.0 3.0 125.1 8.0 105.9 0.0 97.9 6.9 112.4 128.5 95.7 4.5 134.7 167.3 79.5 71.3 83.4 116.2 105.1 1.0 108.3 6.3 85.4 10.3 88.2 73.1 101.9 84.5 88.7 3.3 114.9 5.0 132.9 10.8 92.9 97.5 93.0 108.2 74.0 8.5 98.1 84.0 2.8 87.4 69.0 8.4 10.4 104.6 82.6 11.9 3.4 122.9 116.3 91.3 150.1 93.1 83.4 5.4 Cyflumetofen met.Cyflumetofen 45.3 51.9 4-CPA 2.5 11.6 46.1 47.9 Bentazone 84.2 65.9 8.9 Clopropmet.Spiromesifen Bromoxynil 70.3 62.5 0.5 12.7 29.1 41.6 87.3 77.5 8.6 94.9 106.6 105.2 93.4 DNOC 3.6 3.6 18.4 68.7 79.2 139.3 127.5 54.5 94.8 MCPA 8.1 9.9 20.2 67.4 75.1 5.4 62.2 55.3 15.5 19.6 111.8 96.6 2,4-D 64.5 91.6 47.0 27.7 94.2 80.1 4.3 38.9 44.4 92.5 106.2 66.2 13.4 17.4 Mecoprop 97.0 74.6 6.5 131.4 121.4 97.7 18.4 6.8 77.3 67.8 77.7 11.3 Dichlorprop10.2 60.8 74.5 103.8 115.2 130.7 104.1 16.5 4.6 66.3 87.4 6.8 Ioxynil22.7 111.4 105.2 77.3 60.1 112.8 87.6 89.8 2.1 7.6 64.8 65.6 8.5 65.6 2,4,5-T 12.9 11.6 93.8 89.0 123.9 106.0 2.5 68.4 60.3 7.3 19.6 89.5 58.9 68.4 85.5 5.7 D 3.8 44.2 60.6 95.9 81.1 acid12.3 6.8 119.1 85.5 83.2 63.5 96.7 89.2 16.8 20.6 8.0 1.0 81.1 56.3 90.6 63.9 59.9 8.7 78.1 72.7 Clomeprop 67.0 105.6 73.3 10.0 74.7 66.9 1.7 69.4 54.4 73.5 52.0 9.0 4.0 9.3 12.7 107.0 6.0 112.1 112.5 66.7 84.2 99.8 8.3 83.2 58.8 acid 72.0 76.2 Pindone 3.9 6.4 119.4 10.0 5.2 15.6 110.3 80.9 129.7 115.5 74.2 105.0 78.5 77.1 67.2 6.8 5.5 13.9 94.3 4.8 108.2 9.4 76.2 15.1 13.3 112.3 2.7 81.8 9.8 45.6 64.1 102.5 108.1 0.7 113.5 71.7 Clodinafop 114.8 93.0 79.2 119.9 91.5 68.4 9.8 92.0 11.0 10.2 100.1 82.7 72.6 66.9 138.1 99.9 6.0 F 10.4 124.6 5.7 103.0 1.4 90.3 4.7 91.2 66.2 130.7 8.4 13.4 100.3 5.5 94.7 16.2 105.2 66.0 68.5 107.7 98.6 97.0 13.4 95.0 130.8 98.4 5.8 91.3 67.5 139.7 DA 22.3 57.7 82.5 13.3 79.3 73.0 100.3 74.6 1.0 80.0 51.3 4.0 100.8 64.7 4.1 76.1 63.5 8.6 16.6 96.2 71.2 24.4 40.5 24.0 F BrodifacoumPinoxaden95.9 2.2 104.0 58.8 8.9 71.5 144.4 9.4 99.0 8.2 82.9 102.7 91.4 8.9 68.6 20.9 58.7 83.6 12.7 56.5 47.0 23.8 76.2 4.4 77.3 74.7 8.4 Negative ionization mode Negative Vol. 34, No. 4, 000–000 (2009) Screening method of acidic pesticides by LC/TOF-MS 15 5) (%) RSD n 5) Japanese pear ( (%) RSD n (%) RSD 5) pepper ( Sweet n (Continued) (%) RSD 3) Lettuce ( n Supplemental Table 2. (%) RSD 3) Lemon ( n (%) 3) Spinach ( RSD n Brown rice ( Brown st. st. st. st. st. st. st. st. st. st. st. st. Recovery (%)Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery (%) Recovery Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Matrix Solvent Solvent sticide g/g added)

m Pe (0.1 entachlorophenol 78.0 82.7 2.9 22.6 73.1 81.3 119.6 81.7 3.3 71.0 76.9 7.1 11.5 107.9 72.2 82.1 62.5 6.3 ecloftalam17.5 118.9 5.6 55.7 66.2 133.7 45.8 37.5 5.5 24.1 118.6 78.2 109.0 6.9 90.4 21.8 67.8 56.0 enoxaprop10.3 78.9 6.2 113.4 97.8 7.1 105.4 120.8 met.3.1 130.1 93.5 20.3 116.6 92.4 86.4 5.3 68.8 12.6 enoxaprop 11.3 105.3 85.8 8.5 100.8 98.3 61.4 8.4 101.7 103.5 146.8 20.1 13.5 93.4 87.8 86.0 110.9 78.4 omesafenorchlorfenuron 95.6 111.2 1.6 11.3 53.6 60.3 25.6 129.8 122.9 54.7 61.4 10.4 78.6 75.9 6.9 214.9 162.4 5.3 66.5 58.1 9.2 11.6 142.7 109.8 88.1 89.1 3.5 11.3 59.3 49.0 103.9 106.5 11.0 98.6 109.4 4.2 MCPB12.4 61.6 14.1 121.5 F 142.9 Thidiazuron8.1 103.0 81.9 5.5 112.0 77.1 Haloxyfop11.5 136.4 84.1 Dinoseb1.9 102.4 91.9 91.7 14.9 F 107.1 62.6 10.2 97.7 76.3 Primisulfuron-methyl 80.8 90.5 7.2 74.7 109.8 189.1 116.8 Acifluorfen 1.3 1.0 224.4 130.1 Dinoterb101.7 74.9 2.0 8.7 96.7 85.8 21.0 10.7 74.7 53.2 248.9 144.3 T 135.6 107.6 93.0 66.3 5.1 1.6 6.7 101.4 97.0 6.2 104.7 4.1 F 10.2 163.6 138.0 11.4 120.6 90.0 7.5 106.9 110.5 109.5 103.2 5.9 76.5 118.2 6.3 84.3 7.1 78.2 99.9 86.9 8.7 6.1 95.4 102.3 116.3 86.8 66.5 7.0 P 161.1 122.5 5.1 65.5 80.9 F 18.3 119.1 8.3 119.4 109.3 13.0 96.2 6.0 89.0 113.8 77.6 6.8 92.2 93.9 Flusulfamide84.4 8.5 99.6 95.2 9.2 105.3 85.0 12.4 13.2 72.0 9.3 87.0 39.9 8.2 Dinocap143.8 72.9 5.3 193.2 Fluazinam72.4 4.3 115.1 121.6 5.9 136.4 14.9 117.6 4.7 81.1 7.1 100.1 107.7 0.6 97.3 1.9 132.0 135.8 128.7 131.4 64.0 12.1 231.6 77.5 94.1 17.1 159.9 97.9 146.0 94.5 2.3 103.7 4.4 53.3 109.1 1.9 111.7 1.8 119.2 68.2 2.5 78.0 8.0 99.4 119.3 99.5 134.5