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DOI:10.1021/jf9029892
Pesticide Multiresidue Analysis in Cereal Grains Using Modified QuEChERS Method Combined with Automated Direct Sample Introduction GC-TOFMS and UPLC-MS/MS Techniques† KATERINA MASTOVSKA,*,§,^ KELLY J. DORWEILER,*,# STEVEN J. LEHOTAY,§ JENNIFER S. WEGSCHEID,# AND KELLI A. SZPYLKA# §
Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, and #James Ford Bell Technical Center, Medallion Laboratories, General Mills, 9000 Plymouth Avenue North, Minneapolis, Minnesota 55427. ^ Present address: Nutritional Chemistry and Food Safety, Covance Laboratories, 671 S. Meridian Road, Greenfield, Indiana 46140.
The QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation method was modified to accommodate various cereal grain matrices (corn, oat, rice, and wheat) and provide good analytical results (recoveries in the range of 70-120% and RSDs 3000 (in our case, 10000 rpm equivalent to 12857 rcf was employed, using a 5804 centrifuge from Eppendorf, Westbury, NY); (8) transfer 1 mL of the MeCN extract to a 2 mL minicentrifuge tube containing 150 mg of PSA, 50 mg of C18, and 150 mg of anhydrous MgSO4; (9) mix (vortex) the extract with the sorbent/desiccant for 30 s; (10) centrifuge the tube for 5 min; (11) transfer 300 μL of the supernatant into the chamber of a Mini-UniPrep syringeless filter vial (Whatman, Florham Park, NJ), add 30 μL of the 1 μg/mL QC solution, and mix thoroughly; (12) transfer 125 μL of the extract in the Mini-UniPrep vial into a deactivated glass insert (Agilent, Santa Clara, CA) placed in a GC autosampler vial and cap the vial with a heat-treated septum (overnight at 250 C); and (13) compress the filter (polyvinylidene fluoride, PVDF, 0.2 μm) plunger of the Mini-UniPrep assembly to filter the extracts for the UPLC-MS/MS analysis. Method Validation. The method was validated for each matrix in duplicate at three concentration levels: low, middle, and high, which were equivalent to 12.5, 50, and 125 ng/mL, respectively, for each pesticide in the final extract (assuming 100% recovery). This translates to 25, 100, and 250 ng/g for wheat and rice; 36, 143, and 357 ng/g for oat; and 50, 200, and 500 ng/g for corn. The cereal grain samples were fortified using 250 μL of spiking solutions containing 5000, 2000, and 500 ng/mL, respectively, of each pesticide in MeCN with 0.1% acetic acid. After fortification, the spiked samples were left at room temperature for 30 min prior to the addition of the extraction solvents. Matrix-matched calibration standards were used to calculate the analyte recoveries. Solvent-based standard solutions were also analyzed to assess the matrix effects. Automated DSI-GC-TOF MS Analysis. GC-TOFMS analysis was performed using a Pegasus 4D (Leco Corp., St. Joseph, MI) TOF mass spectrometer combined with an Agilent 6890 GC instrument, which was equipped with a secondary oven and nonmoving quad-jet dual stage modulator for two-dimensional comprehensive GCGC chromatography. Injection was conducted by a CombiPAL autosampler (Leap Technologies, Carrboro, NC) with an automated DSI accessory (LINEX) in combination with an Optic 3 programmable temperature vaporizer (PTV) inlet (both from ATAS-GL International, Veldhoven, The Netherlands). Leco ChromaTOF (version 3.22) software was used for GCTOFMS control and data acquisition/processing, and CombiPAL Cycle Composer with macro editor (version 1.5.2) and ATAS Evolution software (version 1.2a) were used to control the automated DSI process and PTV (including column flow), respectively. The automated DSI involved injection of 10 μL of sample extract from an autosampler vial into a disposable microvial (1.9 mm i.d., 2.5 mm o.d., 15 mm long; Scientific Instrument Services, Ringoes, NJ), which was Siltek deactivated by Restek (Bellefonte, PA), washed with acetone (heated at 250 C), and placed in a LINEX DMI tapered liner. Using a LINEX gripper attached to the CombiPAL head, the liner is then transferred into the Optic inlet equipped with a pneumatically controlled LINEX head that can open and close automatically. A series of macros was designed using the Cycle Composer macro editor to create the DSI method and control the LINEX and CombiPAL mechanics. The optimized Optic 3 PTV conditions involved solvent venting at an injector temperature of 100 C for 4.5 min with an initial column flow of 0.8 mL/min and a split flow of 50 mL/min, followed by a splitless transfer of analytes for 4 min, for which the injector temperature was ramped to 280 C (at 16 C/s) and the column flow changed to 1.5 mL/min (kept constant for the entire GC run). After the splitless period, the split flow was kept at 50 mL/min for 6 min, at which point the split flow was reduced to 25 mL/min and the injector temperature was decreased to 250 C. The GC separation was conducted using a combination of a 20 m 0.25 mm i.d. 0.25 μm film thickness RTX-5 ms column and
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Table 1. Retention Times (tR) and MS Ions Used for Quantitation (Q m/z) in the DSI-LVI-GC-TOFMS Analysis name
MIX
tR (s)
Q m/z
acephate alachlor aldrin ametryn amitraz atrazine azinphos-ethyl azinphos-methyl azoxystrobin BHC, RBHC, R-, 13C6BHC, βBHC, δbifenox bitertanol I bitertanol II bromacil bromophos bromopropylate captan/captafol degradation product (cis-1,2,3,6-tetrahydrophthalimide) carbaryl carbaryl degradation product (1-naphthol) carbofuran carbofuran, 3-hydroxycarbofuran-7-phenol carbophenothion chlordane, cischlordane, transchlordimeform chlorfenson chlorfenvinphos, cischlorfenvinphos, transchlorobenzilate chloroneb chloropropylate chlorothalonil chlorpropham chlorpyrifos chlorpyrifos-methyl chlorthal-dimethyl cinerin I coumaphos cyanazine cyfluthrin I cyfluthrin II cyfluthrin III þ IV cyhalothrin, γcyhalothrin, λcypermethrin I cypermethrin II-IV cyprodinil DDD, o,p0 DDD, p,p0 DDE, o,p0 DDE, p,p0 DDT, o,p0 DDT, o,p0 -, 13C6DDT, p,p0 deltamethrin deltamethrin artifact demeton-O demeton-S diazinon dichlobenil dichlorvos diclofop-methyl dicloran dicofol degradation product (4,40 -dichlorobenzophenone)
1 2 2 2 1 1 1 1 1 2 ISTD 2 2 2 2 2 2 2 2 2 1 1 1 1 1 2 2 2 2 2 1 1 2 2 1 2 2 2 2 2 1 1 2 2 2 2 2 2 2 2 1 2 1 1 1 2 ISTD 1 2 2 2 2 2 2 1 2 2 2
596.9 845.5 895.2 846.8 1331.6 743.1 1348.6 1315.2 1488.0 718.7 718.3 748.0 783.1 1297.5 1366.0 1370.2 888.0 932.1 1276.4 614.1 841.7 625.6 738.2 604.0 539.8 1180.8 1020.5 995.0 693.4 1032.7 952.6 971.7 1117.1 625.0 1117.6 789.6 687.9 905.3 834.5 913.3 1083.7 1378.3 909.7 1391.9 1395.5 1399.5 1329.1 1338.8 1403.2 1409.8 945.5 1073.2 1132.8 1004.2 1059.0 1137.9 1136.3 1200.6 1473.6 1463.7 670.0 729.4 772.2 560.7 517.2 1232.3 731.0 908.7
94 188 263 212 132 200 132 160 344 183 225 183 183 341 170 170 205 331 341 151 144 144 164 180 164 342 373 375 181 175 267 269 251 191 251 266 127 197 286 301 123 362 68 206 206 206 181 181 181 181 224 235 235 246 246 235 247 235 181 181 88 88 179 171 185 253 176 250
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Table 1. Continued name dicrotophos dieldrin dimethoate dioxathion diphenamid diphenylamine disulfoton diuron degradation product (3,4-dichlorophenyl isocyanate) endosulfan R endosulfan β endosulfan sulfate endrin endrin aldehyde endrin ketone EPN ethalfluralin ethion ethoprophos ethoxyquin etridiazole fenamiphos fenarimol fenchlorphos fenitrothion fenobucarb fenobucarb degradation product fenpropathrin fensulfothion fenthion fenvalerate I fenvalerate II fluazifop-p-butyl fluvalinate, τfolpet degradation product (phthalimide) fonofos heptachlor heptachlor epoxide hexachlorobenzene imazalil iprodione iprodione degradation product isazophos isofenphos leptophos lindane malathion metalaxyl methamidophos methidathion methiocarb methiocarb degradation product methoxychlor metolachlor metribuzin mevinphos mirex monocrotophos myclobutanil napropamide nitrapyrin norflurazon omethoate oxadiazon oxyfluorfen paraoxon parathion parathion, d10parathion-methyl
MIX
tR (s)
Q m/z
2 2 1 2 2 2 1 1 2 2 2 2 2 2 1 2 2 2 2 2 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 2 1 2 2 2 2 2 2 1 2 1 2 1 1 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 ISTD 1
700.3 1059.6 732.7 754.4 934.3 675.4 778.9 544.1 1015.3 1113.6 1191.5 1096.7 1168.4 1262.4 1277.4 692.6 1144.2 681.1 733.7 603.6 1037.8 1341.4 854.7 873.7 668.9 527.9 1289.8 1125.9 902.1 1439.8 1448.6 1107.3 1414.5 605.7 763.9 845.4 956.5 727.3 1048.2 1266.9 1130.9 788.8 971.4 1317.9 754.8 889.7 853.0 510.0 997.9 872.8 640.3 1285.2 898.7 824.4 593.1 1323.3 706.6 1074.4 1037.7 603.0 1197.7 664.3 1069.2 1079.7 852.8 906.9 900.2 834.8
127 261 93 270 167 169 88 187 243 195 274 263 345 317 157 276 231 158 202 211 303 251 285 277 150 150 181 293 278 167 167 282 250 147 246 272 353 284 215 316 187 161 213 375 183 173 206 94 145 168 168 227 238 198 127 272 127 179 271 196 303 156 175 252 109 291 301 263
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Table 1. Continued name pendimethalin pentachloronitrobenzene permethrin, cispermethrin, transpermethrin, trans-, 13C6perthane phorate phosalone phosmet phosphamidon piperalin piperonyl butoxide pirimicarb pirimiphos-ethyl pirimiphos-methyl procymidone profenofos profenofos degradation product (4-bromo-2-chlorophenol) profluralin promecarb promecarb degr. product (5-isopropyl-3-methylphenol) prometryn propanil propham propiconazole I propiconazole II propoxur propoxur degradation product propyzamide pyrethrin I quinalphos quizalofop-ethyl resmethrin I resmethrin II simazine simetryn sulfallate sulprofos terbacil terbufos tetrachlorvinphos tetradifon thiabendazole thiobencarb thionazin tolylfluanid degradation product (DMST) triadimefon tribufos (DEF) trifloxystrobin trifluralin trimethacarb trimethacarb degradation product vinclozolin
a 1 m 0.1 mm i.d. 0.1 μm film thickness RTX-pesticide2 column (both from Restek), which translated into a 1.68 m 0.1 mm i.d. “virtual” column setting in the ATAS Evolution software. The oven temperature program (started after a 4.5 min solvent vent period) was as follows: 60 C held for 4 min, ramped to 180 at 20 C/min, then at 5 C/min to 230 C, 20 C/min to 280 C, and finally ramped to 300 at 40 C/min, and held for 12 min. The total run time was 35 min. The MS transfer line and ion source temperatures were held at 280 and 250 C, respectively. The electron energy was 70 eV. The detector voltage was set at about þ200 V above the value obtained in the tuning procedure (at 1750 V during the method validation). The TOF instrument acquired full scan spectra in the range of m/z 45-550 at a data acquisition rate of 10 spectra/s. Agilent’s Pesticide and Endocrine Disruptor Database was converted into NIST format and used for MS library spectra searching and
MIX
tR (s)
Q m/z
2 2 2 2 QC 2 2 2 1 2 2 1 2 2 2 2 2 2 2 1 1 2 1 2 1 1 1 1 2 1 1 2 2 2 2 2 1 2 1 2 2 2 1 1 2 1 2 1 1 2 1 1 2
956.4 760.6 1369.9 1374.3 1381.8 1103.9 712.3 1316.0 1271.8 823.5 1214.3 1243.1 805.9 940.3 875.9 985.9 1053.0 522.6 761.1 709.6 544.4 851.9 820.9 602.8 1195.5 1207.3 670.3 480.3 762.6 1176.6 975.0 1408.5 1238.3 1247.0 736.5 840.2 716.9 1164.6 783.7 758.9 1017.0 1306.1 963.6 889.2 667.7 750.8 911.4 1059.0 1211.1 700.3 693.4 526.0 834.2
252 237 183 183 189 223 260 182 160 264 314 176 166 318 290 283 339 206 318 135 135 184 161 179 173 173 110 110 173 123 146 299 171 171 201 213 188 322 160 231 329 159 201 100 248 214 208 169 116 264 136 136 212
matching. Table 1 gives retention times and MS ions used for the quantitation of the GC-amenable pesticides. UPLC-MS/MS Analysis. UPLC-MS/MS analysis was performed using an Acquity UPLC system (integrated solvent and sample management system with a column heater module) interfaced to a Quattro Premier triple-quadrupole mass spectrometer (both from Waters Corp., Milford, MA). The MassLynx software (version 4.1) was used for instrument control and data acquisition/processing. Sample injection volume was 2 μL. An Acquity UPLC BEH C18 column (50 2.1 mm; 1.7 μm particle size, 130 A˚ pore size) from Waters was employed for the LC separation at 40 C. A binary mobile phase was composed of (A) 10 mM ammonium formate in water (pH 3, adjusted using formic acid) and (B) 10 mM ammonium formate in MeOH. A linear mobile phase gradient started at 30% B (0-4 min) and
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Table 2. Analyte-Specific UPLC-MS/MS Conditions, Including Retention Times (tR) analyte
MIX
tR (min)
precursor ion (m/z)
quantitation product ion (m/z)
confirmation product ion (m/z)
cone voltage (V)
collision energy (V)
dwell time (ms)
acephate acetamiprid aldicarb aldicarb sulfone aldicarb sulfoxide ametryn amitraz atrazine atrazine, d5azinphos-ethyl azinphos-methyl azoxystrobin bifenox bitertanol carbaryl carbofuran carbofuran, 13C6carbofuran, 3-hydroxychloroxuron cyanazine cyprodinil deltamethrin dichlorvos dicrotophos dimethoate dimethoate, d6diuron fenobucarb fensulfothion fluvalinate, τimazalil imidacloprid linuron malathion methamidophos methidathion methiocarb methomyl monocrotophos myclobutanil napropamide norflurazon omethoate oxamyl paraoxon permethrin permethrin, trans-, 13C6phosalone phosmet piperalin profenofos promecarb prometryn propanil propham propiconazole propoxur quizalofop-ethyl resmethrin simazine spinosad A spinosad D tetrachlorvinphos thiabendazole thiobencarb tolylfluanid trifloxystrobin trimethacarb
1 1 1 1 1 2 1 1 ISTD 1 1 1 2 2 1 1 ISTD 1 1 2 1 2 1 2 1 ISTD 1 1 2 2 1 1 1 1 1 2 1 1 2 2 2 2 1 1 1 2 QC 2 1 2 2 1 2 2 1 1 1 2 2 2 1 1 2 1 1 1 1 1
0.38 1.02 1.89 0.44 0.39 7.08 10.31 5.80 5.80 8.15 6.91 7.59 9.66 9.61 4.85 3.95 4.61 1.00 8.05 3.00 8.20 10.38 3.34 0.65 1.00 1.33 6.23 7.19 6.78 10.48 6.60 0.70 7.09 7.79 0.38 6.67 7.39 0.52 0.58 8.04 8.37 6.72 0.39 0.42 6.30 10.56 9.47 9.52 7.00 6.41 9.91 7.64 7.99 7.21 5.71 9.34 3.61 9.89 10.48 3.31 9.76 9.95 9.02 0.95 9.51 9.24 9.79 6.38
184.1 223.3 208.1 223.0 207.1 228.3 294.4 216.5 221.5 346.3 318.1 404.4 359.1 338.4 202.2 222.2 228.1 255.3 291.3 241.4 226.3 523.2 221.1 238.3 230.2 236.0 233.2 208.1 309.2 503.4 297.3 256.1 249.1 331.2 141.9 303.3 226.3 163.1 224.2 289.4 272.4 304.2 214.0 237.3 276.4 408.3 414.8 368.3 318.2 331.4 375.1 208.3 242.3 218.1 180.2 342.4 210.3 373.4 339.4 202.2 732.7 746.7 367.4 202.2 258.3 347.1 409.4 194.0
142.9 125.9 116.0 85.8 132.0 186.1 163.1 174.1 101.1 132.0 132.1 372.2 310.1 70.0 145.0 165.0 171.0 163.0 72.0 214.3 92.9 281.0 109.0 193.0 199.0 204.9 71.9 94.9 281.1 208.1 158.9 175.0 159.9 127.0 93.8 144.9 121.0 87.9 193.0 70.0 129.1 284.2 183.0 72.0 220.0 183.0 189.3 182.0 160.0 173.0 305.0 109.0 157.8 162.0 138.1 159.0 111.0 299.3 171.1 132.0 142.1 142.2 127.0 175.0 124.9 238.1 186.0 137.1
49.0 89.9 88.9 80.9 88.8 95.9 253.3 104.5 179.2 159.9 261.1 344.1 342.2 269.3 127.0 123.0 129.0 181.0 164.1 96.0 107.9 N/A 78.9 193.0 171.0 87.8 159.9 152.0 253.1 180.9 172.9 209.1 182.0 285.1 125.0 84.9 169.0 105.9 97.9 125.0 171.0 160.1 155.0 89.8 173.9 355.3 361.4 322.2 133.0 231.2 347.1 151.1 200.1 127.0 120.0 122.9 168.0 271.2 91.0 124.1 N/A N/A 241.0 131.0 99.9 136.9 206.1 109.1
17 27 10 13 13 32 20 20 20 18 14 21 16 16 20 26 26 13 34 35 49 20 33 21 20 20 26 23 29 20 29 21 30 20 25 19 20 13 21 30 23 45 20 12 24 15 15 22 20 40 29 21 30 28 13 36 17 31 23 32 40 40 27 47 22 12 23 22
16 26 8 18 5 19 13 22 22 14 7 18 10 10 17 13 13 20 16 19 31 12 24 11 9 9 25 12 14 11 25 18 17 11 14 9 16 8 8 21 15 22 10 9 18 9 9 11 30 27 16 14 26 15 9 49 11 19 16 18 21 21 17 24 15 15 18 23
12 25 40 20 20 30 50 30 30 20 10 15 20 60 100 100 100 25 20 70 20 30 90 60 25 25 20 10 30 20 20 5 15 15 15 30 15 20 70 45 40 30 17 30 25 20 20 30 15 40 20 15 40 25 30 30 90 20 30 70 30 30 50 40 15 10 15 17
Article went to 60% B at 7.5 min (held until 8.5 min), followed by a gradient to 100% B at 10.5 min (held until 12.5 min), and concluded by column equilibration at initial conditions of 30% B (12.6-15 min). The flow rate of the mobile phase was 450 μL/min. The MS determination was performed in electrospray (ESI) positive mode (using the optimized MS instrument parameters obtained by the tuning) combined with monitoring of the two most abundant MS/MS (precursor f product) ion transitions. Table 2 gives analyte-specific MS/ MS conditions and LC retention times for the LC-amenable analytes. The MS source conditions were as follows: capillary voltage of 1.7 kV, extractor voltage of 4.0 V, RF lens at 0.9 V, source temperature of 130 C, desolvation temperature of 350 C, collision gas (argon) pressure of 4.31 10-3 mbar, desolvation gas (N2) flow of 600 L/h, and cone gas (N2) flow of 100 L/h. RESULTS AND DISCUSSION
As mentioned in the Introduction, our goal was to modernize a traditional methodology for the analysis of pesticide residues in cereal grains. We started with updating the target list of analytes to add mainly those frequently found in the Pesticide Data Program (PDP), which is a national pesticide residue database program administered by the USDA Agricultural Marketing Service (16). We were interested not only in residues found in cereal grains but also in fruit and vegetable commodities because the target analyte list and instrument methods were also intended for the analysis of pesticide residues in fruits and vegetables using the QuEChERS method with acetate buffer (AOAC International Official Method 2007.01). The target list of analytes for method development and validation is given in Tables 1 and 2. The instrument methods for the DSI-LVI-GC-TOFMS and UPLC-MS/MS analyses had to be developed and optimized first, followed by modification and optimization of the QuEChERS procedure for various cereal grain matrices (corn, oat, rice, and wheat). DSI-LVI-GC-TOF MS Method Development and Optimization. The development of the automated DSI-LVI-GC-TOFMS method involved optimization of each individual component to obtain an overall working system. The GC column setup employed a combination of a 20 m 0.25 mm i.d. 0.25 μm film thickness RTX-5 ms column and a 1 m 0.1 mm i.d. 0.1 μm film thickness RTX-pesticide2 column, for first dimension (1D) and potential second dimension (2D) separations, respectively, if comprehensive two-dimensional GCGC separation was desired or needed. Indisputably, the GCGC analysis has several benefits, including mainly improved GC separation efficiency (thus method selectivity) and increased sensitivity due to the thermal focusing of the peaks eluting from the first dimension (17). However, a routine operation of a GCGC system is rather demanding in terms of the relatively high liquid nitrogen consumption (for thermal modulation) and also when it comes to far more complex data processing as compared to a 1D analysis. Therefore, the optimized method used 1D GC separation for cereals, fruits, and vegetables, but the short secondary column was kept in place for an easy conversion to a 2D system for analysis of more complex samples, such as spices or tea. To facilitate a faster GC system equilibration, the secondary oven and modulator insulations were removed for the 1D operation. Also, the secondary oven and modulator were inactivated in the GC method. As for the TOFMS part, the TOF mass analyzer enables fast acquisition of full mass spectra (up to 500 spectra/s is possible with the Pegasus system). In the MS method, we selected a mass range of m/z 45-550 to cover a typical mass range for pesticide spectra, including mirex with the highest monoisotopic molecular weight (540 g/mol) on our target list. A spectral acquisition rate of 10 spectra/s was used as a sufficient rate for peak characterization and deconvolution in 1D analysis (higher rates led to decreased sensitivity without any significant benefits). An ion source tem-
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perature of 250 C was chosen as a compromise between sensitivity (ionization efficiency) and spectral quality (degree of fragmentation). Lower temperatures gave lower sensitivity (especially for the less volatile analytes), whereas higher temperatures might lead to overfragmented spectra with low abundance of higher ions and poor library match. To obtain deconvoluted reference spectra even for closely eluting peaks, analytes (in total 185 compounds monitored by GC-TOFMS, including important pesticide degradation products) were divided into two groups (designated MIX-1 and MIX-2 in Table 1) for two separate injections into the GC system. The TOFMS instrument does not require presetting of analytespecific conditions for each individual pesticide as opposed to, for example, single ion monitoring with quadrupole or tandem MS with triple-quadrupole or ion trap mass analyzers. Therefore, the analysis (data acquisition) is nontargeted. However, for routine pesticide residue analysis, it is difficult to process the data in a completely nontargeted fashion, relying only on spectral deconvolution, peak finding, and spectral matching algorithms provided by the data processing software. Instead, we preferred to create templates (in the calibration portion of the software) that enabled fast data review for pesticides on our target list by extracting traces of their quantitation ions in expected retention time windows and comparing their deconvoluted and raw MS spectra with library and reference spectra. As mentioned in the Introduction, the DSI technique is a unique form of a LVI, which uses disposable microvials for introduction of samples into the GC system. As opposed to other LVI techniques (18), the nonvolatile matrix components remain in the microvial and are removed from the system after each GC run. Also, it is not necessary to trap the liquid sample in the liner at low temperatures because the microvial holds the liquid in the liner. Therefore, excessive inlet cooling is not required, and the initial inlet temperature can be set at a temperature suitable for effective solvent venting (100 C in our case for MeCN injection). The solvent venting conditions (temperature, vent time, initial column flow, and split flow) were optimized to eliminate 80-90% of MeCN without losing early eluting analytes. It is advisible to leave 1-2 μL of the solvent in the microvial as a keeper (before ramping the injector temperature), but larger volumes should be avoided to prevent peak distortions and potential column bleed (19). Analyte transfer conditions (temperature programming rate, final inlet temperature, and column flow) were optimized to quantitatively transfer analytes (especially the late eluting ones). Different pressure pulses were tested for faster and more effective analyte transfer but did not result in significant improvements in analyte responses. To improve injection reproducibility, the microvials were sent for Siltek deactivation. Also, it was important to rinse each microvial with acetone and heat it at 250 C overnight prior to its use to remove serious background interferences in the GCTOFMS analysis. Another source of interferences were siloxanes from septa in the autosampler vial caps, which were minimized by overnight heating of the septa at 250 C. UPLC-MS/MS Method Development and Optimization. Initially, the LC-MS/MS method development was focused on the conversion of the HPLC-fluorescence method for carbamate insecticides to a modern system that would not require postcolumn derivatization and would offer analyte identification based on MS/MS transitions. However, the list of LC-amenable analytes expanded when the original list of target analytes was updated with some modern, more polar pesticides, such as imidacloprid, acetamiprid, azoxystrobin, trifloxystrobin, or spinosads. Also, some pesticides traditionally analyzed by GCMS but performing far better in LC-MS/MS were included in the LC-MS/MS method, such as more polar organophosphate
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Figure 1. UPLC-MS/MS extracted ion chromatograms of selected pesticides spiked at 25 ng/g in wheat extract, which was obtained by the optimized sample preparation method.
Figure 2. Total ion chromatogram obtained by a DSI-LVI-GC-TOFMS analysis of a corn extract prepared using 5 g of sample, original QuEChERS (with 10 mL of water addition for swelling), and 50 mg of PSA in the dispersive SPE step. The highlighted region of the chromatogram is saturated with fatty acids (mainly linoleic acid), with the dotted trace representing optimized analysis using 2.5 g of corn sample and conducting the dispersive SPE with 150 mg of PSA and 50 mg of C18.
insecticides (e.g., acephate, methamidophos, omethoate, and dimethoate), imidazole fungicides (imazalil and thiabendazole), and even pyrethroid insecticides (e.g., permethrin, deltamethrin, resmethrin, and τ-fluvalinate). The latter group represents less volatile analytes that elute late in the GC analysis and are more problematic in terms of the transfer from the microvial into the GC column. The first two groups include compounds that are susceptible to matrix effects in GC (20, 21). At the time of the validation, the LC-MS/MS method included 64 analytes, 3 isotopically labeled internal standards, and 1 QC standard (see Table 2). Triphenyl phosphate (TPP) was initially chosen as the QC standard (added to the final extract before
GC- and LC-MS analysis) because of its strong signal in both GC- and LC-MS and similarity to organophosporus pesticides. However, TPP was detected in all method and solvent blanks by LC-MS. The source of the contamination was not identified. However, given that TPP is a plasticizer, it is possible that the contamination is due to the use of disposable plastic consumables in the laboratory. We chose to replace TPP with [13C6]-transpermethrin as the QC standard because of its availability as a labeled pesticide amenable to both GC- and LC-MS techniques. The trend of including more analytes into the LC-MS/MS method is likely to continue because most pesticides (except for nonpolar, halogenated hydrocarbons) generally give better or
Article similar results in LC-MS/MS as compared to GC-MS (22). Furthermore, simultaneous analysis by GC-MS and LC-MS/ MS provides confirmatory information for the analytes that are amenable to both of these techniques. The LC-MS/MS analysis employed the UPLC technique using a short, narrow column with 1.7 μm particles for fast yet efficient separation. The method development involved tuning for analyte-specific MS/MS conditions (shown in Table 2) and optimization of analyte separation. Figure 1 shows an example of extracted ion chromatograms of several pesticides spiked at 25 ng/g in a wheat extract, which was obtained by the optimized sample preparation procedure. Sample Preparation Method Development and Optimization. For sample preparation of cereal grains, our goal was to adapt the QuEChERS method, which was originally developed for the analysis of fruits and vegetables. In brief, the original QuEChERS method (4) is based on extraction of a homogenized produce sample (10 g) with MeCN (10 mL). A combination of anhydrous MgSO4 (4 g) and NaCl (1 g) is added to induce separation between the MeCN and aqueous layers (the water originates from the produce sample). The sample is shaken in a tube for 1 min and centrifuged. An aliquot (1 mL) of the upper MeCN layer is cleaned up using dispersive SPE with PSA (25 mg) and anhydrous MgSO4 (150 mg). After brief vortexing/shaking (30 s) and centrifugation, the extract is ready for GC- and LC-MS(/MS) analyses. As opposed to the original method, the buffered AOAC method uses MeCN with 1% acetic acid for sample extraction and sodium acetate instead of NaCl in the salt mixture (5). Also, a double amount of PSA (50 mg) is added to the extract aliquot (1 mL) in the dispersive SPE, in part because the presence of acetic acid reduces the PSA capacity. In comparison with fruits and vegetables, cereal grains represent dry matrices with a high content of fatty acids, which can interfere mainly in the GC-MS analysis. Therefore, several modifications had to be made to accommodate various cereal grain matrices and provide good analytical results for the majority of the target pesticides. These modifications involved mainly optimization of matrix swelling (addition of water), sample to solvent ratio, extraction time, and the sorbent combination and amount in the dispersive SPE cleanup. As for the sample to solvent ratio, we started our optimization experiments with 5 g of sample (23), to which we added 10 mL of water and let the matrix swell for 1 h. After that, the original QuEChERS procedure was followed. For finely milled rice, 15 mL of water per 5 g of sample was necessary for effective swelling. Later, we optimized the sample amount for individual grains (see below) and also found that MeCN (10 mL) should be added to the sample at the same time as water, followed by shaking of the sample for 1 h to facilitate matrix swelling/ extraction and improve analyte recoveries. The addition of MeCN can prevent enzymatic degradation of malathion and some other susceptible pesticides during the matrix swelling process (24). The 5 g sample size seemed to be acceptable for wheat and rice, but it posed a problem for corn (and to a lesser extent for oat). Figure 2 shows a total ion chromatogram (TIC) of a corn extract prepared using 5 g of sample, original QuEChERS (with 10 mL of water addition for swelling), and 50 mg of PSA in the dispersive SPE step. The middle, highlighted, region of the chromatogram is saturated with fatty acids (mainly linoleic acid but also oleic and palmitic acids). To improve the situation, we tested different amounts of PSA (50-200 mg) and also added 50 mg of C18, which was previously demonstrated to be beneficial for samples with a higher fat content, such as milk, eggs, or avocado (25). Up to 150 mg per 1 mL of extract was acceptable in terms of analyte
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Figure 3. Overlays of (A) total ion GC-TOFMS chromatograms of spiked corn extracts obtained using 2.5 g of sample and original and buffered QuEChERS procedures, both with 150 mg of PSA and 50 mg of C18 in the dispersive SPE step. Chromatograms in B and C represent extracted ion chromatograms of m/z 263 and 179, respectively, showing selected pesticide peaks eluting in the region highlighted in (A).
recoveries and volume of the final extract available for the GCand LC-MS analysis. For corn, however, we had to also decrease the sample size to 2.5 g (for oat to 3.5 g) to obtain good analyte peak shapes and rugged method performance. The dotted trace in Figure 2 shows the TIC in the affected region after the original QuEChERS method was optimized for the corn analysis. We also tried the buffered AOAC method but, even with the increased amount of PSA to 150 mg (and addition of 50 mg of C18), the amount of fatty acids left in the final extract was overwhelming due to the reduced capacity of PSA in the presence of acetic acid. Figure 3A provides comparison of the TICs of spiked corn extracts obtained using 2.5 g of sample and original
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Table 3. Pesticide Recoveries and RSDs in Cereal Grains Summarized (i) for the Four Cereal Grain Commodities Based on Different Concentration Levels (Expressed as Concentrations in the Cereal Grain Extracts) and (ii) for the Three Concentration Levels Based on Different Cereal Grain Commoditiesa % recovery (% RSD) concentration level, n = 8 (all commodities) analyte acephate acetamiprid alachlor aldicarb aldicarb sulfone aldicarb sulfoxide aldrin ametryn amitraz atrazine azinphos-ethyl azinphos-methyl azoxystrobin BHC, RBHC, βBHC, δbifenox bitertanol bromacil bromophos bromopropylate carbaryl carbofuran carbofuran, 3-hydroxycarbophenothion chlordane, cischlordane, transchlordimeform chlorfenson chlorfenvinphos, cischlorfenvinphos, transchlorobenzilate chloroneb chloropropylate chlorothalonil chloroxuron chlorpropham chlorpyrifos chlorpyrifos-methyl chlorthal-dimethyl cinerin I coumaphos cyanazine cyfluthrin cyhalothrin cypermethrin cyprodinil DDD, o,p0 DDD, p,p0 DDE, o,p0 DDE, p,p0 DDT, o,p0 DDT, p,p0 deltamethrin demeton-O demeton-S diazinon dichlobenil dichlorobenzophenone dichlorvos diclofop-methyl dicloran dicrotophos
method LC LC GC LC LC LC GC LC LC GC LC LC LC GC GC GC GC LC GC GC GC LC LC LC GC GC GC GC GC GC GC GC GC GC GC LC GC GC GC GC GC GC LC GC GC GC GC GC GC GC GC GC GC LC GC GC GC GC GC LC GC GC LC
high (125 ng/mL) 82 112 99 116 112 98 75 103 69 101 117 116 120 94 98 89 99 103 99 94 104 116 118 114 97 88 85 99 101 106 110 108 100 101 66 112 99 96 98 100 90 106 108 87 101 93 91 92 83 78 81 79 75 99 97 111 100 94 97 97 102 106 98
(7) (3) (10) (5) (7) (6) (5) (4) (16) (13) (3) (8) (8) (14) (6) (15) (12) (5) (12) (18) (10) (4) (5) (4) (9) (9) (9) (16) (11) (13) (9) (10) (13) (12) (13) (5) (8) (6) (15) (5) (19) (11) (6) (27) (19) (24) (13) (6) (12) (18) (24) (9) (12) (9) (16) (11) (12) (16) (9) (11) (9) (9) (5)
middle (50 ng/mL) 66 93 99 93 90 79 70 101 59 95 95 93 98 95 94 88 89 101 97 91 90 95 95 91 89 82 81 118 98 94 94 96 105 90 ND 92 111 90 97 95 101 108 101 87 93 91 90 90 83 69 65 74 73 90 97 100 93 93 86 86 97 105 94
(5) (8) (11) (8) (11) (5) (14) (8) (14) (15) (9) (13) (7) (13) (8) (13) (10) (7) (9) (20) (9) (7) (6) (8) (10) (10) (9) (27) (10) (11) (12) (8) (19) (12) ND (5) (17) (6) (16) (8) (28) (19) (8) (14) (13) (13) (18) (12) (14) (20) (25) (10) (12) (13) (27) (15) (11) (28) (7) (10) (8) (11) (8)
commodity, n = 6 (all concentrations)
low (12.5 ng/mL)
wheat (25-250 ng/g)
rice (25-250 ng/g)
oat (36-357 ng/g)
corn (50-500 ng/g)
60 84 108 83 79 69 70 98 51 104 84 87 88 89 89 93 89 97 110 86 102 87 87 84 93 84 81 100 101 ND 109 100 86 94 ND 84 87 91 91 95 ND 113 102 ND 87 ND 90 82 77 76 70 89 75 100 93 101 89 88 87 81 94 102 94
67 93 109 99 97 83 64 97 51 110 96 96 100 95 93 91 87 97 98 88 92 98 100 96 88 78 76 118 99 110 110 98 101 96 62 96 108 89 99 92 100 114 100 95 97 108 88 81 76 67 62 70 71 90 91 109 94 89 87 84 96 110 92
71 103 97 98 96 83 81 107 72 97 108 109 111 90 99 87 105 108 114 92 101 106 107 101 100 92 89 97 103 96 96 107 93 94 63 98 100 94 96 100 108 102 108 97 97 92 94 93 83 77 77 88 76 103 99 101 108 94 97 102 100 108 98
73 97 105 96 90 81 73 100 56 94 96 87 99 91 89 91 87 99 100 93 100 97 98 95 92 84 81 108 99 92 102 104 93 90 68 96 98 97 95 100 79 102 103 85 100 91 85 90 81 71 67 77 73 92 97 107 91 94 89 84 100 101 97
67 92 96 98 91 80 71 100 58 98 95 102 97 94 95 90 97 98 96 91 102 96 95 92 93 85 83 100 99 100 109 98 99 99 69 94 96 90 93 96 93 119 103 79 91 82 94 89 85 81 78 86 77 101 95 101 88 90 90 82 97 103 95
(13) (8) (8) (9) (12) (7) (24) (5) (18) (17) (9) (17) (11) (21) (19) (16) (14) (9) (20) (16) (13) (7) (7) (6) (11) (18) (13) 39 (10) ND (12) (9) (20) (9) ND (8) (13) (9) (11) (10) ND (10) (5) ND (19) ND (6) (13) (11) (7) (14) (25) (12) (20) (22) (8) (26) (24) (14) (17) (9) (18) (5)
(24) (16) (8) (20) (19) (22) (21) (5) (15) (15) (17) (23) (19) (18) (15) (17) (10) (5) (13) (19) (11) (18) (19) (18) (15) (18) (14) (27) (16) (16) (12) (14) (20) (13) (19) (19) (8) (12) (18) (10) (20) (7) (6) (21) (13) (21) (6) (16) (9) (15) (20) (12) (15) (16) (18) (9) (20) (17) (19) (14) (10) (11) (7)
(14) (10) (4) (20) (15) (16) (13) (3) (15) (19) (10) (15) (11) (12) (9) (15) (7) (6) (19) (14) (12) (11) (12) (14) (9) (9) (9) (6) (7) (15) (14) (10) (13) (16) (27) (9) (10) (5) (9) (7) (32) (22) (3) (27) (5) (5) (19) (9) (17) (22) (28) (9) (13) (13) (15) (8) (18) (18) (11) (9) (11) (5) (4)
(13) (15) (10) (16) (19) (16) (12) (7) (17) (9) (18) (20) (18) (21) (17) (17) (9) (6) (6) (20) (2) (14) (14) (15) (6) (10) (7) (28) (6) (11) (8) (7) (29) (10) (0) (15) (29) (5) (19) (5) (14) (15) (9) (21) (29) (25) (13) (4) (8) (13) (17) (4) (13) (20) (32) (10) (15) 37 (6) (14) (7) (13) (7)
(13) (15) (11) (11) (21) (13) (9) (4) (15) (9) (16) (6) (15) (14) (8) (11) (13) (8) (14) (20) (15) (13) (14) (14) (9) (10) (7) (33) (10) (7) (12) (9) (11) (7) (6) (14) (9) (6) (12) (7) (9) (10) (8) (16) (12) (16) (5) (11) (11) (4) (17) (25) (7) (9) (19) (18) (6) (14) (6) (11) (10) (12) (7)
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Table 3. Continued % recovery (% RSD) concentration level, n = 8 (all commodities) analyte
method
dieldrin dimethoate dioxathion diphenamid diphenylamine disulfoton diuron endosulfan R endosulfan β endosulfan sulfate endrin endrin ketone EPN ethalfluralin ethion ethoprophos ethoxyquin etridiazole fenamiphos fenarimol fenchlorphos fenitrothion fenobucarb fenpropathrin fensulfothion fenthion fenvalerate fluazifop-p-butyl fluvalinate, τfonofos heptachlor heptachlor epoxide hexachlorobenzene imazalil imidacloprid iprodione degradation product isazophos isofenphos leptophos lindane linuron malathion metalaxyl methamidophos methidathion methiocarb methomyl methoxychlor metolachlor metribuzin mevinphos mirex monocrotophos myclobutanil napropamide nitrapyrin norflurazon omethoate oxadiazon oxamyl oxyfluorfen paraoxon parathion parathion-methyl
GC LC GC GC GC GC LC GC GC GC GC GC GC GC GC GC GC GC GC GC GC GC LC GC LC GC GC GC LC GC GC GC GC LC LC GC GC GC GC GC LC LC GC LC LC LC LC GC GC GC GC GC LC LC LC GC LC LC GC LC GC LC GC GC
high (125 ng/mL) 86 116 99 105 99 97 112 92 90 81 83 94 88 103 105 104 114 93 121 100 95 109 117 94 107 103 82 109 100 99 79 87 62 98 114 102 105 107 78 94 116 122 101 79 107 117 114 95 103 102 99 62 96 106 105 97 106 91 100 113 94 117 100 105
(14) (4) (15) (10) (12) (17) (4) (15) (14) (20) (23) (22) (13) (10) (12) (10) (37) (17) (13) (13) (18) (16) (4) (10) (7) (12) (26) (13) (11) (6) (18) (14) (16) (8) (8) (18) (9) (13) (22) (11) (6) (7) (13) (8) (5) (6) (6) (9) (12) (9) (31) (10) (5) (6) (5) (17) (5) (6) (13) (5) (17) (5) (8) (14)
middle (50 ng/mL) 86 91 104 100 97 87 90 90 89 88 86 93 100 97 95 98 107 92 96 93 93 105 96 94 100 98 104 98 97 97 77 86 62 82 90 98 98 99 83 90 94 95 110 64 103 94 92 88 98 98 107 59 92 101 102 95 104 75 89 91 91 95 101 104
(8) (5) (14) (9) (13) (16) (5) (8) (9) (19) (12) (10) (12) (11) (17) (13) (28) (20) (14) (13) (16) (20) (7) (8) (7) (12) (10) (8) (11) (11) (23) (18) (19) (9) (15) (13) (8) (8) (20) (10) (6) (7) (18) (5) (7) (8) (8) (11) (9) (8) (23) (12) (6) (8) (7) (21) (9) (5) (15) (6) (14) (6) (6) (14)
commodity, n = 6 (all concentrations)
low (12.5 ng/mL)
wheat (25-250 ng/g)
rice (25-250 ng/g)
oat (36-357 ng/g)
corn (50-500 ng/g)
86 82 94 102 87 95 82 78 85 92 89 95 94 83 96 97 99 101 104 96 87 104 86 93 101 95 ND 101 89 92 85 89 58 75 88 94 100 104 72 90 83 91 95 54 107 83 86 94 104 105 ND 60 95 101 101 102 102 64 92 80 87 86 99 107
79 96 104 102 96 93 96 87 93 81 84 102 89 92 97 103 77 94 116 100 89 110 98 92 100 101 93 100 76 96 75 84 51 87 99 105 106 104 75 93 94 103 105 65 103 99 98 86 104 101 107 52 95 102 100 93 101 78 92 96 83 99 102 122
92 103 87 105 98 89 97 91 87 73 89 89 100 98 103 104 146 98 102 93 93 100 106 98 109 101 93 109 118 99 85 91 66 93 104 91 105 108 74 93 103 110 113 64 112 108 100 99 105 105 99 70 95 110 108 101 112 77 93 98 91 106 100 100
83 97 101 103 83 89 93 104 93 90 78 97 93 96 104 98 113 93 106 94 93 113 98 97 102 100 108 106 98 92 73 80 57 78 93 118 99 105 80 89 97 99 104 69 103 95 97 95 107 105 97 60 95 101 103 97 103 77 92 92 89 97 100 101
89 91 103 99 99 101 91 84 83 90 91 89 95 95 94 96 116 93 103 97 93 103 95 89 100 95 80 99 91 97 86 93 67 83 94 89 96 99 79 91 98 98 87 65 105 91 95 91 95 97 106 60 92 99 102 100 101 75 96 93 98 95 99 99
(18) (9) (23) (9) (20) (14) (8) (23) (5) 43 (17) (10) (9) (10) (11) (11) (16) (29) (18) (8) (13) (9) (12) (11) (5) (9) ND (12) (41) (17) (11) (8) (26) (15) (15) (13) (10) (10) (10) (18) (12) (8) (14) (10) (9) (14) (5) (13) (7) (10) ND (17) (5) (6) (4) (24) (7) (8) (9) (5) (10) (7) (11) (19)
(17) (20) (19) (13) (20) (12) (17) (12) (15) (14) (20) (12) (11) (9) (18) (14) (24) (17) (22) (7) (16) (21) (18) (14) (6) (16) (25) (18) (30) (12) (3) (7) (16) (16) (23) (13) (11) (16) (22) (14) (25) (17) (15) (25) (8) (19) (18) (16) (12) (8) (22) (15) (6) (6) (5) (15) (5) (21) (12) (21) (7) (18) (6) (12)
(9) (14) (10) (9) (8) (24) (16) (8) (7) (8) (11) (12) (15) (12) (9) (7) (27) (27) (21) (15) (11) (6) (12) (7) (5) (6) (7) (15) (16) (8) (26) (19) (17) (14) (12) (9) (8) (11) (10) (17) (12) (12) (12) (21) (4) (10) (10) (9) (7) (9) (13) (6) (5) (6) (4) (25) (5) (15) (17) (13) (17) (13) (11) (17)
(17) (14) (18) (6) (19) (11) (15) (16) (11) (30) (18) (19) (12) (22) (11) (14) (31) (30) (9) (14) (16) (16) (15) (6) (7) (8) (24) (5) (13) (15) (13) (13) (26) (17) (19) (6) (9) (5) (25) (16) (15) (19) (14) (13) (6) (18) (16) (6) (5) (7) 51 (4) (6) (4) (4) (27) (5) (16) (12) (16) (14) (16) (10) (10)
(9) (18) (15) (9) (10) (10) (15) (6) (8) (28) (20) (13) (8) (10) (17) (9) (12) (11) (14) (11) (22) (14) (17) (8) (6) (13) (20) (7) (15) (11) (9) (3) (13) (11) (15) (13) (6) (10) (19) (8) (15) (15) (12) (12) (9) (20) (15) (8) (10) (12) (24) (9) (6) (8) (8) (17) (8) (15) (13) (15) (11) (13) (2) (11)
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Table 3. Continued % recovery (% RSD) concentration level, n = 8 (all commodities) analyte pendimethalin pentachloronitrobenzene permethrin perthane phorate phosalone phosmet phosphamidon phthalimide piperalin piperonyl butoxide pirimicarb pirimiphos-ethyl pirimiphos-methyl procymidone profenofos profluralin promecarb prometryn propanil propham propiconazole propoxur propyzamide pyrethrin I quinalphos quizalofop-ethyl resmethrin simazine simetryn spinosad A spinosad D sulfallate sulprofos terbacil terbufos tetrachlorvinphos tetradifon tetrahydrophthalimide thiabendazole thiobencarb thionazin tolylfluanid triadimefon tribufos (DEF) trifloxystrobin trifluralin trimethacarb vinclozolin
method GC GC LC GC GC LC LC GC GC LC GC GC GC GC GC LC GC LC LC GC LC LC LC GC GC GC LC LC LC GC LC LC GC GC GC GC LC GC GC LC LC GC LC GC GC LC GC LC GC
high (125 ng/mL) 104 84 92 95 99 107 108 93 86 35 99 106 107 103 105 97 108 117 105 97 117 112 118 102 104 103 105 98 105 102 87 82 96 97 103 101 106 93 104 97 111 104 2 104 92 120 102 115 103
(11) (5) (9) (9) (10) (7) (9) (13) (22) (13) (12) (10) (12) (11) (17) (16) (10) (4) (9) (12) (5) (5) (5) (16) (17) (8) (11) (7) (6) (9) (6) (5) (18) (9) (10) (11) (9) (9) (20) (7) (6) (12) (68) (14) (16) (6) (6) (6) (12)
middle (50 ng/mL) 91 83 90 88 98 102 90 125 79 35 91 99 92 99 104 95 93 96 99 101 99 91 95 95 92 95 101 89 102 99 71 67 86 84 93 95 104 91 83 80 91 102 2 99 79 96 94 96 100
(10) (15) (12) (9) (11) (8) (9) (30) (14) (17) (21) (10) (8) (16) (8) (7) (10) (7) (7) (8) (8) (10) (6) (15) (5) (10) (8) (8) (7) (9) (6) (5) (23) (8) (14) (10) (9) (9) (10) (6) (10) (13) (100) (9) (16) (7) (12) (11) (10)
commodity, n = 6 (all concentrations)
low (12.5 ng/mL)
wheat (25-250 ng/g)
rice (25-250 ng/g)
oat (36-357 ng/g)
corn (50-500 ng/g)
93 83 91 90 85 102 80 ND 80 41 94 99 97 100 103 96 89 87 98 97 87 84 89 97 ND 101 97 94 100 102 67 64 91 88 101 86 102 89 74 74 83 92 23 101 81 89 86 85 91
90 75 88 84 89 98 91 131 84 36 97 105 100 101 109 91 100 98 97 101 99 90 99 105 98 106 94 85 99 99 73 68 79 87 110 94 99 89 99 84 94 104 38 103 80 99 94 96 105
112 89 92 98 100 112 103 87 88 43 97 103 101 105 108 104 98 109 110 98 109 106 107 103 110 98 109 98 106 104 81 73 95 98 101 99 111 94 94 91 103 101 3 107 84 110 98 106 98
94 83 90 91 92 102 90 127 67 32 91 100 102 104 101 93 99 98 98 101 97 91 99 95 90 97 103 92 104 104 74 70 85 83 93 88 102 92 87 81 90 101 1 98 82 99 91 96 99
97 85 94 91 95 102 87 91 85 36 92 99 96 95 100 97 94 96 99 94 99 96 97 91 107 98 98 99 100 97 73 71 101 92 90 97 103 91 80 78 93 96 2 99 88 98 95 95 93
(12) (16) (20) (14) (23) (7) (10) ND (21) (25) (9) (8) (10) (9) (9) (5) (10) (11) (6) (7) (15) (15) (6) (11) ND (18) (7) (10) (4) (14) (8) (8) (24) (15) (16) (15) (5) (11) (33) (8) (7) (14) (245) (11) (7) (11) (14) (11) (7)
(11) (8) (17) (13) (28) (6) (21) (30) (26) (31) (7) (13) (18) (15) (18) (8) (18) (19) (6) (12) (13) (22) (19) (13) (2) (18) (6) (8) (3) (14) (18) (17) (23) (14) (12) (20) (6) (15) (32) (16) (15) (15) (193) (17) (16) (21) (10) (18) (13)
(14) (12) (16) (12) (7) (4) (12) (8) (12) (14) (23) (10) (11) (7) (8) (5) (14) (9) (7) (6) (10) (10) (12) (9) (8) (12) (6) (5) (4) (10) (11) (13) (27) (11) (8) (11) (8) (7) (15) (12) (14) (3) (70) (10) (20) (10) (10) (14) (11)
(8) (15) (14) (7) (18) (6) (12) (24) (18) (12) (13) (8) (9) (13) (11) (13) (13) (15) (5) (6) (20) (16) (14) (12) (12) (10) (12) (4) (7) (8) (13) (14) (11) (15) (14) (15) (8) (7) (9) (12) (14) (17) (28) (10) (11) (16) (21) (15) (11)
(8) (8) (9) (9) (8) (6) (12) (17) (14) (9) (14) (9) (11) (11) (8) (9) (9) (15) (6) (11) (18) (14) (12) (21) (19) (9) (7) (9) (6) (10) (8) (8) (13) (5) (14) (7) (6) (8) (28) (14) (17) (13) (63) (7) (15) (14) (9) (17) (7)
a Recoveries 120% are highlighted in bold. The method indicates which technique (“GC” for the DSI-LVI-GC-TOFMS and “LC” for the UPLC-MS/MS) was used for the determination (“ND” means not determined).
and buffered QuEChERS procedures, both with 150 mg of PSA and 50 mg of C18 in the dispersive SPE step. Panels B and C of Figure 3 show comparison of selected pesticide peaks eluting in the region highlighted in Figure 3A, which mainly represents elution of a large amount of linoleic acid in the corn extract obtained with the buffered procedure. The peaks eluting in the affected region show apparent retention time shifts, peak shape distortions, and also reduced signal intensity due to the detector saturation with the large amount of fatty acids. For the above reasons, we do not recommend using the QuEChERS procedure
with acetate buffer for cereal grains and other samples with a higher content of fatty acids. The optimized procedure for cereals is summarized under Materials and Methods. After the dispersive SPE, centrifugation, and addition of a QC solution, an aliquot of the final extract can be directly injected into the GC-MS system. Filtration is not necessary because any potential particles will remain in the DSI microvial. For UPLC-MS/MS, filtration is highly recommended to prevent column clogging. For fast and convenient filtration, we employed Mini-UniPrep syringeless filters for the filtration of the
Article final extracts instead of syringe filters (26). The syringeless filters consist of two parts: a chamber and a filter plunger that together form an autosampler vial that can be used for sample storage and for sample introduction using common autosamplers. Method Validation. The analyte recoveries and RSDs obtained in the method validation are summarized on the basis of concentration levels and cereal grain commodities in Table 3. Figure 4 shows distribution of the recoveries and RSDs obtained in different cereal grains, demonstrating that recoveries in the range of 70-120% were obtained for 93-97% of the analytes, with RSDs being
