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Home , Butocarboxim, Ethiofencarb, Fenoxycarb

TechnicalNOTE (low pH), raisin (high sugar) and wheat flour (dry). (low pH),raisin(highsugar)andwheatflour(dry). matrix): tomato(highwater),avocadofat),lemon were validatedforfivecommodities(representative Theextractionand analyticalmethods was performed. metabolites. Agenericextractionprocedureandclean-up the quantificationof81pesticidesandpesticide Using theMRMtechnique,amethodwasdevelopedfor Multiple ReactionMonitoring(MRM)mode. inthe using triplequadrupolemassspectrometry improvements inanalyticalselectivitymaybeachieved resulting inamorecomplexsamplematrix.Significant clean-up stageofsamplepreparationiscompromised, target analytesisincreased,theselectivityof analyzed inthisstudy. Asthenumberanddiversityof triazines, cyclohexanedioneoximesandureaswere organophosphorus, oximecarbamates,sulfonylureas, 81 pesticidesincluding:,benzimidazoles, as manypossibleduringasingleanalysis. be foundinfoodstuffs,itisadvantageoustodetermine Given thelargenumberofpesticideresiduesthatmay Maximum ResidueLimit(MRL)ofanygivenpesticide. consumption mustcontainlessthanthestatutory agricultural produce.Foodproduceusedforhuman pesticide userequirestheregularmonitoringof fungi. Thelegalenforcementofregulationsgoverning use tocontrolundesirableweeds,insects,rodentsand Worldwide, thereareover800pesticidescurrentlyin Introduction Waters Micromass Quattro Premier Quattro Micromass Waters ™ MS System. MS Peter Hancock Peter 2 RESIDUES INFRUITANDVEGETABLES USINGTHE Federal Institute for Risk Assessment, P.O. Box 33 00 13, D-14191 Berlin, Germany Berlin, D-14191 13, 00 33 Box P.O. Assessment, Risk for Institute Federal 1 1 QUATTRO PREMIERMASSSPECTROMETER Waters Corporation, Atlas Park, Simonsway, Manchester, M22 5PP, UK UK 5PP, M22 Manchester, Simonsway, Park, Atlas Corporation, Waters AN ENHANCEDLC/MS/MSMETHODFOR , Gordon Kearney Gordon , a Waters using Liquid chromatographyseparationswereperformed Experiments were performed onanAlliance Experiments wereperformed system coupledtoaWaters Micromass normal produce or 0.5 g/mL for dry produce. produceor0.5g/mLfor dry normal HPLC vialandhasamatrixequivalent of1g/mLfor filtered througha0.45µmsyringe filterintoaglass diluted with1000µLwater.further Thefinalextractis methanol withthehelpofanultrasonic bathand residueisredissolved in250µL nitrogen. Thedry The eluateisgentlyevaporatedunderastreamofdry the columniselutedwith16mLofdichloromethane. Aftera5minute waitingperiod, diatomaceous earth. is transferredtoacolumncontaining5mLof samplematerial,respectively) or0.625gdry normal (which containsthepesticidesresiduesof1.25g NaCl (20gin100mLwater).Analiquotof5 6 mLoftheextractismixedwith2asolution aliquot iscentrifugedat6000rpmfor5minutes. turbidextracts,an is 30mL.Inthecaseofvery extract for 2minutes.Thetotalvolumeofsupernatant addition of20mLmethanol,thesampleisblended 10 minutesaftertheadditionofwater. Afterafurther towait samplematerials,itisnecessary case ofdry mL and9.5ofwaterisadded,respectively. Inthe content 20%)andwheatflour(water10%)9 are added,respectively. To 5gofraisin(water (water content70%)0.5mL,1mLand3ofwater content 95%),lemon(water90%)oravocado natural andaddedwater. To 10gtomato(water is addedtoallsamplesobtain10mLasasumof isweighedintothecup.Waterhomogenized portion sample materials,e.g.raisinorwheatflour, a5g g istransferredintoablendercup.Forthedry The testsampleischoppedavoidinglossofjuice.10 Extraction Procedure Method tandem quadrupolemassspectrometer. THE DETERMINATION OF81PESTICIDE ® Atlantis 1 , Anthony Newton Anthony , ™ dC 18 column 2.1mmi.d.x100mm. 1 , Lutz Alder Lutz , ® 2 Quattro Premier and Jeannette Klein Jeannette and ® HPLC ™ 2 , , TechnicalNOTE Injection Volume =10µL Flow rate=0.3mL/min mm, 3µm Multiplier =650V Collision GasPressure=Argonat3.2e Desolvation GasFlow=850l/hr Cone GasFlow=50l/hr Desolvation Temperature =450˚C Source Temperature =120˚C RF Lens=0V Extractor =5V VoltageCapillary =0.6kV Electrospray modewithpositivepolarity Waters QuattroPremiermassspectrometer MS Method 0%B Time 40min 0%B 100%B Time 29.1min 100%B Time 29min 0%B Time 15min Time 0min Gradient Column =Waters AtlantisdC 5 mMCH Mobile phaseB=Methanol/Water (9:1,v/v)+ 5 mMCH Mobile phaseA=Methanol/Water (1:9,v/v)+ Waters Alliance2795HPLCSystem HPLC Method Guard Column=Waters AtlantisdC µm at30˚C 3 3 CO CO 2 2 NH NH 4 4 18 , 2.1x100mm,3 18 , 2.1x20 -3 mBar whilst a short overallscancycletimeismaintained. whilst ashort can beincreasedbytheuseoflongerdwelltimes the signal-to-noise(S/N)ratiooflessintensepeaks allows theflexibleuseofMRMdwelltimes,where based onanalyteretentiontimes.Thissystem were distributedintoelevenfunctionwindows, pesticide arelistedinTable 1.TheMRMtransitions collision energiesanddwelltimesforeach The MRMtransitions,alongwiththeconevoltages, illustrated inFigures2,4,6,8and10,respectively. imazalil inraisinandaldicarbwheatflourare inavocado,monocrotophoslemon, tomato, forquinmeracin Representative calibrationcurves then processedusingWaters QuanLynx™ software. each injectedfourtimesinatypicalbatchanalysisand levels forraisinandwheatflour. Thesestandardswere and lemon,atthe1,2,510,2040µg/kg 1, 5,10,20and40µg/kglevelsfortomato,avocado Matrix matchedstandardsweregeneratedatthe0.5, extract spikedat10µg/kgisillustratedinFigure1. The Total IonChromatogram(TIC)foranavocado Results andDiscussion Figure 1. TIC of 81 pesticides divided into 11 into divided pesticides 81 of TIC 1. Figure MRM function windows, 10µg/kg in avocado. in 10µg/kg windows, function MRM 100 Matrix Standard 10 QPAvocado007 % 0 .040 .080 00 20 40 60 80 00 22.00 20.00 18.00 16.00 14.00 12.00 10.00 8.00 6.00 4.00 2.00 pp b Avocado 11: MRM of 3 Channels ES+ ES+ 11: MRM 3Channels of Time 9.02e6 TIC TechnicalNOTE Table 1. MRM Method Parameters. Method MRM 1. Table Clethodim-sulfoxid Clethodim-sulfon Triasulfuron Flazasulfuron Dimethoat Metamitron Cinosulfuron -3-hydroxy Clethodim-imin-sulfoxid Vamidothion Chlorsulfuron 5-Hydroxy-clethodim-sulfon Clethodim-imin-sulfon -sulfon Rimsulfuron Thifensulfuron-methyl Ethiofencarbsulfoxid Metsulfuron-methyl Thiofanox-sulfoxid Ethiofencarbsulfon 6-Cl-4-OH-3-phenylpyridazin Amidosulfuron Nicosulfuron Pymetrozin Quinmerac -S-methyl-sulfon Oxydemeton-methyl Aldoxycarb Butoxycarboxim -sulfoxid Butoxycarboxim-sulfoxid Omethoat Pesticide 288.0 145.8 357.9 140.8 408.0 203.9 256.0 208.9 432.0 181.9 388.0 166.8 242.0 106.8 382.0 166.8 252.1 103.8 275.1 106.8 370.0 261.0 223.9 126.7 411.0 181.9 218.0 104.8 221.9 140.8 263.0 168.8 247.0 168.8 189.0 101.8 240.0 105.8 206.9 131.8 206.9 131.8 213.9 182.8 183.8 142.8 252.9 125.7 376.1 206.0 392.1 300.1 402.0 166.9 408.0 181.9 229.9 124.7 202.9 174.9 414.0 182.9 220.0 162.9 286.1 208.0 141.8 93.8 302.1 97.8 268.1 75.8 206.8 76.8 162.8 87.8 237.0 71.8 240.0 85.8 MRM Transition Cone Voltage 20 26 10 18 26 11 13 11 16 16 20 16 24 30 22 22 28 25 16 30 26 26 25 16 26 22 36 10 24 26 20 15 22 10 12 38 20 18 26 28 Collision Energy 35 18 10 15 20 13 22 16 12 14 25 16 15 18 21 22 18 18 12 18 15 21 24 34 12 18 26 18 20 18 14 22 34 16 18 20 22 7 7 9 Dwell Time/ms 200 100 200 200 200 200 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 TechnicalNOTE Table 1. MRM Method Parameters. Method MRM 1. Table Furathiocarb Quizalofop-ethyl Fluazifop-P-butyl Haloxyfop-ethoxyethyl Spiroxamine Haloxyfop-methyl Imazalil Cyprodinil Tebuconazol Tebufenozid Metolachlor Fenhexamid Pyrimethanil Linuron Azoxystrobin Clethodim Diuron 3,4,5-Trimethacarb Isoxaflutole Isoproturon Metalaxyl Atrazin Bensulfuron-methyl Thiodicarb Triflusulfuron-methyl Carbaryl Prosulfuron Carbofuran Aldicarb Pesticide Fenpropimorph Pyridate 304.2 146.9 379.0 206.9 489.0 157.8 383.1 194.9 373.0 299.0 384.1 282.0 434.0 315.9 298.2 143.9 376.0 315.9 297.0 158.8 353.1 132.8 284.1 175.9 208.0 150.8 243.1 120.8 199.9 106.7 249.0 159.8 404.1 372.0 360.0 163.8 193.9 136.8 377.0 250.9 280.1 220.0 216.0 173.8 411.0 148.8 493.0 264.0 225.9 106.8 201.8 144.8 420.0 140.8 224.0 108.8 221.9 164.8 210.0 110.8 208.0 115.8 191.8 159.8 239.1 71.8 308.1 69.8 302.1 87.8 302.1 96.9 232.9 71.8 206.9 71.8 355.0 87.8 212.9 74.8 226.0 92.8 MRM Transition Cone Voltage 32 26 15 28 26 15 17 26 16 19 14 24 26 44 22 25 22 34 30 26 30 30 32 44 30 12 22 20 36 18 40 26 21 22 25 18 10 26 20 8 8 Collision Energy 19 23 15 21 23 18 10 22 20 14 16 16 20 30 18 27 21 21 24 24 22 20 25 35 24 20 22 28 26 10 26 28 20 16 23 20 13 22 20 16 8 Dwell Time/ms 200 200 100 100 100 200 300 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 NOTE

The MRM extracted chromatogram for these Compound name: Carbaryl Correlation coefficient: r = 0.999743, r^2 = 0.999486 compounds at the lowest calibrated level of 0.5 µg/kg Calibration curve: 3583.69 * x + 89.1887 Response type: External Std, Area in tomato, avocado and lemon, and 1 µg/kg in raisin Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 143455 and wheat flour are illustrated in Figures 3, 5, 7, 9 and 11, respectively.

Compound name: Quinmerac Response Correlation coefficient: r = 0.999737, r^2 = 0.999475 Calibration curve: 9969.54 * x + 1311.77 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

400143

0 ppb 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 Technical

Response Figure 4. Calibration curve for carbaryl in avocado.

QPAvocado111 F6:MRM of 8 channels,ES+ Matrix Standard 0.5ppb Avocado 201.8 > 144.8 Carbaryl 1.518e+004 100 12.54 1792.42

0 ppb 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

Figure 2. Calibration curve for quinmerac in tomato.

%

QPTomato011 F2:MRM of 8 channels,ES+ Matrix Standard 0.5ppb Tomato 221.9 > 140.8 Quinmerac 6.428e+004 100 7.05 5789.78

0 min 11.50 11.75 12.00 12.25 12.50 12.75 13.00 13.25

% Figure 5. MRM chromatogram for carbaryl in avocado at 0.5µg/kg.

For each matrix the lowest level calibration standard was used to estimate the Limits of 0 min 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 Detection (LODs) for all 81 pesticides. The LODs are defined as the concentrations at which the S/N Figure 3. MRM chromatogram for quinmerac in ratio is ≥3:1. The results are illustrated in Figure tomato at 0.5µg/kg. 12, with the least sensitive compounds in the most complex matrices giving LODs at least an order of magnitude less than those specified by the MRLs. TechnicalNOTE Matrix Standard 0.5ppb Lemon QPLemon011 Figure 8. Calibration curve for imazalil in raisin. in imazalil for curve Calibration 8. Figure monocrotophos for chromatogram MRM 7. Figure monocrotophos for curve Calibration 6. Figure Response in lemon at 0.5µg/kg. at lemon in lemon. in Response Curve type: Linear, Origin: Exclude, type:Response Weighting: External 1/x, Std, Axis Area trans: None Calibration curve: 7011.91 *x + -781.274 Correlation coefficient: r = 0.999854, r^2 = Monocrotophos name: 0.999708Compound Curve type: Linear, Origin: Exclude, type:Response Weighting: External 1/x, Std, Axis Area trans: None Calibration curve: 3358.83 *x + 1492.35 Correlation coefficient: r = 0.999657, r^2 = 0.999313 Imazalil name: Compound 136234 100 279730 % 0 -781 0 . . 001. 002. 003. 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 . . 001. 002. 003. 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 .072 .076 7.80 7.60 7.40 7.20 7.00 Monocrotophos 2676.20 7.48 F2:MRM of 8channels,ES+ 223.9 > 126.7 2.367e+004 Matrix Standard 1ppbRaisin QPRaisin004 Smooth(Mn,1x2) Matrix Standard 1ppbFlour QPFlour004 Smooth(Mn,1x2) min Figure 10. Calibration curve for aldicarb in in aldicarb for curve Calibration 10. Figure Figure 11. MRM chromatogram for aldicarb in aldicarb for chromatogram MRM 11. Figure Figure 9. MRM chromatogram for imazalil in imazalil for chromatogram MRM 9. Figure wheat flour. wheat Response wheat flour at 1µg/kg. at flour wheat raisin at 1µg/kg. at raisin Curve type: Linear, Origin: Exclude, type:Response Weighting: External 1/x, Std, Axis Area trans: None Calibration curve: 1709.08 *x + -22.2636 Correlation coefficient: r = 0.999610, r^2 = Aldicarb name: 0.999220Compound 100 100 % 5801.0 6201.0 16.600 16.400 16.200 16.000 15.800 34168 0 % 0 ppb ppb -22 . . . . 001. 501. 20.0 17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0 .51.01.51.01.51.011.25 11.00 10.75 10.50 10.25 10.00 9.75 4650.06 Imazalil 16.28 1619.19 Aldicarb 10.52 F5:MRM of 9channels,ES+ F9:MRM of 7channels,ES+ 1.683e+004 208 > 115.8 6.095e+004 297 > 158.8 b min pp min NOTE wheat flour matrix is illustrated in Figure 14. For 1.2 the concentrations of 1, 2, 5 and 10 µg/kg, the 1.0 mean values for all 81 pesticides were calculated 0.8 Tomato to be 0.92, 1.97, 5.38 and 10.44 µg/kg, with Avocado 0.6 Lemon percent relative standard deviations of 7.7, 5.2, Raisin ug/kg 4.0 and 3.5, respectively. These results indicate 0.4 Flour that the method is reproducible at concentration 0.2 levels that are significantly lower than the 0.0 1 9 17 25 33 41 49 57 65 73 81 detection limits required by law. 81 Pesticides

Compound name: Methiocarb Figure 12. Estimated LODs for all 81 pesticides in Correlation coefficient: r = 0.999576, r^2 = 0.999153 Calibration curve: 963.533 * x + 24.7907

Technical Response type: External Std, Area all five matrices, µg/kg. Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

38873 The S/N ratios for a 5 pg/µL standard in solvent were compared to a 50 pg/µL standard injected on the Quattro micro API using the conditions specified in Waters Application Note Response 720000686EN. An example of the sensitivity difference between the Quattro micro API and the Quattro Premier for propoxur is illustrated in

0 ppb Figure 13. In this example, 500 pg was injected 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 on column with the Quattro micro API, compared to 50 pg with the Quattro Premier – the S/N ratios Figure 14. Overlaid calibration curves for are comparable. The average increase in methiocarb in wheat flour. sensitivity across all 81 pesticides between the two instruments was calculated to be 7.2. With the sensitivity and reproducibility achieved by the Quattro Premier, the results indicate either

Tomato001 6: MRM of 8 Channels ES+ an increased number of pesticides could be 100 S/N:PtP=2991.63 210 > 111 2.51e5 analyzed in a single run, and/or confirmatory

Propoxur, 500pg on column, transitions for each pesticide could be added to Quattro micro API % the method. A suitable number of data points across any of the chromatographic peaks are still 0 Setup005 6: MRM of 8 Channels ES+ 100 S/N:PtP=2816.39 210 > 110.8 1.49e6 required for good quantification, but more pesticides or confirmatory transitions would lead to

Propoxur % 50pg on column an increase in the number of MRM transitions in a Quattro Premier function and the overall cycle time. To overcome

0 Time 11.20 11.40 11.60 11.80 12.00 12.20 12.40 12.60 12.80 this the dwell time of each transition and the inter- channel delay must be decreased.

Figure 13. Sensitivity difference between the Quattro The Quattro Premier incorporates a T-Wave Micro API and the Quattro Premier for propoxur. (Travelling Wave) collision cell that minimizes ion transit times and provides optimum performance for Four replicate batch analyzes were carried out on narrow chromatographic peaks or, in this case, matrix-matched samples spiked with all 81 multiple MRM transitions. This T-Wave cell maintains pesticides. The calibration curves were overlaid signal intensity and minimizes interchannel crosstalk and a representative curve for methiocarb from the even with dwell and interchannel delay times of 5ms. NOTE To study this the original experiment, containing Setup031 Sm (Mn, 1x10) 2: MRM of 27 Channels ES+ x1000 10.22 100 5 ms dwell 252.9 > 125.7 8.25e5 eleven MRM function windows, was changed to 9.33 9.44 9.63 %

0 three and the inter-channel and inter-scan delays Setup030 Sm (Mn, 1x6) 2: MRM of 27 Channels ES+ x1000 10.23 100 252.9 > 125.7 9.51e5 9.27 9.47 9.69 10 ms dwell were standardised on 5ms for each function. All the %

0 Setup029 Sm (Mn, 1x3) 2: MRM of 27 Channels ES+ dwell times for all the pesticides were changed from x1000 10.23 100 252.9 > 125.7 9.58e5 9.35 9.50 9.63 20 ms dwell those listed in Table 1 to 40, 30, 20, 10 and 5ms. %

0 Setup028 Sm (Mn, 1x2) 2: MRM of 27 Channels ES+ x1000 10.23 100 252.9 > 125.7 Therefore, each function contained approximately 9.16e5 9.34 9.55 30 ms dwell 27 MRM transitions so the overall cycle time was % 0 Setup027 Sm (Mn, 1x2) 2: MRM of 27 Channels ES+ x1000 10.22 100 252.9 > 125.7 1.22, 0.95, 0.68, 0.41 and 0.275s, respectively. 9.42 8.18e5 % Thiacloprid 40ms dwell, 1.22s cycle time

The results are illustrated in figure 15 for thiacloprid 0 Time 8.75 9.00 9.25 9.50 9.75 10.00 10.25 10.50 10.75 11.00 11.25 11.50 11.75 12.00 where the overall cycle time has decreased by a

Technical factor of 4.4 but the peak area has only decreased Figure 16. S/N of smoothed data versus by 5.5% between 40 and 5 ms. Similarly, the S/N decreasing dwell time. ratios for the smoothed data between 40 and 5 ms have not significantly changed, as shown in figure Therefore, the method could be extended to further 16. This feature of the T-Wave collision cell allows pesticides and/or confirmatory transitions for each. fast switching between MRM transitions.

Conclusions Setup031 2: MRM of 27 Channels ES+ 10.21 100 252.9 > 125.7 89497 5 ms dwell 1.22e6 Area % A generic extraction and LC/MS/MS method, valid 0 Setup030 2: MRM of 27 Channels ES+ 10.22 100 252.9 > 125.7 98582 1.31e6 for a wide range of compound classes in a 10 ms dwell Area % representative set of matrix types, was validated and 0 Setup029 2: MRM of 27 Channels ES+ 10.22 100 252.9 > 125.7 97494 1.24e6 Area shown to be suitable for the screening of 81 pesticide % 20 ms dwell

0 residue compounds in fruit and vegetables. The limits Setup028 2: MRM of 27 Channels ES+ 10.23 100 252.9 > 125.7 92639 1.20e6 Area of detection achieved for the pesticides analyzed are % 30 ms dwell

0 Setup027 2: MRM of 27 Channels ES+ well below that required for surveillance monitoring 10.22 100 252.9 > 125.7 94709 1.24e6 Area % Thiacloprid 40ms dwell, 1.22s cycle time in the European Union. Therefore, the method is

0 Time 9.25 9.50 9.75 10.00 10.25 10.50 10.75 11.00 11.25 11.50 11.75 12.00 12.25 clearly extendable to greater numbers of pesticide targets and or confirmatory transitions in a single run. Figure 15. Peak area versus decreasing dwell time.

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Waters, Alliance, Micromass, Atlantis, Quattro Premier, QuanLynx and Quattro micro are trademarks of Waters Corporation. All other trademarks are property of their respective owners. ©2004 Waters Corporation Printed in the USA May 2004 720000840EN MB-UL