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Screening of Multi Pesticide Residues in Vegetables using LC/MS/MS Quantitation Enhanced Data Dependent Scan

Kaori Saito* Yoko Yamagishi Michiko Kanai Saito*, Yamagishi, Thermo Fisher Scientific K.K

Introduction

The aim of this study is to easily and precisely screen for potential residues of known y y p y p p pesticides in vegetables using the Quantitation Enhanced Data-Dependent (QED) scan g g p ( ) of triple stage q p g quadrupole mass spectrometer (LC-MS/MS). p p ( ) The Quantitation Enhanced Data-Dependent (QED) scan function on a regular triple p ( ) g p q quadrupole instrument delivers an information rich MS/MS in real time. When using p g QED, a "full scan MS/MS" mass spectrum is obtained by data dependent scanning , p y p g during the selected reaction monitoring (SRM) experiment. Therefore QED-MS/MS g g( ) p p provides MS/MS spectrum at low levels followed by a fast, highly specific full MS/MS p y , g y p scan for confirmation. In addition, a unique technology called Reversed Energy Ramp , q gy gy p ( (RER) was used for the collision energy setting during the analysis. The RER was ) gy g g y devised for improving product ion sensitivity when using a triple stage q p gp y g p g quadrupole mass p spectrometer (TSQ) and as a means for increasing the information content for better p ( Q) g structural elucidation. Ap pesticide library was made using RER and acquired data was automatically identified y g q y from the detected spectrum using unique software called ToxID, which conducts a p g q , compound search of a library containing mass spectrum of standards. p y g p FIGURE 1. QED with RER QED (Q tit ti Enhanced Data-Dependent ) (Quantitation E h dD t D d t

SRM Chromatogram

100 90 RT:10.0

Results

The QED method was made by FIGURE 5. MS/MS Library 5 LC/MS method package for Multi pesticide residue (ThermoFisher Scientific.K.K) and a pesticide library that was made using RER MS/MS spectrum (Figure 5 shown). A configration file including g g information of RT and p precurser ion ( (m/z) for ToxID was made by ) y analysis of standard p y pesticides. Then analysis was conducted on y 10 pp of five commonly detected ppb y p pesticides in Japan added to p vegetables prepared with the Rapid g p p p Multiresidue Method. The five p pesticides were Imazalil, Carbaryl, , y, Imidacloprid, Azoxystrobin, Thiacloprid. Figure 6 is shown a QED data of 10 ppb p , y , p g pp Standards added sample. a) is TIC, it were found five compounds. b) is QED p ) , p ) chromatogram of Carbaryl (RT:6.12), black bar is shown the SRM data points and red bar g y ( ), p is shown the MS/MS data p point. c) is RER MS/MS spectrum of Carbaryl. ) p y FIGURE 6. QED Data

RT: 0.00 - 21.86 100

RER (Reverse Enegy Ramp)

MS/MS Spectrum

MS/MS Spectrum

Intensity

C Collisi ion Energy Q2 y

80 70 Relative Abundance e 60 50 40 30 20 10 0 9.8 10.0 10.2 min 10.4 10.6

a)

TIC

4.44

13.16

90 80 R Relative Abun e ndance e 70 60 50 40 30 20 4.28 6.08 6.05 0.52 0 0 1 1.58 2 2.54 3 3.64 4 5 5.87 6 6.15 6 15 6.52 7.47 7 8 8.53 9 9.91 9 91 10.00 9.73 10 16.05 16.65 16 17 16.03

NL: 1.04E6 TIC MS 0305_35 0305 35

Signal Threshold

m/z

m/z

SRM

An SRM scan (Event 1) above ( ) the threshold value will trigger an MS/MS in Scan Event 2 2.

Linearly ramps the collision energy in Q2 from high to low low.

10

11.43 12.28 13

13.57 14

14.78 15

18.04 18.93 18 19

19.92 20.56 20 21

11 12 Time (min)

RT: 8.76 11.17 SM: RT 8 76 - 11 17 SM 7G 100 9.91 9.93 9.95 9.85 NL: 8.99E4 TIC F: + c ESI sid=7.00 SRM ms2 202.090 202 090 [144.500-145.500] MS 0305_35 _ NL: 1.17E5 TIC F: + c ESI sid=-7.00 d Full ms2 202.090 [30.000-207.090] MS 0305_35

0305_35 0305 35 #61884 RT 9 88 AV 1 NL 6 05E4 RT: 9.88 AV: NL: 6.05E4 F: + c ESI sid=-7.00 d Full ms2 202.090 [30.000-207.090] 100 90 80 R Relative Abun e ndance e 70 60 50 40 30 20 117 152 120 m/z 140 160 167 185 145

Methods

Relativ Abu ve undanc ce

90 80 70 60 50 40 30 20 10 0

b) )

c) )

RER MS/MS spectrum p

127

9.97

Workflow : Step1 : Extract Analytes from Vegetables Step2 : Analyze the samples with QED with RER Method Step3 : Automated library search and reports for idendentification with ToxID Chemicals : This experiment was conducted using chemicals distributed by Hayashi Pure Chemical Ind., Ltd These chemicals are in accordance with the multi residue Ind Ltd. method I and II for Agricultural Chemicals by LC/MS established by Japanese LC/MS, Ministry of Health, Labour, and Welfare [1] (FIGURE 2) . Health Labour Library : A pesticide library was made using RER (Ramp 0 60 eV) 0-60 eV). Sample preparation : Spinach was prepared with the Rapid Multi residue Method [2] (FIGURE 3). LC : Accela Gradient Method for LC/MS simultaneous analysis established by Japanese Ministry of Health Labour and Health, Labour, Welfare [1] [1]. MS : TSQ Quantum Access Using QED (Threshold 5*e4) with RER (Ramp 0 60eV). 5 e4) 0-60eV) Figure 4 shown MS Method. Method FIGURE 2. Target Compounds 2

Method I

Abamectin Acibenzolar-S-methyl Acibenzolar S methyl Aldicarb Aldoxycarb Anilofos Aramite Azafenidin Azamethiphos Azinphos_methyl Azoxystrobin Bendiocarb Benzofenap Boscalid Butafenacil Carbaryl Carbofuran Carpropamid Chloridazon Chlorxuron Chromafenozide Clofentezine Clomeprop Cloquintocet_mexyl Clothianidin Cumyruron Cyazofamid Cycloate Cycloprothrin Cyflufenamid Cyprodinil Di-allate Diflubenzuron Dimethirimol Dimethomorph_E Dimethomorph E Dimethomorph_Z Diuron Dymuron Epoxiconazole p Fenamidone Fenobucarb Fenoxaprop-ethyl Fenoxycarb Fenpyroximate_E Fenpyroximate_Z Fenpyroximate Z Ferimzone_E Ferimzone_Z Ferimzone Z Flufenacet Flufenoxuron Fluridon Furametpyr Furathiocarb Hexaflumuron Hexythiazox Imazalil Imidacloprid Indanofan Indoxacarb_MP Iprovalicarb p Isoxaflutol Lactofen Linuron Lufenuron Mepanipyrim Methabenzthiazuron Methiocarb Methomyl Methoxyfenozide Monolinuron Naproanilide Novalron Oryzalin Oxamyl Oxaziclomefone Oxycarboxine Pencycuron Pentoxazone Phenmedipham Pirimicarb Propaquizafop Pyraclostrobin y Pyrazolynate Pyriftalid Quizalofop-ethyl Silafluofen Simeconazole Spinosyn_A Spinosyn A Spinosyn_D Tebufenozide Tebuthiuron Teflubenzuron Tetrachlorvinphos Thiabendazole Thiacloprid Thiamethoxam Thiodicarb Tralkoxydim Tridemorph Triflumuron Triticonazole

10.00 10 00 9.83 9 83 10.02 10.04 10.06

9.81 9.78 9.17 9.0 9.5

10.0 Time ( (min) )

QED 10.5 11.0 Chromatogram

10.73 11.03

10 0 40 60 80

115 91 100

180

200

The ToxID report shown in Figure 7 a) is summary report that includes LC 7,a) chromatograms data as well as a list of pesticides that were found and identified in the data, sample. sample b) is Long report that have an LC chromatogram the MS2 spectra acquired chromatogram, from the sample the matched spectra in the library and a delta spectrum The delta sample, spectrum. spectrum is the difference between the acquired spectra and the library spectra. In spectra addition, addition search indexes and structures are also included In the long report each included. report, pesticides that is detected has one full page dedicated to it That is a result of Carbaryl. it. Carbaryl FIGURE 7. ToxID Reports p a) b)

Method II

1-Naphthylacetic acid 2,4-D 2 2 4 D 4 PA 2,4-PA 4-Chlorophenoxyacetic acid Acifluorfen Azimsulfuron Bensulfuron_methyl Bensulfuron methyl Bromoxynil Chlorimuron ethyl Chlorsulfuron Cinosulfuron Clodinafop acid Cloprop Cloransulam-methyl Cyclanilide Cyclosulfamuron Dichloroprop racemate Diclomezine Diclosuram Ethametsulfyron-methyl Ethoxysulfuron y Fenhexamid Flazasulfuron Florasuram Fluazifop Flumetsulam Fluroxypyr Fomesafen Foramsulfuron Forchlorfenuron Gibberellic acid Halosulfuron_methyl Haloxyfop Imazaquin Imazosulfuron Iodosulfuron methyl Ioxynil MCPA MCPB Mecoprop Mesosulfuron-methyl Metosulam Metsulfuron methyl Naptalam Penoxsulam Primisulfuron_methyl Primisulfuron methyl Propoxycarbazone Na Prosulfuron Pyrazosulfuron-ethyl Sulfentrazone Sulfosulfuron Thidiazauron Thidiazuron Thifensulfuron methyl Triasulfuron Tribenuron methyl Triclopyr Trifloxysulfuron-Na y Triflusulfron methyl

O HN O

FIGURE 3 Sample Preparation 3.

Sample 10g Add 10ml of water Homogenize Add 20ml of acetonitrile Add 1g of NaCl, 4g of MgSO4 Centrifuge Take 8 L T k 8mL acetonitrile l t it il layer Envi carb/PSA Envi-carb/PSA Elute with 50ml acetonitrile/tolene (3:1) Concentrate & Dissolve C t t Di l Dissolve with 10ml of MeOH

FIGURE 4. MS Method 4

Event SRM

Conclusion

Screening of Multi Pesticide Residues methods has been developed to characterize the pesticides in vegetables using the QED with RER scan of TSQ Quantum Access and ToxID And analysis was conducted on five commonly detected pesticides added ToxID. to vegetables prepared with the Rapid Multi residue Method All five additives were Method. easily detected using this screening method. method

Acknowledgment

Event2Dependent Scan

Author thank Masahiro Okihashi (O k Prefectual I tit t of P bli H lth) A th th k M hi Okih hi (Osaka P f t l Institute f Public Health) preparation of Vegetable sample. ti fV t bl l

for f

Reference

Signal Threshold g

5.0 60 0

RER energy e e gy

[1] The Japanese Ministry of Health, Labour, and Welfare can be found on theWorld Wide Web at: Health Labour http://www.mhlw.go.jp/index.html http://www mhlw go jp/index html (Japanese) http://www.mhlw.go.jp/english/index.html ( g p g jp g (English) ) [2] M.Okihashi, et al, EPRW2006 Poster presentation Rapid Multiresidue Method for the Determination of Pesticide Residues in Food by GC/MS, GC/FPD and LC/MS/MS d

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