analytical validation

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TRADE SECRET

Study Title

ANALYTICAL METHOD FOR THE DETERMINATION OF CYMOXANIL AND IN-KQ960 IN SPINACH (LEAFY VEGETABLES) USING LC/MS

Test Guidelines EEC Directive 91/414/EEC, Annex IIA 4.2.1 as amended by EC Directive 96/46/EC; SANCO/825/00 rev.6 (20/06/00) Guidance Document on Residue Analytical Methods

U.S. EPA Residue Chemistry Test Guidelines, August 1996 OPPTS 860.1340 Residue Analytical Method

Authors Joseph P. McClory Robert M. Henze

Date Study Completed January 22, 2004

Performing Laboratory E.I. du Pont de Nemours and Company DuPont Crop Protection Global Technology Division Stine-Haskell Research Center Newark, Delaware 19714-0030

Laboratory Project ID DuPont-13753

Cymoxanil +IN-KQ960, Leafy Veg. Only

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PAGE RESERVED

STATEMENT OF CONFIDENTIALITY This report is the property of E.I. du Pont de Nemours and Company and contains confidential and trade secret information. Except as required by law, this report should not be partially or fully (i) photocopied or released in any form to an outside party without the prior written consent of E.I. du Pont de Nemours and Company or its affiliates, or (ii) used by a registration authority to support the registration of any other product without the prior written consent of E.I. du Pont de Nemours and Company or its affiliates.

JPM/grs

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GOOD LABORATORY PRACTICE STATEMENT The work described in this report is not required to be conducted in compliance with U.S. EPA FIFRA (40 CFR Part 160) Good Laboratory Practice Standards, which are compatible with the OECD Principles of Good Laboratory Practice (as revised 1997), ENV/MC/CHEM(98)17, OECD, Paris, 1998. However, work was conducted in a GLP compliant facility following Standard Operating Procedures. The lack of compliance does not affect the validity of the study.

Applicant/Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.

Applicant/Sponsor DuPont Representative Date

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CERTIFICATION

ANALYTICAL METHOD FOR THE DETERMINATION OF CYMOXANIL AND IN-KQ960 IN SPINACH (LEAFY VEGETABLES) USING LC/MS We, the undersigned, declare that the work described in this report was performed under our supervision, and that this report provides an accurate record of the procedures and results.

Date Study Initiated: September 23, 2003 (first set of validation samples prepared)

Date Study Completed: January 22, 2004

Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.

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LIST OF ABBREVIATIONS AND SYMBOLS % percent °C degrees centigrade APCI atmospheric pressure chemical ionization interface Cat. No. Catalog Number ESI electrospray interface HPLC high performance liquid chromatography LC/MS liquid chromatography/mass spectrometry LOQ limit of quantitation kg kilogram µg microgram min minute MS/MS tandem mass spectrometry (2-stage mass analysis experiment), MS2 m/z mass/charge ratio n number ng nanogram ppb parts per billion ppm parts per million Rec recovery RF Response Factor (analyte peak area / analyte concentration) RSD relative standard deviation (StDev / mean) SAX Strong anion exchange sec second SPE Solid phase extraction StDev standard deviation

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TABLE OF CONTENTS Title Page .......................................................................................................................1 Page Reserved ................................................................................................................2 Good Laboratory Practice Statement .............................................................................3 Certification ...................................................................................................................4 List of Abbreviations and Symbols................................................................................5 Table of Contents...........................................................................................................6 1.0 Abstract .................................................................................................................8 2.0 Introduction ...........................................................................................................8 3.0 Materials................................................................................................................9

3.1 Equipment ......................................................................................................10 3.2 Reagents and Standards..................................................................................11

3.2.1 Reagents ................................................................................................11 3.2.2 Reference Analytical Standards.............................................................11

3.3 Safety and Health............................................................................................12 4.0 Methods...............................................................................................................12

4.1 Principle of the Analytical Method ................................................................12 4.2 Analytical Procedure ......................................................................................13

4.2.1 Glassware & Equipment Cleaning Procedures......................................13 4.2.2 Preparation & Stability of Reagent Solutions........................................13 4.2.3 Stock Standard Preparation and Stability ..............................................13 4.2.4 Fortification Standard Preparation and Stability ...................................13 4.2.5 Chromatographic Standard Preparation and Stability ...........................14 4.2.6 Source (& Characterization) of Samples ...............................................14 4.2.7 Storage & Preparation of Samples ........................................................14 4.2.8 Sample Fortification Procedure.............................................................14 4.2.9 Analyte Extraction Procedure................................................................15 4.2.10 Cymoxanil Purification Procedure ........................................................15 4.2.11 IN-KQ960 Purification Procedure.........................................................16

4.3 Instrumentation...............................................................................................16 4.3.1 Chromatography ....................................................................................16 4.3.2 LC/MS Analysis ....................................................................................17 4.3.3 Calibration Procedure and Sample Analysis .........................................18

4.4 Calculations ....................................................................................................18 4.4.1 Methods .................................................................................................18 4.4.2 Example.................................................................................................19

5.0 Results and Discussion........................................................................................20 5.1 Method Validation Results .............................................................................20

5.1.1 Detector Response .................................................................................20

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5.1.2 Control Samples ....................................................................................20 5.1.3 Recoveries (Accuracy & Precision).......................................................20 5.1.4 Extraction Efficiency.............................................................................21 5.1.5 Limit of Quantitation and Limit of Detection .......................................21

5.2 Timing ............................................................................................................21 5.3 Modifications or Special Precautions.............................................................21 5.4 Method Ruggedness .......................................................................................21

5.4.1 Stability..................................................................................................21 5.4.2 Specificity/Potential Interference ..........................................................22 5.4.3 Confirmatory Method............................................................................22

6.0 Conclusions .........................................................................................................22 7.0 Retention of Records...........................................................................................22 8.0 References ...........................................................................................................22

TABLE Table 1 Summary of Cymoxanil and IN-KQ960 Fortification (Recovery)

Data in Spinach ....................................................................................23

FIGURES Figure 1 Flow Diagram of Analytical Method....................................................24

Figure 2 Full Scan Spectrum for Cymoxanil and IN-KQ960 .............................26

Figure 3 Cymoxanil Representative Curve and Standards .................................27

Figure 4 IN-KQ960 Representative Curve and Standards..................................30

Figure 5 Cymoxanil - Example Chromatograms of Control and Fortified Spinach Samples...................................................................................33

Figure 6 IN-KQ960 - Example Chromatograms of Control and Fortified Spinach Samples...................................................................................34

Figure 7 Signal-to-Noise Ratios .........................................................................35

Figure 8 Cymoxanil LC/MS/MS Confirmation..................................................36

Figure 9 IN-KQ960 LC/MS/MS Confirmation ..................................................37

APPENDIX Appendix 1 LC/MS Experimental Conditions.........................................................38

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ANALYTICAL METHOD FOR THE DETERMINATION OF CYMOXANIL AND IN-KQ960 IN SPINACH (LEAFY VEGETABLES) USING LC/MS

Joseph P. McClory and Robert M. Henze

1.0 ABSTRACT The purpose of this study was to develop an analytical method for the detection, quantitative analysis, and confirmation of cymoxanil and IN-KQ960 in spinach. Cymoxanil and its metabolite, IN-KQ960, were extracted from samples of spinach with a mixture of acetonitrile and water. This solvent mixture has been shown to be effective at extracting cymoxanil from plant matrices.

For cymoxanil, NaCl was added to an aliquot to separate the aqueous phase from the organic phase. The aqueous phase was discarded, and the acetonitrile layer containing cymoxanil was passed through a SAX SPE column. The extract is then further cleaned up using a hexane liquid/liquid extraction followed by Envi Carb SPE column. Cymoxanil is not retained on either of these columns.

IN-KQ960 does not partition quantitatively when salted out, so a separate aliquot of extract is used for the analysis of the metabolite. The extract is cleaned up using a hexane liquid/liquid extraction followed by passing through SAX and Envi Carb SPE columns. IN-KQ960 is not retained on either of these columns

The LOQ by LC/MS analysis was determined to be 0.050 µg/g (ppm) for both cymoxanil and IN-KQ960. During method validation, acceptable recoveries were generated for spinach samples fortified at the LOQ through the highest levels anticipated in field treated samples as indicated in the following table:

Average Recovery

LEVEL (PPM)

CYMOXANILAVG ± RSD (%)

IN-KQ960 AVG% (RSD) N

0.050 (LOQ) 92 ± 4.3 83 ± 3.6 5

0.50 88 ± 2.6 83 ± 6.2 5 The mean recovery of cymoxanil from 10 freshly fortified spinach samples was 90% with a RSD of 4.3%. The mean recovery of IN-KQ960 from 10 freshly fortified spinach samples was 83% ± 4.8% (RSD). Unfortified control samples showed no quantifiable residues of cymoxanil and IN-KQ960.

2.0 INTRODUCTION Cymoxanil is a fungicide used for control of various fungal diseases in crops, such as grapes, potatoes, tomatoes, cucurbits, and leafy vegetables. IN-KQ960 is a metabolite identified in a cymoxanil lettuce metabolism study (Reference 1). The objective of this study is to provide a detailed and validated method to monitor for cymoxanil and

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IN-KQ960 in leafy vegetables. The results may be used in the generation of data for submission for regulatory monitoring and control.

Ground samples are extracted with a mixture of acetonitrile/water. The metabolism study (Reference 1) demonstrated that the acetonitrile/water mixture, used in this residue method, extracts the total toxic residue from the lettuce matrix. Additional sample cleanup is performed with hexane liquid/liquid extraction, SAX and Envi-Carb SPE columns. Analysis is performed by LC/MS with an LOQ of 0.050 ppm for both cymoxanil and IN-KQ960.

3.0 MATERIALS Equivalent equipment and materials may be substituted unless otherwise specified; note any specifications in the following descriptions before making substitutions. Substitutions should only be made if equivalency/suitability has been verified with acceptable control and fortification recovery data.

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3.1 Equipment

Equipment Description Product ID Supplier

Freezer Labline Frigid-Cab Labline Instruments, Inc. (Melrose Park, IL)

Refrigerator 6FAR Marvel Industries, Inc. (Richmond, IN)

Commercial Food Processor Model 31FP93 Waring Products (New Hartford, CT)

Analytical Balance AE163 Dual Range Balance; PM460 Toploading Balance; PM400 Toploading Balance; AE163 Dual Range Balance

Mettler Instrument Corp. (Hightstown, NJ)

Analytical Evaporator N-Evap Model 111 with stainless steel luer fit needles

Organomation Assoc. (South Berlin, MA)

Homogenizer Tissumizer homogenizer Model SDT-20 equipped with Model SDT-182EN shaft (Teflon bearing)

Tekmar Company (Cincinnati, OH)

Sonication Bransonic 52 or 2200 Ultrasonic Cleaner, 0.75 gal. capacity

Branson Ultrasonics Corp. (Danbury, CT)

Vortex Mixer Vortex Genie K-550-G or Vortex-2 Genie VWR, Inc. (West Chester, PA)

Filtration Gelman Acrodisc 13 CR, 0.2-µm PTFE 13 mm dia. membrane syringe filter, Cat. No. 4423

VWR (Bridgeport, NJ)

Solid Phase Extraction Supelclean ENVI-Carb SPE cartridge, 0.5 g/6 mL, Cat. No. 57094; Visiprep DL SPE Manifold, Cat. No. 5-7030M;

Supelco (Bellefonte, PA)

Solid Phase Extraction

Bond Elut SAX SPE cartridge, 6 cc/1 g, Cat. No. 1225-6013; 75-mL Plastic Reservoirs, Cat. No. 12131012; union adapter for 6-mL, Cat. No: 12131001; union adapter for 60 mL columns, Cat. No. 12131004; Reservoir Adapters, Cat. No. 12131003;

Varian, Inc. (Palo Alto, CA)

Centrifuge Sorvall Centrifuge, Model RC 3B Sorvall Instruments (Wilmington, DE)

Centrifuge Sorvall Centrifuge, Model GLC-2B(bench top) Sorvall Instruments (Wilmington, DE)

Analytical Evaporator N-Evap Model 111(with stainless steel luer fit needles)

Organomation Assoc. (South Berlin, MA.)

pH Meter Beckman φ 10 pH meter with combination pH electrode

Beckman Instruments, Inc. (Fullerton, CA)

Labware

250 mL, Nalgene Cat. No. 16129 028 Polypropylene Centrifuge Bottles; Boroscilicate glass scintillation vials with cap, 20 mL, Cat. No. 66022-004; Pyrex Brand Single Metric Scale Graduated Cylinders, 10-mL and 100-mL capacity, Cat. No. 24709-715 and 24709-748, respectively; Glass wool - PYREX brand glass fiber, Cat. No. 32848.003168; VWR brand Disposable Pasteur Pipettes, Borosilicate Glass, 9 in, Cat. No. 53283-914 equipped with 2 mL, 13 X 32 mm rubber bulbs, Cat. No. 56310-240

VWR (Bridgeport, NJ)

Labware Electronic 1000-µL and 10-mL Pipettors Rainin (Walnut Creek, CA)

Labware Mechanical, positive displacement, 25-µL, 50-µL and 250-µL Pipettors

Gilson Inc. (Middletown, WI)

Labware

Falcon 2098 (50 mL), 2096 (15 mL) Polypropylene Centrifuge Tubes; 3-mL Disposable Syringe, Cat. No. BD309585; 60-mL Disposable Syringe, Cat. No. BD309663

Becton Dickinson (Franklin Lakes, NJ)

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HPLC/MS System

HPLC

HP1100: G1322A degasser, G1312A binary pump, G1311A quaternary pump; G1313A autosampler; G1316A column unit; G1314A variable wavelength detector

Agilent Technologies, Inc. (Palo Alto, CA)

Autosampler Vials Target DP Amber Kit, T/S/T Septa, 100 PK, Cat. No. 5182-0556


HPLC Column Eclipse XDB-C8; 4.6 mm × 150 mm, 5 µm particle size diameter


Splitter tee Valco zero dead-volume tee (split-flow to MS), Cat. No. ZT1C

Valco Instruments, Inc. (Houston, TX)

Switching Valve Valco 6 Port Electrically Actuated Valve, Cat. No. 1384

Valco Instruments, Inc. (Houston, TX)

Triple Quadrupole MS

MicroMass Quattro II triple quadrupole mass spectrometer using an electrospray (ESI) or atmospheric chemical ionization (ACPI) interface and MassLynx NT version 3.1 software

Waters Corporation (Milford, MA)

3.2 Reagents and Standards

3.2.1 Reagents The equivalency/suitability of substituted reagents should be verified.

Reagents Product Description Product ID Supplier

Formic Acid GR, ACS, 98% FX0440-11 EM Science (Gibbstown, NJ)

Methanol OmniSolv, 4L MX0488-1 EM Science (Gibbstown, NJ)

Hexane OmniSolv, 4L HX0296-1 EM Science (Gibbstown, NJ)

Acetonitrile OmniSolv, 4L AX0142-1 EM Science (Gibbstown, NJ)

Water OmniSolv, 4L WX0004-1 EM Science (Gibbstown, NJ)

Methylene Chloride OmniSolv, 1L DX0831-6 EM Science (Gibbstown, NJ)

3.2.2 Reference Analytical Standards Reference analytical standards of cymoxanil (Lot No. DPX-T3217-151, purity 99.6%), and IN-KQ960 (Lot No. 3, purity 96.2%), were synthesized at E.I. du Pont de Nemours and Company, DuPont Crop Protection, Newark, Delaware. Characterization data are archived by DuPont Crop Protection, E.I. du Pont de Nemours and Company, Newark, Delaware. The structures and specific information for cymoxanil and IN-KQ960 follow:

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NNH

O

O

N

CH3

NH

O

CH3

Cymoxanil

DuPont Code: DPX-T3217

CAS Chemical Name: 2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyimino)acetamide

CAS Registry Number: 57966-95-7

IUPAC Chemical Name: 1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea

Molecular weight = 198.2 g/mole

NHN

O

O NHOCH3

CONH2

C2H5

KQ960

DuPont Code: IN-KQ960

CAS Chemical Name: 3-Ethyl-4-(methoxyamino)-2,5-dioxo-4-imidazolidinecarboxamide

CAS Registry Number: none

Molecular weight = 216 g/mole

3.3 Safety and Health Each analyst must be acquainted with the potential hazards of the reagents, products and solvents used in this method before commencing laboratory work. All appropriate material safety data sheets should be read and followed, and proper personal protective equipment should be used.

4.0 METHODS

4.1 Principle of the Analytical Method Samples are extracted using an acetonitrile/water mixture as described in DuPont Report No. AMR 3705-95, Revision No. 2, “Analytical Method for the Determination of DPX-JE874 and Cymoxanil Residues in Various Matrices” (EPA MRID No. 44579102, Reference 2). Changes incorporated in the current study include the addition of IN-KQ960 as an analyte. Also, LC/MS is used for the analysis of both cymoxanil and IN-KQ960. Based on the sensitivity and selectivity of LC/MS, sample cleanup procedures were simplified.

For cymoxanil, NaCl is added to an aliquot to separate the aqueous phase from the organic phase. The aqueous phase is discarded, and the acetonitrile layer containing cymoxanil is passed through a SAX SPE column. The extract is then further cleaned up using a hexane liquid/liquid extraction followed by Envi Carb SPE column. Cymoxanil is not retained on either of these columns.

IN-KQ960 does not partition quantitatively when salted out, so a separate aliquot of extract is used for the analysis of the metabolite. The extract is cleaned up using a hexane liquid/liquid extraction followed by passing through SAX and Envi Carb SPE columns. IN-KQ960 is not retained on either of these columns.

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4.2 Analytical Procedure

4.2.1 Glassware & Equipment Cleaning Procedures The effectiveness of any cleaning procedure used should be demonstrated by preparation and analysis of reagent blanks. In general, all reusable glass and plasticware should be washed in hot tap water with laboratory grade, non-phosphate detergent, rinsed several times with tap water, rinsed several times with deionized water, rinsed once with acetone, and allowed to fully dry before use. Care should be taken to avoid working with high levels of the analyte being monitored in the same laboratory where samples are being extracted and analyzed.

4.2.2 Preparation & Stability of Reagent Solutions 90/10 methylene chloride/methanol (v/v)- 100 mL of a 90/10 methylene chloride/methanol (v/v) is prepared on the day of analysis by adding 90 mL of methylene chloride to 10 mL of methanol. 15 mL of this solution is required per sample; if analyzing more than six samples adjust volume of solution prepared accordingly.

0.02% formic Acid (v/v)- 2.0 L of the solution is prepared by adding 0.4 mL of formic acid to 2.0 liter of de-ionized water. Solution is stored at room temperature and is stable for 1 month.

4.2.3 Stock Standard Preparation and Stability

Use Class A volumetric flasks when preparing standard solutions. Prepare standard stock solutions by accurately weighing 10 ± 0.01 mg of cymoxanil and IN-KQ960 into separate 100-mL volumetric flasks using an analytical balance. Record the accurate weight of the standard. Dissolve the standard in approximately 50 mL of HPLC-grade methanol. After dissolving, bring the solution to a volume of 100 mL, using HPLC-grade methanol and invert the volumetric flask to mix the solution to homogeneity. These standard solutions are stable for approximately 6 months when stored at approximately 4°C immediately after each use. The concentration of each analyte, cymoxanil and IN-KQ960, in solution is 100 µg/mL in methanol.

4.2.4 Fortification Standard Preparation and Stability

Use Class A volumetric flasks when preparing standard solutions. Intermediate standard solutions containing of 10.0, 1.00, and 0.100 µg/mL of both cymoxanil and IN-KQ960 were prepared by combining 10.0, 1.00, and 0.100 mL, respectively, of the cymoxanil and IN-KQ960 stock solutions and diluting to 100 mL in methanol. These standard solutions are stable for approximately 6 months when stored at approximately 4°C immediately after each use.

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4.2.5 Chromatographic Standard Preparation and Stability The calibration standards were prepared by pipetting volumes of the 10.0 µg/mL or the 1.0-µg/mL intermediate standard solutions, as shown in the following table, into separate 10.0-mL volumetric flasks and diluting to the mark with 5% methanol:95% 0.02% formic acid. Alternate or additional standards may be prepared as needed. These standard solutions should be freshly prepared monthly and stored at approximately 4°C immediately after each use.

Desired Standard Concentration (µg/mL)

Volume of 10.0 µg/mL Intermediate Standard Required (mL)

Volume of 1.0 µg/mL Intermediate Standard Required (mL)

0.10 0.10 x 0.050 0.05 x 0.010 x 0.10

0.0075 x 0.075 0.0050 x 0.05

4.2.6 Source (& Characterization) of Samples Spinach samples were purchased fresh from the local supermarket.

4.2.7 Storage & Preparation of Samples Upon arrival, the samples were stored frozen at –20 ± 5°C prior to sample preparation, extraction, and analysis. In preparation for analysis, samples were removed from frozen storage and ground frozen with dry ice using a Hobart Mixer. Each sample was mixed extensively during the grinding process to ensure homogeneity. Samples were returned to the freezer for storage until extraction and analysis. The samples remained frozen throughout sample preparation. Control samples should be processed first to prevent cross-contamination.

4.2.8 Sample Fortification Procedure For the samples fortified at the 0.050 ppm (LOQ) level, 1.0 mL of the 1.00 µg/mL intermediate standard was used. For the samples fortified at the 0.50 ppm (10XLOQ) level, 1.0 mL of the 10.0 µg/mL intermediate standard was used.

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4.2.9 Analyte Extraction Procedure 1. Weigh out 20 grams of Spinach into a 250-mL centrifuge bottle.

2. For fresh fortification samples, spike sample at the appropriate level and let sit for 5 minutes for the solvent to evaporate.

3. Add 60 mL of water and 120 mL of acetonitrile to the sample.

4. Blend with a tissuemizer for 5 minutes on medium speed. Centrifuge for 10 minutes at 3000 rpm.

5. Remove an 8-mL aliquot, transfer into a 15-mL centrifuge tube, and take this aliquot through the purification procedure.

4.2.10 Cymoxanil Purification Procedure 1. Take the 8-mL aliquot from the extraction and add 4 grams of sodium chloride.

Shake vigorously for 1 minute and then centrifuge at 3000 rpm for 5 minutes.

2. Remove the upper layer (acetonitrile) and put into a new 15-mL centrifuge tube. Add 3 mL of acetonitrile to the tube containing the NaCl layer from Step 1. Shake vigorously for 1 minute and then centrifuge at 3000 rpm for 3 minutes. Remove the upper layer and add this to the tube containing the acetonitrile from this step.

3. Condition a 1-gram/6 mL SAX cartridge using 5 mL of acetonitrile, discard conditioning solution.

4. Add the sample to the SAX cartridge, collecting the solution in a 15-mL centrifuge tube. After the sample has gone through, add 3 mL of acetonitrile to the tube from Step 3, vortex, and also pass that through the cartridge.

5. Remove tube from manifold, cap, vortex, and put on the N-Evap to reduce the volume to approximately 9 mL.

6. Take sample (from Step 5) and add 5 mL of hexane. Vortex and centrifuge at 3000 rpm, then remove upper layer (hexane) and discard. Repeat this step again.

7. Condition a 1-gram/6 mL Envir- Carb carbon column using 5 mL of a 90% methylene chloride/10% acetonitrile and discard solution.

8. Add the sample (from Step 6) to the column and collect this elution in another 15-mL centrifuge tube. Rinse tube (from Step 6) with 4 mL of the (90% methylene chloride/10% acetonitrile) solution, vortex and pass through the Enviro Carb column and collect also.

9. Remove sample from the manifold, cap vortex, remove the cap and put on the N-Evap to be evaporated to about 0.75 mL at 38 to 40°C. (After every 3 to 4 mL is evaporated, remove sample from N-Evap, cap, vortex, and return to keep the analyte in the solution.)

10. Dilute to a final volume of 4mL with 0.02% formic acid, cap, vortex, and sonicate for 2 minutes. Filter through a syringe filter and analyze by LC/MS.

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4.2.11 IN-KQ960 Purification Procedure 1. Take the 8-mL aliquot from the extraction and add 5 mL of hexane. Shake

vigorously for 1 minute and then centrifuge at 3000 rpm for 5 minutes. Remove the upper layer (hexane) and discard.

2. Repeat Step1. Discard top hexane layer. Save lower layer (water and acetonitrile).

3. Condition a 1-gram/6 mL SAX cartridge using 5 mL of acetonitrile. Discard this conditioning solution.

4. Add the sample from Step 2 to the cartridge, collecting the elution in a 15- mL centrifuge tube. After the sample has gone through add an additional 3 mL of acetonitrile to the tube from Step 2, vortex, and also pass that through the cartridge.

5. Condition a 1-gram/6 mL Envir-Carb carbon column using 5 mL of 90% methylene chloride/10% acetonitrile. Discard solution.

6. Add the sample (from Step 4) to the column and collect this elution in another 15-mL centrifuge tube. Rinse tube (from Step 4) with 5 mL of the (90% methylene chloride/10% acetonitrile) solution, vortex, and pass through the Carbon column and collect also.

7. Remove sample from the manifold, cap vortex, remove the cap and put on the N-Evap to be evaporated to about 0.75 mL at 38 to 40°C. (After every 3 to 4 mL is evaporated, remove sample from N-Evap, cap, vortex, and return to keep the analyte in the solution.)

8. Dilute to a final volume of 4 mL with 0.02% formic acid, cap, vortex, and sonicate for 2 minutes.

9. Filter through a syringe filter and analyze by LC/MS.

A flow diagram for both analytes is shown in Figure 1.

4.3 Instrumentation

4.3.1 Chromatography Reversed-phase liquid chromatography was used to separate cymoxanil and IN-KQ960 from co-extractants. An Agilent Eclipse XDB-C8 HPLC column was selected. The column choice reflected experimental results indicating preferred separation of cymoxanil and IN-KQ960 from co-extractants. Since the sample cleanup was performed on two separate aliquots, separate injections were made for cymoxanil and IN-KQ960.

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System: Hewlett-Packard HP1100 HPLC

Column: 2.1 mm i.d. × 10 cm, Agilent Eclipse XDB-C8, 3 µm diameter packing

Column Temperature: 30°C

Auto-sampler Temperature 4°C

Injection Volume: 25-30 µL

Flow Rate: 1.0 ml/min

Conditions: A: 0.02% Formic Acid B: Methanol

Time %A %B Time %A %B

------Cymoxanil---- ------IN-KQ960---

0.0 95 5 0.0 98.0 2.0

17.0 50 50 2.0 98.0 2.0

19.9 10 90 15.0 80 20.0

22.0 95 5 16.0 1.0 99.0

27.00 95 5 18.0 1.0 99.0

19.0 70.0 30.0

22.0 98.0 2.0

26.0 98.0 2.0

IN-KQ960 Retention Time:

Cymoxanil Retention Time:

~ 9.3 min

~ 14.8 min

Total Run Time: 27 min

A six-port electronically activated switching valve was used to direct column effluent flow to waste prior to and following elution of the compounds of interest. The use of this valve reduced source contamination and enabled additional samples to be analyzed before the ion source required cleaning. The valve switching times are given in the following table.

TIME (MINUTES) COLUMN ELUATE FLOW 0.00-8.0 Waste 8.0-17.0 MS source

17-27 Waste Since electrospray LC/MS systems perform optimally at low flow rates, but a flow rate of 1.0 mL/min was used for sample analysis, the LC should be configured with a splitter, which diverts approximately 90% of the flow to waste.

4.3.2 LC/MS Analysis Analysis of cymoxanil and IN-KQ960 was performed using a Micromass Quattro II triple quadrapole LC/MS/MS instrument with an electrospray ionization (ESI) source operated in MS (SIR) negative ion mode. A summary of representative experimental conditions is provided in the following table:

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Micromass Quattro LC ESI-LC/MS Mass Spectrometer Conditions

Analytes Ions Monitored Cone

Voltage Source Temp.

Dwell (Seconds)

Cymoxanil 197.0 ± 0.1 AMU 17V 125°C 0.02

IN-KQ960 215.0 ± 0.1 AMU 15V 125°C 0.02

Electrospray Voltage: 3.50 kV Detector Voltage: 700 V Nebulizing Gas Flow: 15 L/h Drying Gas Flow: 300 L/h

A complete list of the experimental parameters is given in Appendix 1. A typical LC/MS full scan spectrum is shown Figure 2.

The instrument was operated in MS (SIR) negative ion mode for quantitative analysis. Peak area was used for quantitation. For confirmation MS/MS was used. The relative ratio of the fragment ions was evaluated to confirm the presence of an analyte in an unknown sample.

4.3.3 Calibration Procedure and Sample Analysis A 0.01-µg/mL chromatographic standard should be analyzed prior to the start of analyses to establish that the instrument is working properly. If a signal-to-noise ratio of approximately 10 to 1 is not attained, the instrument must be tuned or cleaned prior to sample analysis. Operating parameters must be tailored to the particular instrument used, especially if it is to be an alternate vendor’s instrument, and should be checked daily. Note that some ion channels, other than those used for development of this method, may need to be added or eliminated when utilizing this method on other instrumentation. Each ion channel used for sample analysis/quantitation must be checked to insure it is free of interference. The control will be used to demonstrate that baseline interference is less than signal-to-noise 3:1. Begin each sample set by injecting a minimum of 2 calibration standards. The first injection should always be disregarded.

4.4 Calculations

4.4.1 Methods The response factor, RF, for each analytical standard is the ratio of the analyte concentration to the analyte peak area.

areapeak Analyte(µg/mL) analyte ofion Concentrat=RFstd

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The average response factor, RFave, calculated from all standards analyzed in an analytical set containing control, fortified or treated samples was used to calculate the concentration of cymoxanil and IN-KQ960 in these samples.

Injected Standards ofNumber Total)RFstdn.......3RFstd2RFstdstd1RF(=RFave

+++

The concentration (µg/g or ppm) of analyte found in each sample was calculated as follows:

µg/g analyte Found = (mL)Taken Aliquot x(g) Wt.Sample

])(.[ xRFave] x Areaeak [ solvmLxmLVolFinalP

Where:

Total Extract Volume (mL solv) = 180 mL Final Extract Volume (Final Vol.) = 5.0 mL Aliquot Taken = 4.0 mL Sample Weight = 20.0 grams

The percent recovery found was calculated as follows:

100% x µg/g) level,tion (Fortifica

Found) (µg/g =Recovery %

4.4.2 Example The calculation below shows the concentration of cymoxanil in a fortified sample MV1-LOQ1, see data in Table 1 and chromatogram in Figure 5:

g/mL0.00638 784

g/mL)( 0.005 RFstd µµ==

RFave =

7E6)-(6.69)671.6(6)-(6.72E6)-(6.81E6)-(6.59E6)-(6.60E6)-(6.38E +−+++++ E

RFave = 6.64E-6 g/mLµ

Peak Area Cymoxanil: = 1175

mL Solvent: = 180 mL

mL Aliquot 1: = 8.0 mL

Final Volume: = 5.0 mL

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Sample Weight: = 20.0 grams

ppm Cymoxanil = g20.0 8.0mL

) mL 180 x mL (5.0 x ug/mL) 6-6.64E (1175×

x = 0.0439 µg/g

100% x µg/g 0.050

µg/g 0.0439 =Recovery % = 88%

5.0 RESULTS AND DISCUSSION

5.1 Method Validation Results

5.1.1 Detector Response Standard calibration solutions used for quantitative analysis ranged from 5.0 to 100.0 ng/mL for both cymoxanil and IN-KQ960. Typical experimental conditions for each analyte are provided in Appendix 1. Typical LC/MS chromatograms for standards analyzed during method validation are provided in Figure 3 and Figure 4. The response of the MS detector was linear over the range of standards analyzed, as evidenced by relative standard deviation of the response factors consistently being less than 15%.

Control samples fortified at 0.050 µg/g to 0.50 µg/g were successfully extracted, cleaned up, and analyzed using this method. Representative chromatograms of fortified and unfortified samples are shown in Figure 5 for cymoxanil and Figure 6 for IN-KQ960.

5.1.2 Control Samples Interference peaks in unfortified sample chromatograms were less than the LOQ at the retention time for both cymoxanil and IN-KQ960.

5.1.3 Recoveries (Accuracy & Precision) Unfortified controls and controls fortified at 0.050, and 0.50 ppm of cymoxanil and IN-KQ960 were analyzed to verify method performance. The fortification levels tested bracketed the range of residue values expected in treated samples encountered from the field. All results are provided in Table 1 and summarized below:

Average Recovery

Level (ppm) Cymoxanil

Avg ± RSD (%)IN-KQ960

Avg ± RSD (%) n

0.050 (LOQ) 92 ± 4.3 83 ± 3.6 5

0.50 88 ± 2.6 83 ± 6.2 5

Overall Mean 90 ± 4.3 83 ± 4.8 10

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The mean percent recovery of cymoxanil from 10 freshly fortified control spinach samples was 90% with a RSD of 4.3%. The mean percent recovery of IN-KQ960 from 10 freshly fortified control spinach samples was 83% ± 4.8% (RSD). Unfortified control samples showed no quantifiable residues of cymoxanil and IN-KQ960.

5.1.4 Extraction Efficiency In metabolism studies with radiolabeled 14C test substance, cymoxanil was readily extracted into an organic solvent when the plant tissue was macerated using high speed mixing. The metabolism study (Reference 1) demonstrated that the acetonitrile/water mixture used in the residue method is valid for the extraction of the total toxic residue from lettuce matrix. IN-KQ960 was isolated in an aqueous surface wash fraction. The acetonitrile/water mixture used in the residue method should be effective for IN-KQ960 as well.

5.1.5 Limit of Quantitation and Limit of Detection The LOQ by LC/MS analysis was determined to be 0.050 µg/g for cymoxanil and IN-KQ960. This quantitation limit is defined as the lowest fortification level evaluated at which acceptable average recoveries (70-120%, RSD <20%) were achieved. This quantitation limit also reflects the fortification level at which analyte peaks were consistently generated at a level approximately 10-20 times the signal at the retention time of each analyte in an untreated control sample. An example of the signal-to-noise calculation is provided in Figure 7.

The limit of detection is estimated to be 0.02 µg/g, which is one-third the value of the corresponding LOQ value.

5.2 Timing Typically six to eight samples can be prepared during the course of an eight-hour day. LC/MS analyses were run unattended overnight. The sample extraction and cleanup procedure is the rate-determining step.

5.3 Modifications or Special Precautions The analysis of cymoxanil is sensitive to mobile phase pH. LC systems that have previously run alkaline mobile phases must be thoroughly flushed with water prior to starting sample analysis.

5.4 Method Ruggedness

5.4.1 Stability The stability of the analytes and reagent solutions has been stated in the respective sections of this report. Analytes are stable for a minimum of two weeks when stored in a refrigerator when not in use.

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5.4.2 Specificity/Potential Interference Due to the selective and specific nature of LC/MS detection method a single peak was observed at the retention time of cymoxanil and IN-KQ960. As a result of the selective detection used, interference testing is not necessary for this method.

5.4.3 Confirmatory Method Only one parent daughter transition was available for both cymoxanil and IN-KQ960. For confirmation the transition monitored for cymoxanil was (M-1) 197→42 and for IN-KQ960 (M-1) 215→140 was observed. Figure 8 and Figure 9 show that the chromatograms constructed from these transitions are identical for the standards prepared in reagent solvents versus fortified spinach matrix. Therefore, these transitions are adequate to confirm the identity of both cymoxanil and IN-KQ960 in spinach (leafy vegetables).

6.0 CONCLUSIONS This method for determination of cymoxanil and IN-KQ960 residues extracted from spinach (leafy vegetables) meets U.S. EPA and EU guidelines.

This LC/MS method with mass selective detection is free of interference above the LOQ of 0.050 ppm at the retention times corresponding to cymoxanil and IN-KQ960 in unfortified samples. This method generated acceptable recoveries over concentration levels expected in the samples tested.

7.0 RETENTION OF RECORDS Originals or exact copies of all raw data and pertinent information, and the final report will be retained at:

E.I. du Pont de Nemours and Company DuPont Crop Protection Global Technology Division Stine-Haskell Research Center Newark, Delaware 19714-0030

8.0 REFERENCES 1. Fox, G. C., “Metabolism of [2-14C]Cymoxanil in Lettuce”; DuPont Report

No. AMR 4375-97, E.I. du Pont de Nemours and Company, Wilmington, Delaware; MRID No. 44944605.

2. Nathan, E.C. III, “Analytical Method for the Determination of DPX-JE874 and Cymoxanil Residues in Various Matrices”; DuPont Report No. AMR 3705-95, Rev. 2, E.I. du Pont de Nemours and Company, Wilmington, Delaware; MRID No. 44579102.

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TABLE 1 SUMMARY OF CYMOXANIL AND IN-KQ960 FORTIFICATION (RECOVERY) DATA IN SPINACH

Set No.

Fortification Level (ppm)1

Cymoxanil

PK Area ppm % Recovery

IN-KQ960

PK Area ppm % Recovery

MV1-LOQ1 0.050 1175 0.0439 882 992 0.429 86

MV1-LOQ2 0.050 1294 0.0483 97 966 0.0418 84

MV2-LOQ1 0.050 1274 0.0479 96 924 0.0420 84

MV2-LOQ2 0.050 1199 0.0450 90 867 0.0394 79

MV2-LOQ3 0.050 1217 0.0457 91 884 0.0402 80

Mean % Recovery ± SD (n = 4) = RSD =

92 ± 3.9 4.3

83 ± 3.0 3.6

MV1-10LOQ1 0.50 11406 0.426 85 8970 0.388 78

MV1-10LOQ2 0.50 12168 0.454 91 9137 0.395 79

MV1-10LOQ3 0.50 12084 0.451 90 9431 0.408 82

MV2-10LOQ1 0.50 11493 0.432 86 9608 0.437 87

MV2-10LOQ2 0.50 11660 0.446 88 9861 0.449 90

Mean % Recovery ± SD (n = 3) = RSD =

88 ± 2.3 2.6

83 ± 5.2 6.2

Overall Mean % Recovery ± SD (n = 2) = RSD =

90 ± 3.9 4.3

83 ± 4.0 4.8

1 Limit of quantitation (LOQ) for determination of both cymoxanil and IN-KQ960 in spinach was 0.050 ppm. Residue values

carried to an excessive number of significant figures were used to calculate % Recovery. After calculation, % Recovery values were rounded to the nearest whole number and reported.

2 Additional data necessary to calculate % recoveries (see calculation on page 19) sample wt. Extract Vol. Aliquot Final Vol. Res. Factor(MV-1) Res. Factor(MV-2) Cymoxanil 20.0g 180ml 8.0ml 5.0ml 0.00664 0.00668

IN-KQ960 20.0g 180ml 8.0ml 4.0ml 0.00962 0.01011

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FIGURE 1 FLOW DIAGRAM OF ANALYTICAL METHOD

Envi Carb SPE and Analysis1) Condition a Envi Carb SPE, with 5 ml of 90/10 MeCl2/MeOH

2) Pass sample thru column rinse with 4 ml 90/10, evap to 0.75 ml3) Dilute to 4ml with 0.02% Formic acid, sonicate and filter into LC vial

SAX SPE and Hexane L/L Extraction1) Condition a SAX SPE, pass sample thru column

2) Pass an additional 3 ml thru column and evaporate to 9ml3) Add 5 ml hexane to sample, shake, remove hexane, repeat

Salt Out1) Add 4 g of NaCl to 8.0 ml of extract, Shake 1 min.

2) Centrifuge 10 min, retain top layer 3) Add additional 8 ml ACN to sample and repeat steps 1 and 2

Cymoxanil Extraction1) To 20g sample add 60ml H20 and 120ml ACN

2) Blend withTissuemizer for 5 minutes, centrifuge 10 minutes

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FIGURE 1 FLOW DIAGRAM OF ANALYTICAL METHOD (CONTINUED)

Envi Carb SPE and Analysis1) Condition a Envi Carb SPE, with 5 ml of 90/10 MeCl2/MeOH

2) Pass sample thru column rinse with 4 ml 90/10, evap to 0.75 ml3) Dilute to 4ml with 0.02% Formic acid, sonicate and filter into LC vial

Hexane L/L Extraction and SAX SPE1) To 8.0 ml extract, add 5 ml hexane, shake, remove hexane, repeat

2) Condition a SAX SPE, pass sample thru column 3) Pass an additional 3 ml thru column

IN-KQ960 Extraction1) To 20g sample add 60ml H20 and 120ml ACN

2) Blend withTissuemizer for 5 minutes, centrifuge 10 minutes

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FIGURE 2 FULL SCAN SPECTRUM FOR CYMOXANIL AND IN-KQ960

A) Cymoxanil

10.0ug/ml std.

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z0

100

%

11190307 371 (14.784) Cm (369:371-(169:264+444:475)x3.000) Scan ES- 2.64e5196

41.5 166

197

242

B) IN-KQ960

10.0ug/ml std.

50 100 150 200 250 300 350 400m/z0

100

%

11130307 117 (9.235) Cm (106:119-(2:58+166:254)x4.000) 1: Scan ES- 1.11e4215

140 261216

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FIGURE 3 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS

Compound 1 name: CymoxanilCorrelation coefficient: r = 0.999990, r^2 = 0.999979Calibration curve: 149538 * x + 3.13622Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

0.0 0.1Conc0

1.50e4

Response

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FIGURE 3 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS (CONTINUED)

18:03:48 29-Sep-2003

13.00 14.00 15.00 16.00Time 0

100

C

0

100

B

0

100

A

09260301B Sm (Mn, 3x1) SIR of 1 Channel ES- 197.00 1.52e4

Area

14.79 758


Area

14.80 1138


Area

14.81 1469

A Cymoxanil Standard

B Cymoxanil Standard

C Cymoxanil Standard

0.005 µg/mL Standard 0.0075 µg/mL Standard 0.01 µg/mL Standard

Peak Area: 758 Peak Area: 1138 Peak Area: 1469

Analysis Date:

29 Sep 2003

Analysis Date:

29 Sep 2003

Analysis Date:

29 Sep 2003

Set No.: 1 Set No.: 1 Set No.: 1

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FIGURE 3 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS (CONTINUED)

23:13:45 29-Sep-2003

13.00 14.00 15.00 16.00Time 0

100

B

0

100

A


Area

14.81 7448


Area

14.79 14974

A Cymoxanil Standard

B Cymoxanil Standard

0.050 µg/mL Standard 0.10 µg/mL Standard

Peak Area: 7448 Peak Area: 14974

Analysis Date:

29 Sep 2003

Analysis Date:

29 Sep 2003

Set No.: 1 Set No.: 1

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FIGURE 4 IN-KQ960 REPRESENTATIVE CURVE AND STANDARDS

Compound 1 name: KQ960Correlation coefficient: r = 0.999953, r^2 = 0.999906Calibration curve: 105285 * x + -60.5998Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

0.0 0.1Conc-60.6

1.05e4

Response

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FIGURE 4 IN-KQ960 REPRESENTATIVE CURVE AND STANDARDS (CONTINUED)

14:10:08 24-Sep-2003 0.005ug/ml std.

8.00 9.00 10.00 11.00Time 0

100

C

0

100

B

0

100

A

09240300A Sm (Mn, 3x1) SIR of 1 Channel ES- 215.00 1.49e4

Area

9.43 537


Area

9.41 691


Area

9.39 963

A IN-KQ960 Standard

B IN-KQ960 Standard

C IN-KQ960 Standard

0.005 µg/mL Standard 0.0075 µg/mL Standard 0.01 µg/mL Standard

Peak Area: 537 Peak Area: 691 Peak Area: 963

Analysis Date:

24 Sep 2003

Analysis Date:

24 Sep 2003

Analysis Date:

24 Sep 2003


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FIGURE 4 IN-KQ960 REPRESENTATIVE CURVE AND STANDARDS (CONTINUED)

19:35:58 24-Sep-2003 0.05 ug/ml std.

8.00 9.00 10.00 11.00Time 0

100

B

0

100

A

09240312A Sm (Mn, SIR of 1 Channel ES-215.00 5.13e4

Area

9.375213

09240315A Sm (Mn, SIR of 1 Channel ES-215.00 9.09e4

Area

9.3410467

A IN-KQ960 Standard

B IN-KQ960 Standard

0.050 µg/mL Standard 0.10 µg/mL Standard

Peak Area: 5213 Peak Area: 10467

Analysis Date:

24 Sep 2003

Analysis Date:

24 Sep 2003

Set No.: 2 Set No.: 2

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FIGURE 5 CYMOXANIL - EXAMPLE CHROMATOGRAMS OF CONTROL AND FORTIFIED SPINACH SAMPLES

19:28:15 29-Sep-2003 Control Spinach

13.00 14.00 15.00 16.00Time 0

100

C

0

100

B

0

100

A

09260304B Sm (Mn, SIR of 1 Channel ES-197.00 1.09e4

13.73 13.28 14.07 14.32 14.74 15.08 15.68 16.56 16.15


Area

14.801175


Area

14.8111406

A Control B Cymoxanil LOQ Fort.

C Cymoxanil 10XLOQ Fort.

Peak Area- Peak Area: 1175 Peak Area: 11406

<0.0.05 ppm 0.044 ppm (89% Recovery)

4.30 ppm (86% Recovery)

Analysis Date:

29 Sep 2003

Analysis Date:

29 Sep 2003

Analysis Date:

29 Sep 2003


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FIGURE 6 IN-KQ960 - EXAMPLE CHROMATOGRAMS OF CONTROL AND FORTIFIED SPINACH SAMPLES

15:57:49 24-Sep-2003Control Spinach

8.00 9.00 10.00 11.00 Time 0

100

C

0

100

B

0

100

A

09240304A Sm (Mn, 3x1) SIR of 1 Channel ES-215.001.11e4

10.94 8.87 8.80 8.11 10.61 8.93 10.52 10.03


Area

9.38 924


Area

9.38 9608

A Control B IN-KQ960 LOQ Fort.

C IN-KQ960 10XLOQ Fort.

Peak Area- Peak Area: 924 Peak Area: 9608

<0.0.05 ppm 0.044 ppm (88% Recovery)

4.14 ppm (83% Recovery)

Analysis Date:

24 Sep 2003

Analysis Date:

24 Sep 2003

Analysis Date:

24 Sep 2003


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FIGURE 7 SIGNAL-TO-NOISE RATIOS

A) Cymoxanil

20:24:35 29-Sep-2003Spinach LOQ 1

13.00 14.00 15.00 16.00 Time 27

100

%

09260306B SIR of 1 Channel ES-197.002.00e4

Area 45

S/N=45/8=5.6

8

B) IN-KQ960

16:52:17 24-Sep-2003Spinach LOQ 1

8.00 9.00 10.00 11.00 Time 2

100

%

09240306A SIR of 1 Channel ES-215.002.00e4

Area 30

S/N=30/6=5

6

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FIGURE 8 CYMOXANIL LC/MS/MS CONFIRMATION

20-Nov-2003

13.00 14.00 15.00 16.00Time 0

100

B

0

100

A

11200302 MRM of 2 Channels ES197.00 > 42.00

6.76e3 Area

14.78 669

11200310 MRM of 2 Channels ES197.00 > 42.00

4.64e3 Area

14.79 474

A 0.10 ppm Cymoxanil Standard

B Cymoxanil 10 X LOQ

Fort. Spinach

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FIGURE 9 IN-KQ960 LC/MS/MS CONFIRMATION

14-Nov-2003

7.00 8.00 9.00 10.00 11.00 12.00Time 0

100

B

0

100

A

11140302 MRM of 1 Channel ES-214.80 > 140.00

1.42e3 Area

9.21 99

11140310 MRM of 1 Channel ES-214.80 > 140.00

1.30e3 Area

9.19 97

A 0.10 ppm IN-KQ960 Standard

B IN-KQ960 10 X LOQ

Fort. Spinach

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APPENDIX 1 LC/MS EXPERIMENTAL CONDITIONS

CYMOXANIL Acquisition Experiment Report File: g:\je874.pro\data\09260309b Header Acquired File Name: 09260309B Acquired Date: 29-Sep-2003 Acquired Time: 21:49:08 Job code: 092603CymoxanilValidationSet1 Task code: User Name: Administrator Laboratory Name: Lab Instrument: Inst Conditions: Submitter: SampleID: Spinach 10X LOQ 1 Bottle Number: 18 Description: Spinach 10X LOQ 1

Instrument Calibration Parameters MS1 Static: Mass 85 Da to 596 Da. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : STATMS1 MS1 Scanning: Mass 80 Da to 600 Da. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : SCNMS1 MS1 Scan Speed: Scan 64 to 473 amu/sec. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : FASTMS1 MS2 Static: Mass 85 Da to 596 Da. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : STATMS2 MS2 Scanning: Mass 80 Da to 600 Da. Resolution : 15.0/15.0 Ion Energy : 0.8

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Reference File : peghnh4 Acquisition File : SCNMS2

MS2 Scan Speed: Scan 64 to 473 amu/sec. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : FASTMS2 Calibration Time: 10:09 Calibration Date: 10/31/01 Coefficients MS1 Static: -0.000000000023*x^4 + 0.000000036395*x^3 + -0.000021182443*x^2 + 1.005305892780*x +-0.410940902914 MS2 Static: -0.000000000032*x^4 + 0.000000046280*x^3 + -0.000021460490*x^2 + 1.003089283521*x +-0.039381138254 Function 1: None

Instrument ID: OCP -v3.1_4 -QUAT2 4000 Tuning Parameters: ES- Source Page (ESI) Capillary: 3.50 kVolts HV Lens: 0.87 kVolts Cone: 15 Volts Skimmer Offset: 5 Volts Skimmer: 1.6 Volts RF Lens: 0.3 Volts Source Temp: 125 øC

MS1 Ion Energy: 2.0 Volts Ion Energy Ramp: 0.0 Volts LM Resolution: 15.0 HM Resolution: 15.0 Lens 5: 100 Volts Lens 6: 5 Volts Multiplier 1: 700 Volts

MS2 Ion Energy: 2.0 Volts Ion Energy Ramp: 0.0 Volts LM Resolution: 15.0 HM Resolution: 15.0 Lens 7: 250 Volts Lens 8: 40 Volts Lens 9: 0 Volts Multiplier: 700 Volts

Pressures Analyser Vacuum: 2.2e-5 mBar Gas Cell: 1.8e-3 mBar

Acquisition Threshold SIR or MRM Data Baseline level: 1.0 General Ion count threshold: 0

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Prescan Statistics Zero Level: 25 ADC zero: 82.54 ADC standard deviation: 1.14

Acquisition Threshold MS2

SIR or MRM Data Baseline level: 1.0 General Ion count threshold: 0 Prescan Statistics Zero Level: 24 ADC zero: 70.48 ADC standard deviation: 1.20

ACE Experimental Record

HP1100 LC Pump Initial Conditions Solvents A% 95.0 B% 5.0 C% 0.0 D% 0.0 Flow (ml/min) 1.000 Stop Time (mins) 27.0 Min Pressure (bar) 0 Max Pressure (bar) 400 Oven Temperature Left(°C) 40.0 Oven Temperature Right(°C) 40.0 HP1100LC Pump Gradient Timetable The gradient Timetable contains 5 entries which are :

Time A% B% C% D% Flow Pressure 0.00 95.0 5.0 0.0 0.0 1.000 400 17.00 50.0 50.0 0.0 0.0 1.000 400 19.99 10.0 90.0 0.0 0.0 1.000 400 22.00 95.0 5.0 0.0 0.0 1.000 400 27.00 95.0 5.0 0.0 0.0 1.000 400

HP1100 LC Pump External Event Timetable The Timetable contains 4 entries which are :

Time Column Switch Contact 1 Contact 2 Contact 3 Contact 4 Initial On Off Off Off Off 0.00 On Off Off On Off 8.00 On Off Off Off On 17.00 On On Off Off Off

HP1100 Autosampler Initial Conditions Injection Volume(µl) 25.0 Draw Speed 200.0 Eject Speed (µl/min) 200 Draw Position (mm) 0.00 Stop Time (mins) 27.00

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End of experimental record. Solvent Delay None Function 1 Scans in function: 10379 Cycle time (secs): 0.050 Inter Channel delay (secs): 0.00 Retention window (mins): 8.000 to 17.000 Ionization mode: ES- Data type: SIR or MRM data Function type: SIR of 1 channel Chan Mass Dwell(secs) Cone Volt. 1 : 197.00 0.02 17.0

IN-KQ960 Acquisition Experiment Report File: g:\je874.pro\data\09260309b

Header Acquired File Name: 09260309B Acquired Date: 29-Sep-2003 Acquired Time: 21:49:08 Job code: 092603CymoxanilValidationSet1 Task code: User Name: Administrator Laboratory Name: Lab Instrument: Inst Conditions: Submitter: SampleID: Spinach 10X LOQ 1 Bottle Number: 18 Description: Spinach 10X LOQ 1

Instrument Calibration Parameters MS1 Static: Mass 85 Da to 596 Da. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : STATMS1 MS1 Scanning: Mass 80 Da to 600 Da. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : SCNMS1 MS1 Scan Speed: Scan 64 to 473 amu/sec.

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Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : FASTMS1 MS2 Static: Mass 85 Da to 596 Da. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : STATMS2 MS2 Scanning: Mass 80 Da to 600 Da. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : SCNMS2 MS2 Scan Speed: Scan 64 to 473 amu/sec. Resolution : 15.0/15.0 Ion Energy : 0.8 Reference File : peghnh4 Acquisition File : FASTMS2 Calibration Time: 10:09 Calibration Date: 10/31/01 Coefficients MS1 Static: -0.000000000023*x^4 + 0.000000036395*x^3 + -0.000021182443*x^2 + 1.005305892780*x +-0.410940902914 MS2 Static: -0.000000000032*x^4 + 0.000000046280*x^3 + -0.000021460490*x^2 + 1.003089283521*x +-0.039381138254 Function 1: None

Instrument ID: OCP -v3.1_4 -QUAT2 4000 Tuning Parameters: ES- Source Page (ESI) Capillary: 3.50 kVolts HV Lens: 0.87 kVolts Cone: 15 Volts Skimmer Offset: 5 Volts Skimmer: 1.6 Volts RF Lens: 0.3 Volts Source Temp: 125 øC

MS1 Ion Energy: 2.0 Volts Ion Energy Ramp: 0.0 Volts LM Resolution: 15.0 HM Resolution: 15.0 Lens 5: 100 Volts Lens 6: 5 Volts Multiplier 1: 700 Volts

MS2 Ion Energy: 2.0 Volts Ion Energy Ramp: 0.0 Volts

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LM Resolution: 15.0 HM Resolution: 15.0 Lens 7: 250 Volts Lens 8: 40 Volts Lens 9: 0 Volt Multiplier: 700 Volts

Pressures Analyser Vacuum: 2.2e-5 mBar Gas Cell: 1.8e-3 mBar

Acquisition Threshold SIR or MRM Data Baseline level: 1.0 General Ion count threshold: 0 Prescan Statistics Zero Level: 25 ADC zero: 82.54 ADC standard deviation: 1.14 Acquisition Threshold MS2 SIR or MRM Data Baseline level: 1.0 General Ion count threshold: 0 Prescan Statistics Zero Level: 24 ADC zero: 70.48 ADC standard deviation: 1.20

ACE Experimental Record

--------------------- Run method parameters ----------------

HP1100 LC Pump Initial Conditions Solvents A% 95.0 B% 5.0 C% 0.0 D% 0.0 Flow (ml/min) 1.000 Stop Time (mins) 27.0 Min Pressure (bar) 0 Max Pressure (bar) 400 Oven Temperature Left(°C) 40.0 Oven Temperature Right(°C) 40.0

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HP1100 LC Pump Gradient Timetable

The gradient Timetable contains 5 entries which are :

Time A% B% C% D% Flow Pressure 0.00 95.0 5.0 0.0 0.0 1.000 400 17.00 50.0 50.0 0.0 0.0 1.000 400 19.99 10.0 90.0 0.0 0.0 1.000 400 22.00 95.0 5.0 0.0 0.0 1.000 400 27.00 95.0 5.0 0.0 0.0 1.000 400

HP1100 LC Pump External Event Timetable

The Timetable contains 4 entries which are :

Time Column Switch Contact 1 Contact 2 Contact 3 Contact 4 Initial On Off Off Off Off 0.00 On Off Off On Off 8.00 On Off Off Off On 17.00 On On Off Off Off

HP1100 Autosampler Initial Conditions

Injection Volume(µl) 25.0 Draw Speed 200.0 Eject Speed (µl/min) 200 Draw Position (mm) 0.00 Stop Time (mins) 27.00 Vial Number 18

---------------------------- oOo -----------------------------

End of experimental record.

Solvent Delay None Function 1 Scans in function: 10379 Cycle time (secs): 0.050 Inter Channel delay (secs): 0.00 Retention window (mins): 8.000 to 17.000 Ionization mode: ES- Data type: SIR or MRM data Function type: SIR of 1 channel Chan Mass Dwell(secs) Cone Volt. 1 : 197.00 0.02 17.0

analytical validation

Documents

statms2 ms2 scanning

fastms1 ms2 static

pressures analyser vacuum

statms1 ms1 scanning

peghnh4 acquisition file

total toxic residue

092603cymoxanilvalidationset1 task code

prior written consent