Analytical Validation

Cymoxanil +IN-KQ960, Leafy Veg.Only DuPont-13753 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 Page 1 of 44 DuPont-13753 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 2 DuPont-13753 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 3 DuPont-13753 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. 4 DuPont-13753 LIST OF ABBREVIATIONS AND SYMBOLS % °C APCI Cat. No. ESI HPLC LC/MS LOQ kg µg min MS/MS m/z n ng ppb ppm Rec RF RSD SAX sec SPE StDev percent degrees centigrade atmospheric pressure chemical ionization interface Catalog Number electrospray interface high performance liquid chromatography liquid chromatography/mass spectrometry limit of quantitation kilogram microgram minute tandem mass spectrometry (2-stage mass analysis experiment), MS2 mass/charge ratio number nanogram parts per billion parts per million recovery Response Factor (analyte peak area / analyte concentration) relative standard deviation (StDev / mean) Strong anion exchange second Solid phase extraction standard deviation 5 DuPont-13753 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 6 DuPont-13753 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 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Flow Diagram of Analytical Method....................................................24 Full Scan Spectrum for Cymoxanil and IN-KQ960 .............................26 Cymoxanil Representative Curve and Standards .................................27 IN-KQ960 Representative Curve and Standards..................................30 Cymoxanil - Example Chromatograms of Control and Fortified Spinach Samples...................................................................................33 IN-KQ960 - Example Chromatograms of Control and Fortified Spinach Samples...................................................................................34 Signal-to-Noise Ratios .........................................................................35 Cymoxanil LC/MS/MS Confirmation..................................................36 IN-KQ960 LC/MS/MS Confirmation ..................................................37 APPENDIX Appendix 1 LC/MS Experimental Conditions.........................................................38 7 DuPont-13753 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) 0.050 (LOQ) 0.50 CYMOXANIL IN-KQ960 AVG ± RSD (%) AVG% (RSD) 92 ± 4.3 88 ± 2.6 83 ± 3.6 83 ± 6.2 N 5 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 8 DuPont-13753 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. 9 DuPont-13753 3.1 Equipment Equipment Description Freezer Refrigerator Product ID Labline Frigid-Cab 6FAR Supplier Labline Instruments, Inc. (Melrose Park, IL) Marvel Industries, Inc. (Richmond, IN) Waring Products (New Hartford, CT) Commercial Food Processor Model 31FP93 Analytical Balance AE163 Dual Range Balance; PM460 Toploading Balance; PM400 Toploading Balance; AE163 Dual Mettler Instrument Corp. (Hightstown, NJ) Range Balance N-Evap Model 111 with stainless steel luer fit needles Tissumizer homogenizer Model SDT-20 equipped with Model SDT-182EN shaft (Teflon bearing) Bransonic 52 or 2200 Ultrasonic Cleaner, 0.75 gal. capacity Vortex Genie K-550-G or Vortex-2 Genie Gelman Acrodisc 13 CR, 0.2-µm PTFE 13 mm dia. membrane syringe filter, Cat. No. 4423 Supelclean ENVI-Carb SPE cartridge, 0.5 g/6 mL, Cat. No. 57094; Visiprep DL SPE Manifold, Cat. No. 5-7030M; 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; Sorvall Centrifuge, Model RC 3B Sorvall Centrifuge, Model GLC-2B(bench top) N-Evap Model 111(with stainless steel luer fit needles) Beckman φ 10 pH meter with combination pH electrode Organomation Assoc. (South Berlin, MA) Analytical Evaporator Homogenizer Tekmar Company (Cincinnati, OH) Sonication Vortex Mixer Filtration Branson Ultrasonics Corp. (Danbury, CT) VWR, Inc. (West Chester, PA) VWR (Bridgeport, NJ) Solid Phase Extraction Supelco (Bellefonte, PA) Solid Phase Extraction Varian, Inc. (Palo Alto, CA) Centrifuge Centrifuge Analytical Evaporator pH Meter Sorvall Instruments (Wilmington, DE) Sorvall Instruments (Wilmington, DE) Organomation Assoc. (South Berlin, MA.) 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; VWR (Bridgeport, NJ) 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 Electronic 1000-µL and 10-mL Pipettors Mechanical, positive displacement, 25-µL, 50-µL and 250-µL Pipettors Rainin (Walnut Creek, CA) Gilson Inc. (Middletown, WI) Labware Labware Labware Falcon 2098 (50 mL), 2096 (15 mL) Polypropylene Centrifuge Tubes; 3-mL Disposable Becton Dickinson (Franklin Lakes, NJ) Syringe, Cat. No. BD309585; 60-mL Disposable Syringe, Cat. No. BD309663 10 DuPont-13753 HPLC/MS System HP1100: G1322A degasser, G1312A binary pump, G1311A quaternary pump; G1313A autosampler; Agilent Technologies, Inc. (Palo Alto, CA) G1316A column unit; G1314A variable wavelength detector Target DP Amber Kit, T/S/T Septa, 100 PK, Cat. No. 5182-0556 Eclipse XDB-C8; 4.6 mm × 150 mm, 5 µm particle size diameter Agilent Technologies, Inc. (Palo Alto, CA) Agilent Technologies, Inc. (Palo Alto, CA) HPLC Autosampler Vials HPLC Column Splitter tee Switching Valve Valco zero dead-volume tee (split-flow to MS), Cat. Valco Instruments, Inc. (Houston, TX) No. ZT1C Valco 6 Port Electrically Actuated Valve, Cat. No. 1384 MicroMass Quattro II triple quadrupole mass spectrometer using an electrospray (ESI) or atmospheric chemical ionization (ACPI) interface and MassLynx NT version 3.1 software Valco Instruments, Inc. (Houston, TX) Triple Quadrupole MS Waters Corporation (Milford, MA) 3.2 3.2.1 Reagents and Standards Reagents The equivalency/suitability of substituted reagents should be verified. Reagents Formic Acid Methanol Hexane Acetonitrile Water Methylene Chloride Product Description GR, ACS, 98% OmniSolv, 4L OmniSolv, 4L OmniSolv, 4L OmniSolv, 4L OmniSolv, 1L Product ID FX0440-11 MX0488-1 HX0296-1 AX0142-1 WX0004-1 DX0831-6 Supplier EM Science (Gibbstown, NJ) EM Science (Gibbstown, NJ) EM Science (Gibbstown, NJ) EM Science (Gibbstown, NJ) EM Science (Gibbstown, NJ) 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: 11 DuPont-13753 N O N H3C O N H O N H CH3 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 Cymoxanil O HN O N C2H5 CONH2 NHOCH3 Molecular weight = 198.2 g/mole 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 KQ960 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 4.1 METHODS 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. 12 DuPont-13753 4.2 4.2.1 Analytical Procedure 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. 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. 4.2.2 13 DuPont-13753 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 0.050 0.010 0.0075 0.0050 0.10 0.05 x x x x x 0.10 0.075 0.05 4.2.6 Source (& Characterization) of Samples Spinach samples were purchased fresh from the local supermarket. 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. 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. 4.2.7 4.2.8 14 DuPont-13753 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. 15 DuPont-13753 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 4.3.1 Instrumentation 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. 16 DuPont-13753 System: Column: Column Temperature: Auto-sampler Temperature Injection Volume: Flow Rate: Conditions: Hewlett-Packard HP1100 HPLC 2.1 mm i.d. × 10 cm, Agilent Eclipse XDB-C8, 3 µm diameter packing 30°C 4°C 25-30 µL 1.0 ml/min A: 0.02% Formic Acid B: Methanol Time %A %B Time %A ------IN-KQ960--0.0 2.0 15.0 16.0 18.0 19.0 22.0 26.0 IN-KQ960 Retention Time: Cymoxanil Retention Time: Total Run Time: ~ 9.3 min ~ 14.8 min 27 min 98.0 98.0 80 1.0 1.0 70.0 98.0 98.0 2.0 2.0 20.0 99.0 99.0 30.0 2.0 2.0 %B ------Cymoxanil---0.0 17.0 19.9 22.0 27.00 95 50 10 95 95 5 50 90 5 5 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) 0.00-8.0 8.0-17.0 17-27 COLUMN ELUATE FLOW Waste MS source 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: 17 DuPont-13753 Micromass Quattro LC ESI-LC/MS Mass Spectrometer Conditions Analytes Cymoxanil IN-KQ960 Electrospray Voltage: Detector Voltage: Nebulizing Gas Flow: Drying Gas Flow: Ions Monitored 197.0 ± 0.1 AMU 215.0 ± 0.1 AMU 3.50 kV 700 V 15 L/h 300 L/h Cone Voltage 17V 15V Source Temp. 125°C 125°C Dwell (Seconds) 0.02 0.02 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. Calculations Methods The response factor, RF, for each analytical standard is the ratio of the analyte concentration to the analyte peak area. RFstd = Concentration of analyte (µg/mL) Analyte peak area 4.4 4.4.1 18 DuPont-13753 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. RFave = (RFstd1 + RFstd 2 + RFstd3 + .......RFstdn ) Total Number of Standards Injected The concentration (µg/g or ppm) of analyte found in each sample was calculated as follows: µg/g analyte Found = Where: Total Extract Volume (mL solv) Final Extract Volume (Final Vol.) Aliquot Taken Sample Weight = = = = 180 mL 5.0 mL 4.0 mL 20.0 grams [ Peak Area x RFave] x [ Final Vol. (mL) x mL solv ] Sample Wt. (g) x Aliquot Taken (mL) The percent recovery found was calculated as follows: % Recovery = (µg/g Found) x 100% (Fortification level, µg/g) 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: RFstd = 0.005 ( µ g/mL) 784 = 0.00638 µ g/mL RFave = (6.38E- 6) + (6.60E- 6) + (6.59E- 6) + (6.81E- 6) + (6.72E- 6) + (6.71E − 6) + (6.69 - E6) 7 RFave = 6.64E-6 µg/mL Peak Area Cymoxanil: mL Solvent: mL Aliquot 1: Final Volume: = = = = 1175 180 mL 8.0 mL 5.0 mL 19 DuPont-13753 Sample Weight: ppm Cymoxanil = % Recovery = = 20.0 grams (1175 x 6.64E - 6 ug/mL) x (5.0 mL x 180 mL ) = 0.0439 µg/g 8.0mL × 20.0 g 0.0439 µg/g x 100% = 88% 0.050 µg/g 5.0 5.1 5.1.1 RESULTS AND DISCUSSION Method Validation Results 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. 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) 0.050 (LOQ) 0.50 Overall Mean Cymoxanil IN-KQ960 Avg ± RSD (%) Avg ± RSD (%) 92 ± 4.3 88 ± 2.6 90 ± 4.3 83 ± 3.6 83 ± 6.2 83 ± 4.8 n 5 5 10 5.1.3 20 DuPont-13753 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. 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. 5.1.5 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. 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. Method Ruggedness 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. 5.3 5.4 5.4.1 21 DuPont-13753 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. 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). 5.4.3 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. 22 DuPont-13753 TABLE 1 SUMMARY OF CYMOXANIL AND IN-KQ960 FORTIFICATION (RECOVERY) DATA IN SPINACH Fortification Level (ppm)1 0.050 0.050 0.050 0.050 0.050 Cymoxanil PK Area 1175 1294 1274 1199 1217 ppm 0.0439 0.0483 0.0479 0.0450 0.0457 % Recovery 88 2 IN-KQ960 PK Area 992 966 924 867 884 ppm 0.429 0.0418 0.0420 0.0394 0.0402 % Recovery 86 84 84 79 80 83 ± 3.0 3.6 Set No. MV1-LOQ1 MV1-LOQ2 MV2-LOQ1 MV2-LOQ2 MV2-LOQ3 97 96 90 91 92 ± 3.9 4.3 Mean % Recovery ± SD (n = 4) = RSD = MV1-10LOQ1 MV1-10LOQ2 MV1-10LOQ3 MV2-10LOQ1 MV2-10LOQ2 0.50 0.50 0.50 0.50 0.50 11406 12168 12084 11493 11660 0.426 0.454 0.451 0.432 0.446 85 91 90 86 88 88 ± 2.3 2.6 90 ± 3.9 4.3 8970 9137 9431 9608 9861 0.388 0.395 0.408 0.437 0.449 78 79 82 87 90 83 ± 5.2 6.2 83 ± 4.0 4.8 Mean % Recovery ± SD (n = 3) = RSD = Overall Mean % Recovery ± SD (n = 2) = RSD = 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. Additional data necessary to calculate % recoveries (see calculation on page 19) Extract Vol. Aliquot Final Vol. Res. Factor(MV-1) sample wt. Cymoxanil 20.0g 180ml 8.0ml 5.0ml 0.00664 IN-KQ960 20.0g 180ml 8.0ml 4.0ml 0.00962 Res. Factor(MV-2) 0.00668 0.01011 2 23 DuPont-13753 FIGURE 1 FLOW DIAGRAM OF ANALYTICAL METHOD Cymoxanil Extraction 1) To 20g sample add 60ml H20 and 120ml ACN 2) Blend withTissuemizer for 5 minutes, centrifuge 10 minutes Salt Out 1) 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 SAX SPE and Hexane L/L Extraction 1) Condition a SAX SPE, pass sample thru column 2) Pass an additional 3 ml thru column and evaporate to 9ml 3) Add 5 ml hexane to sample, shake, remove hexane, repeat Envi Carb SPE and Analysis 1) 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 ml 3) Dilute to 4ml with 0.02% Formic acid, sonicate and filter into LC vial 24 DuPont-13753 FIGURE 1 FLOW DIAGRAM OF ANALYTICAL METHOD (CONTINUED) IN-KQ960 Extraction 1) To 20g sample add 60ml H20 and 120ml ACN 2) Blend withTissuemizer for 5 minutes, centrifuge 10 minutes Hexane L/L Extraction and SAX SPE 1) 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 Envi Carb SPE and Analysis 1) 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 ml 3) Dilute to 4ml with 0.02% Formic acid, sonicate and filter into LC vial 25 DuPont-13753 FIGURE 2 FULL SCAN SPECTRUM FOR CYMOXANIL AND IN-KQ960 A) Cymoxanil 10.0ug/ml std. 11190307 371 (14.784) Cm (369:371-(169:264+444:475)x3.000) 196 100 197 % 41.5 0 40 60 80 100 120 140 242 180 200 220 240 260 280 300 320 340 360 380 m/z 400 Scan ES2.64e5 166 160 B) IN-KQ960 10.0ug/ml std. 11130307 117 (9.235) Cm (106:119-(2:58+166:254)x4.000) 215 100 1: Scan ES1.11e4 % 140 0 50 100 150 200 216 261 250 300 350 m/z 400 26 DuPont-13753 FIGURE 3 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS Compound 1 name: Cymoxanil Correlation coefficient: r = 0.999990, r^2 = 0.999979 Calibration curve: 149538 * x + 3.13622 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None 1.50e4 Response 0 0.0 Conc 0.1 27 DuPont-13753 FIGURE 3 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS (CONTINUED) 18:03:48 09260301B Sm (Mn, 3x1) 100 A 0 09260302B Sm (Mn, 3x1) 100 B 0 09260305B Sm (Mn, 3x1) 100 C 0 14.81 1469 14.80 1138 14.79 758 29-Sep-2003 SIR of 1 Channel ES197.00 1.52e4 Area SIR of 1 Channel ES197.00 1.82e4 Area SIR of 1 Channel ES197.00 2.16e4 Area 13.00 14.00 15.00 16.00 Time A Cymoxanil Standard B Cymoxanil Standard C Cymoxanil Standard 0.01 µg/mL Standard Peak Area: 1469 Analysis Date: 29 Sep 2003 Set No.: 1 0.005 µg/mL Standard Peak Area: 758 Analysis Date: 29 Sep 2003 Set No.: 1 0.0075 µg/mL Standard Peak Area: 1138 Analysis Date: 29 Sep 2003 Set No.: 1 28 DuPont-13753 FIGURE 3 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS (CONTINUED) 23:13:45 09260312B Sm (Mn, 3x1) 100 14.81 7448 29-Sep-2003 SIR of 1 Channel ES197.00 7.06e4 Area A 0 09260315B Sm (Mn, 3x1) 100 14.79 14974 SIR of 1 Channel ES197.00 1.34e5 Area B 0 13.00 14.00 15.00 16.00 Time A Cymoxanil Standard B Cymoxanil Standard 0.050 µg/mL Standard Peak Area: 7448 Analysis Date: 29 Sep 2003 Set No.: 1 0.10 µg/mL Standard Peak Area: 14974 Analysis Date: 29 Sep 2003 Set No.: 1 29 DuPont-13753 FIGURE 4 IN-KQ960 REPRESENTATIVE CURVE AND STANDARDS Compound 1 name: KQ960 Correlation coefficient: r = 0.999953, r^2 = 0.999906 Calibration curve: 105285 * x + -60.5998 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None 1.05e4 Response -60.6 0.0 Conc 0.1 30 DuPont-13753 FIGURE 4 IN-KQ960 REPRESENTATIVE CURVE AND STANDARDS (CONTINUED) 0.005ug/ml std. 09240300A Sm (Mn, 3x1) 100 A 0 09240302A Sm (Mn, 3x1) 100 B 0 09240305A Sm (Mn, 3x1) 100 C 0 14:10:08 9.43 537 24-Sep-2003 SIR of 1 Channel ES215.00 1.49e4 Area 9.41 691 SIR of 1 Channel ES215.00 1.57e4 Area 9.39 963 SIR of 1 Channel ES215.00 1.66e4 Area 8.00 9.00 10.00 11.00 Time A IN-KQ960 Standard B IN-KQ960 Standard C IN-KQ960 Standard 0.01 µg/mL Standard Peak Area: 963 Analysis Date: 24 Sep 2003 Set No.: 2 0.005 µg/mL Standard Peak Area: 537 Analysis Date: 24 Sep 2003 Set No.: 2 0.0075 µg/mL Standard Peak Area: 691 Analysis Date: 24 Sep 2003 Set No.: 2 31 DuPont-13753 FIGURE 4 IN-KQ960 REPRESENTATIVE CURVE AND STANDARDS (CONTINUED) 0.05 ug/ml std. 09240312A Sm (Mn, 100 19:35:58 9.37 5213 24-Sep-2003 SIR of 1 Channel ES215.00 5.13e4 Area A 0 09240315A Sm (Mn, 100 9.34 10467 SIR of 1 Channel ES215.00 9.09e4 Area B 0 8.00 9.00 10.00 11.00 Time A IN-KQ960 Standard B IN-KQ960 Standard 0.050 µg/mL Standard Peak Area: 5213 Analysis Date: 24 Sep 2003 Set No.: 2 0.10 µg/mL Standard Peak Area: 10467 Analysis Date: 24 Sep 2003 Set No.: 2 32 DuPont-13753 FIGURE 5 CYMOXANIL - EXAMPLE CHROMATOGRAMS OF CONTROL AND FORTIFIED SPINACH SAMPLES Control Spinach 19:28:15 29-Sep-2003 SIR of 1 Channel ES197.00 15.68 16.15 16.56 1.09e4 09260304B Sm (Mn, 13.28 13.73 14.07 14.32 14.74 15.08 100 A 0 09260306B Sm (Mn, 100 B 0 09260309B Sm (Mn, 100 C 0 13.00 14.00 15.00 14.80 1175 SIR of 1 Channel ES197.00 1.79e4 Area 14.81 11406 SIR of 1 Channel ES197.00 1.05e5 Area 16.00 Time A Control B Cymoxanil LOQ Fort. C Cymoxanil 10XLOQ Fort. Peak Area: 11406 4.30 ppm (86% Recovery) Analysis Date: 29 Sep 2003 Set No.: 1 Peak Area<0.0.05 ppm Analysis Date: 29 Sep 2003 Set No.: 1 Peak Area: 1175 0.044 ppm (89% Recovery) Analysis Date: 29 Sep 2003 Set No.: 1 33 DuPont-13753 FIGURE 6 IN-KQ960 - EXAMPLE CHROMATOGRAMS OF CONTROL AND FORTIFIED SPINACH SAMPLES Control Spinach 09240304A Sm (Mn, 3x1) 100 A 0 09240306A Sm (Mn, 3x1) 100 B 0 09240310A Sm (Mn, 3x1) 100 C 0 8.11 8.80 15:57:49 8.87 8.93 24-Sep-2003 SIR of 1 Channel ES215.00 10.03 10.52 10.61 10.94 1.11e4 9.38 924 SIR of 1 Channel ES215.00 1.66e4 Area 9.38 9608 SIR of 1 Channel ES215.00 9.16e4 Area 8.00 9.00 10.00 11.00 Time A Control Peak Area- B IN-KQ960 LOQ Fort. C IN-KQ960 10XLOQ Fort. Peak Area: 9608 4.14 ppm (83% Recovery) Analysis Date: 24 Sep 2003 Set No.: 2 Peak Area: 924 0.044 ppm (88% Recovery) Analysis Date: 24 Sep 2003 Set No.: 2 <0.0.05 ppm Analysis Date: 24 Sep 2003 Set No.: 2 34 DuPont-13753 FIGURE 7 A) Cymoxanil SIGNAL-TO-NOISE RATIOS Spinach LOQ 1 09260306B 20:24:35 29-Sep-2003 SIR of 1 Channel ES197.00 2.00e4 Area 45 100 S/N=45/8=5.6 % 8 27 13.00 14.00 15.00 16.00 Time B) IN-KQ960 Spinach LOQ 1 09240306A 16:52:17 24-Sep-2003 SIR of 1 Channel ES215.00 2.00e4 Area 30 100 S/N=30/6=5 % 6 2 8.00 9.00 10.00 11.00 Time 35 DuPont-13753 FIGURE 8 CYMOXANIL LC/MS/MS CONFIRMATION 20-Nov-2003 11200302 100 14.78 669 MRM of 2 Channels ES 197.00 > 42.00 6.76e3 Area A 0 11200310 100 14.79 474 MRM of 2 Channels ES 197.00 > 42.00 4.64e3 Area B 0 13.00 14.00 15.00 16.00 Time A 0.10 ppm Cymoxanil Standard B Cymoxanil 10 X LOQ Fort. Spinach 36 DuPont-13753 FIGURE 9 IN-KQ960 LC/MS/MS CONFIRMATION 14-Nov-2003 11140302 100 9.21 99 MRM of 1 Channel ES214.80 > 140.00 1.42e3 Area A 0 11140310 100 9.19 97 MRM of 1 Channel ES214.80 > 140.00 1.30e3 Area B 0 7.00 8.00 9.00 10.00 11.00 12.00 Time A 0.10 ppm IN-KQ960 Standard B IN-KQ960 10 X LOQ Fort. Spinach 37 DuPont-13753 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 38 DuPont-13753 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: ESSource 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 Ion Energy Ramp: 0.0 LM Resolution: 15.0 HM Resolution: 15.0 Lens 5: 100 Lens 6: 5 Multiplier 1: 700 MS2 Ion Energy: Ion Energy Ramp: LM Resolution: HM Resolution: Lens 7: Lens 8: Lens 9: Multiplier: Pressures Analyser Vacuum: Gas Cell: 2.0 0.0 15.0 15.0 250 40 0 700 2.2e-5 1.8e-3 Volts Volts Volts Volts Volts Volts Volts Volts Volts Volts Volts mBar mBar Acquisition Threshold SIR or MRM Data Baseline level: General Ion count threshold: 1.0 0 39 DuPont-13753 Prescan Statistics Zero Level: ADC zero: ADC standard deviation: 25 82.54 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% B% C% D% Flow (ml/min) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Left(°C) Oven Temperature Right(°C) 95.0 5.0 0.0 0.0 1.000 27.0 0 400 40.0 40.0 HP1100LC Pump Gradient Timetable The gradient Timetable contains 5 entries which are : Time 0.00 17.00 19.99 22.00 27.00 A% 95.0 50.0 10.0 95.0 95.0 B% 5.0 50.0 90.0 5.0 5.0 C% 0.0 0.0 0.0 0.0 0.0 D% 0.0 0.0 0.0 0.0 0.0 Flow 1.000 1.000 1.000 1.000 1.000 Pressure 400 400 400 400 400 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) Draw Speed Eject Speed (µl/min) Draw Position (mm) Stop Time (mins) 25.0 200.0 200 0.00 27.00 40 DuPont-13753 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: ESData 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. 41 DuPont-13753 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: ESSource 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 Ion Energy Ramp: 0.0 LM Resolution: 15.0 HM Resolution: 15.0 Lens 5: 100 Lens 6: 5 Multiplier 1: 700 MS2 Ion Energy: 2.0 Ion Energy Ramp: 0.0 Volts Volts Volts Volts Volts Volts Volts 42 DuPont-13753 LM Resolution: HM Resolution: Lens 7: Lens 8: Lens 9: Multiplier: Pressures Analyser Vacuum: Gas Cell: Acquisition Threshold SIR or MRM Data Baseline level: 15.0 15.0 250 40 0 700 Volts Volts Volt Volts 2.2e-5 1.8e-3 mBar mBar 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% B% C% D% Flow (ml/min) Stop Time (mins) Min Pressure (bar) Max Pressure (bar) Oven Temperature Left(°C) Oven Temperature Right(°C) 95.0 5.0 0.0 0.0 1.000 27.0 0 400 40.0 40.0 43 DuPont-13753 HP1100 LC Pump Gradient Timetable The gradient Timetable contains 5 entries which are : Time 0.00 17.00 19.99 22.00 27.00 A% 95.0 50.0 10.0 95.0 95.0 B% 5.0 50.0 90.0 5.0 5.0 C% 0.0 0.0 0.0 0.0 0.0 D% 0.0 0.0 0.0 0.0 0.0 Flow 1.000 1.000 1.000 1.000 1.000 Pressure 400 400 400 400 400 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) Draw Speed Eject Speed (µl/min) Draw Position (mm) Stop Time (mins) Vial Number ---------------------------- 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: ESData type: SIR or MRM data Function type: SIR of 1 channel Chan Mass Dwell(secs) Cone Volt. 1 : 197.00 0.02 17.0 25.0 200.0 200 0.00 27.00 18 44