Transcript
Cymoxanil +IN-KQ960, Leafy Veg. Only DuPont-13753
TRADE SECRET
Study Title
ANALYTICAL METHOD FOR THE DETERMINATION OF C YMOXANIL 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. 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 was conducted in a GLP compliant facility facility following following Standard Operating Procedures. 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 C YMOXANIL 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 BBREVIATIONS AND S YMBOLS %
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 MS/MS
minute tandem mass spectrometry (2-stage (2-stage mass analysis experiment), MS
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
2
5
DuPont-13753
TABLE OF CONTENTS Title Page ...................................................... ............................................................................................................. .................................................................1 ..........1 Page Reserved................................................................................................................2 Good Laboratory Practice Statement ................................................... ..........................3 Certification ....................................................... .............................................................................................................. ............................................................4 .....4 List of Abbreviations and Symbols................................................................................5 Table of Contents...........................................................................................................6 1.0 Abstract ...................................................... .................................................................................................................8 ...........................................................8 2.0 Introduction ................................................... ....................................................... ........................................................8 .8 3.0 Materials................................................................................................................9 3.1 Equipment ............................................. ................................................... ......10 3.2 Reagents and Standards...................................................... Standards ..................................................................................11 ............................11 3.2.1 Reagents ................................................ ................................................11 3.2.2 Reference Analytical Standards............................................ Standards.............................................................11 .................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 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 ................................................... .................................................1 8 4.4.1 Methods ................................................. ................................................18 4.4.2 Example.................................................................................................19 5.0 Results and Discussion........................... ................................................... ..........20 5.1 Method Validation Results................................................... 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.................................................. 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 .......................................22 8.0 References .................................................. .........................................................22
TABLE Table 1
Summary of Cymoxanil and IN-KQ960 Fortification (Recovery) Data in Spinach ..................................................... ....................................................................................23 ...............................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 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.......................................... Confirmation. ......................................... ........36
Figure 9
IN-KQ960 LC/MS/MS Confirmation ......................................... .........37
APPENDIX Appendix 1
LC/MS Experimental Conditions ............................................ .............38
7
DuPont-13753
ANALYTICAL METHOD FOR THE DETERMINATION OF C YMOXANIL 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 ex tracted from samples of spinach with a mixture of acetonitrile 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, discarded, and the acetonitrile layer layer containing cymoxanil was 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 analysis of the metabolite. The extract is cleaned up using a hexane liquid/liquid extraction followed by passing through SAX and Envi Ca rb SPE columns. IN-KQ960 is not retained on either of these these columns The LOQ by LC/MS analysis was determined to be 0.050 µg/g (ppm) for both cymoxanil and IN-KQ960. 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)
CYMOXANIL IN-KQ960 AVG RSD (%) 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 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 metabolite identified in a cymoxanil 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. acetonitrile/water. The metabolism study (Reference 1) demonstrated that the acetonitrile/water mixture, used in this residue method, extracts the total total toxic residue from the the lettuce matrix. Additional sample cleanup is performed with hexane liquid/liquid extraction, SAX and Envi-Carb SPE columns. Analysis is is performed by LC/MS 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
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 Mettler Instrument Corp. (Hightstown, NJ) Range Balance
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; 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 m m rubber bulbs, Cat. No. 56310-240
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 Becton Dickinson (Franklin Lakes, NJ) Syringe, Cat. No. BD309585; 60-mL Disposable Syringe, Cat. No. BD309663
10
DuPont-13753
HPLC/MS System
HPLC
HP1100: G1322A degasser, G1312A binary pump, G1311A quaternary pump; G1313A autosampler; Agilent Technologies, Inc. (Palo Alto, CA) G1316A column unit; G1314A variable wavelength detector
Autosampler Vials
Target DP Amber Kit, T/S/T Septa, 100 PK, Cat. No. 5182-0556
Agilent Technologies, Inc. (Palo Alto, CA)
HPLC Column
Eclipse XDB-C8; 4.6 mm × 150 mm, 5 µm particle size diameter
Agilent Technologies, Inc. (Palo Alto, CA)
Splitter tee
Valco zero dead-volume tee (split-flow to MS), Cat. Valco Instruments, Inc. (Houston, TX) No. ZT1C
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 s oftware
Waters Corporation (Milford, MA)
3.2
Reagents and Standards
3.2.1
Reagents The equivalency/suitability of substituted reagents should be verified.
3.2.2
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)
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
DPX-T3217
CAS Chemical Name:
N H
O N
DuPont Code:
O N H
CH3
2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyim 2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyimino)acetamide ino)acetamide
CAS Registry Number:
H3C
57966-95-7
IUPAC Chemical Name: 1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea
Cymoxanil O HN
DuPont Code: N
C2H5 CONH2
O
NHOCH3
KQ960 3.3
Molecular weight =
198.2 g/mole
IN-KQ960
CAS Chemical Name: 3-Ethyl-4-(methoxyamino)-2,5-dioxo-4-imidazolidinecarboxamide
CAS Registry Number: none
Molecular weight =
216 g/mole
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 the current study include the addition of IN-KQ960 as an analyte. analyte. Also, LC/MS is used for the the analysis of both cymoxanil and IN-KQ960. IN-KQ960. Based on the sensitivity and selectivity of LC/MS, 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, discarded, and the acetonitrile layer layer containing cymoxanil is passed passed through a SAX SPE column. The extract is then further 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 analysis of the metabolite. The extract is cleaned up using a hexane liquid/liquid extraction followed by passing through SAX and Envi Ca rb SPE columns. IN-KQ960 is not retained on either of these these columns.
12
DuPont-13753 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 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. 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 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 solution to a volume of 100 mL, using HPLC-grade methanol and invert the volumetric flask to mix the solution to homogeneity. homogeneity. These standard solutions are stable for approximately 6 months when stored at approximately 4° 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 are stable for approximately 6 months when stored at approximately 4° 4°C immediately after each use.
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-µ 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° 4°C immediately after each use.
4.2.6
Desired Standard
Volume of 10.0 g/mL Intermediate
Volume of 1.0 g/mL Intermediate
Concentration ( g/mL)
Standard Required (mL)
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
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. analysis. In preparation for analysis, 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 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 fortified at the 0.50 ppm (10XLOQ) level, 1.0 mL of the 10.0 µg/mL intermediate standard was used.
14
DuPont-13753 4.2.9
Analyte Extraction Procedure 1. Weigh out 20 grams of Spinach into into a 250-mL centrifuge bottle. 2. For fresh fortification samples, samples, spike sample at the appropriate appropriate level and let let sit for 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 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 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. again. 7. Condition a 1-gram/6 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 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 the N-Evap to be evaporated to about 0.75 mL at 38 to 40° 40°C. (After every 3 to 4 mL 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 o f 4mL with 0.02% formic acid, cap, vortex, and sonicate for 2 minutes. Filter through a syringe syringe filter and analyze analyze by LC/MS.
15
DuPont-13753 4.2.11
IN-KQ960 Purification Procedure 1. Take the 8-mL aliquot from from the extraction and add 5 mL of hexane. Shake vigorously for 1 minute and then centrifuge centrifuge at 3000 rpm for 5 minutes. Remove the upper layer (hexane) and discard. 2. Repeat Step1. Discard top hexane layer. layer. Save lower layer (water and acetonitrile). 3. Condition a 1-gram/6 mL SAX cartridge cartridge using 5 mL of acetonitrile. acetonitrile. Discard this conditioning solution. 4. Add the sample from Step 2 to the cartridge, 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 methylene chloride/10% acetonitrile. acetonitrile. Discard solution. 6. Add the sample (from Step 4) to the column and collect this elution elution in another 15-mL centrifuge 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 the N-Evap to be evaporated to about 0.75 mL at 38 to 40° 40°C. (After every 3 to 4 mL 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 formic acid, cap, vortex, vortex, and sonicate for 2 minutes. 9. Filter through through a syringe filter and analyze 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 from co-extractants. An Agilent Eclipse XDB-C8 HPLC column was selected. The column choice reflected experimental results indicating preferred preferred separation of cymoxanil 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:
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
------Cymoxanil----
%A
%B
------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:
~ 9.3 min
Cymoxanil Retention Time:
~ 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 prior to and following elution of the compounds of interest. The use of this valve reduced source contamination and enab led additional samples to be analyzed before the ion source required cleaning. 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) (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
Ions Monitored
Cone Voltage
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: Drying Gas Flow:
Source Temp.
Dwell (Seconds)
15 L/h 300 L/h
A complete list of the experimental parameters is 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. quantitation. For confirmation 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-µ 0.01-µg/mL chromatographic standard should be analyzed prior to the start of analyses to establish that the instrument is working properly. 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. analysis. Operating parameters must be tailored 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 e liminated when utilizing this method on other instrumentation. Each ion channel used for sample sample analysis/quantitation analysis/quantitation must be checked to insure it is free of interference. interference. The control will be used to demonstrate that baseline interference is is less than signal-to-noise signal-to-noise 3:1. Begin each sample set by injecting a minimum of 2 calibration standards. The first injection should always 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.
RFstd =
Concentration of analyte (µg/mL) Analyte peak area
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 (µ (µg/g or ppm) of analyte found in each sample was calculated as follows: µg/g analyte Found =
[ P eak Area x RFave] x [ Final Vol . (mL) x mL solv ] Sample Wt. (g) x Aliquot Taken (mL)
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
The percent recovery found was calculated as follows: % Recovery =
4.4.2
(µg/g Found) (Fortification level, µg/g)
x 100%
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) + (6.71 E − 6) + (6.69- E6) 7 RFave = 6.64E-6
µ g/mL
Peak Area Cymoxanil:
=
1175
mL Solvent:
=
180 mL
mL Aliquot 1:
=
8.0 mL
Final Volume:
=
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 ) 8.0mL × 20.0 g
0.0439 µg/g 0.050 µg/g
= 0.0439 µg/g
x 100% = 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 cymoxanil and IN-KQ960. Typical experimental conditions for each analyte are provided in Appendix 1. Typical LC/MS 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 o f 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 analyzed to verify method performance. performance. The fortification levels tested bracketed the range of residue values expected in treated samples encountered from the field. All results are are provided in Table 1 and summarized summarized below: Average Recovery Level (ppm)
Cymoxanil IN-KQ960 Avg RSD (%) 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
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 IN-KQ960 from 10 freshly fortified control spinach samples was 83% ± 4.8% (RSD). Unfortified control samples showed no quantifiable qu antifiable residues of cymoxanil and IN-KQ960. 5.1.4
Extraction Efficiency 14
In metabolism studies with radiolabeled C 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 surface wash fraction. The acetonitrile/water mixture used in the residue method 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 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 0.0 2 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 analyses were run unattended overnight. overnight. The sample extraction and cleanup procedure is the rate-determining step. 5.3
Modifications or Special Precautions
The analysis of cymoxanil cymoxanil is sensitive to mobile phase pH. LC systems systems that have previously run alkaline mobile phases must b e 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 stable for a minimum of two weeks when stored in a refrigerator when not in use.
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 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→ 197→42 and for IN-KQ960 (M-1) 215→ 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 solvents versus fortified fortified spinach matrix. Therefore, these transitions are adequate to confirm co nfirm 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 “Metabolism of [2-14C]Cymoxanil in Lettuce”; 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, III, “Analytical “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 C YMOXANIL AND IN-KQ960 FORTIFICATION (RECOVERY) DATA IN SPINACH Cymoxanil
IN-KQ960
Fortification 1
Set No.
Level (ppm)
PK Area
MV1-LOQ1
0.050
1175
0.0439
88
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
SD (n = 4) = RSD =
92 ± 3.9 4.3
Mean % Recovery
ppm
% Recovery 2
PK Area
ppm
% Recovery
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 in spinach was 0.050 ppm. Residue values values carried to an excessive number of significant figures were used to calculate % Recovery. After calculation, calculation, % Recovery values were rounded to the nearest whole number and reported.
2
Additional data data necessary necessary to calculate calculate % recoveries (see calculation on page 19) sample wt. Extract Vol. Aliquot Final Vol. Res. Factor(MV-1) 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
23
DuPont-13753
FIGURE 1
FLOW DIAGRAM OF ANALYTICAL METHOD
Cymoxanil Extraction 1) To 20g sample add 60ml H 20 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. 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 col umn rinse with 4 ml 90/10, evap to 0.75 ml 3) Dilute to 4ml with 0.02% Formic acid, sonic ate 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 H 20 and 120ml ACN 2) Blend withTissuemize withTissuemizerr for 5 minutes, centrifuge 10 minutes
Hexane L/L Extraction and SAX SPE 1) To 8.0 ml extract, extract, add 5 ml hexane, shake, shake, remove hexane, repeat 2) Condition a SAX SPE, pass sample thru column 3) Pass an additional 3 ml thru colu mn
Envi Carb SPE and Analysis 1) Condition a Envi Carb SPE, with 5 ml of 90/10 MeCl2/MeOH 2) Pass sample thru column col umn rinse with 4 ml 90/10, evap to 0.75 ml 3) Dilute to 4ml with 0.02% Formic acid, sonic ate and filter into LC vial
25
DuPont-13753
FIGURE 2
FULL SCAN SPECTRUM FOR C YMOXANIL AND IN-KQ960
A) Cymoxanil
10.0ug/ml std. 1 1190307 371 (14.784) Cm (369:371-(169:264+444:475)x3.000)
Scan ES2.64e5
196
100
197 %
41.5
242
166
0 40
60
80
100
120
140
160
180
200
220
240
260
280
300
320 340
360
380
m/z 400
B) IN-KQ960
10.0ug/ml std. 11130307 117 (9.235) C m (106:119-(2:58+166:254)x4.000) 215 100
1: Scan ES1.11e4
% 216
140
261
0 50
100
150
200
2 50
300
350
m/z 400
26
DuPont-13753
FIGURE 3
C YMOXANIL REPRESENTATIVE CURVE AND STANDARDS
Compound 1 name: C ymoxanil ymoxanil Correlation coefficient: r = 0.999990, r^2 = 0.999979 Calibration curve: 149538 * x + 3.13622 Response type: External Std, Area Curve type: Linear, Origi n: Exclude, Exclude, Wei ghting: Null, Axis trans: None 1.50e4
Response
0 0.0
Conc 0.1
27
DuPont-13753
FIGURE 3
C YMOXANIL REPRESENTATIVE CURVE AND STANDARDS (CONTINUED )
18:03:48 09260301B Sm (Mn, 3x1) 100
14.79 758
SIR of 1 Channel ES197.00 1.52e4 Area
14.80 1138
SIR of 1 Channel ES197.00 1.82e4 Area
14.81 1469
SIR of 1 Channel ES197.00 2.16e4 Area
A 0 09260302B Sm (Mn, 3x1) 100
29-Sep-2003
B 0 09260305B Sm (Mn, 3x1) 100 C 0 13.00
14.00
A
Cymoxanil Standard
15.00
B
Cymoxanil Standard
16.00
Time
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:
Analysis Date:
Analysis Date:
29 Sep 2003
29 Sep 2003
29 Sep 2003
Set No.: 1
Set No.: 1
Set No.: 1
28
DuPont-13753
FIGURE 3
C YMOXANIL REPRESENTATIVE CURVE AND STANDARDS (CONTINUED )
23:13:45
29-Sep-2003
09260312B Sm (Mn, 3x1) 100
14.81 7448
SIR of 1 Channel ES197.00 7.06e4 Area
14.79 14974
SIR of 1 Channel ES197.00 1.34e5 Area
A
0 09260315B Sm (Mn, 3x1) 100
B
0 13.00
14.00
A
15.00
Cymoxanil Standard
16.00
B
Time
Cymoxanil Standard
0.050 µg/mL Standard
0.10 µg/mL Standard
Peak Area: 7448
Peak Area: 14974
Analysis Date:
Analysis Date:
29 Sep 2003
29 Sep 2003
Set No.: 1
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 Calibratio n curve: curve: 105285 * x + -60.5998 Response type: External Std, Area Curve type: Linear, Origi n: 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.
14:10:08
24-Sep-2003
09240300A Sm (Mn, 3x1) 100
9.43 537
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
A 0 09240302A Sm (Mn, 3x1) 100 B 0 09240305A Sm (Mn, 3x1) 100 C 0 8.00
A
IN-KQ960 Standard
9.00
10.00
B
IN-KQ960 Standard
11.00
Time
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:
Analysis Date:
Analysis Date:
24 Sep 2003
24 Sep 2003
24 Sep 2003
Set No.: 2
Set No.: 2
Set No.: 2
31
DuPont-13753
FIGURE 4
IN-KQ960 REPRESENTATIVE CURVE AND STANDARDS (CONTINUED )
0.05 ug/ml std.
19:35:58
24-Sep-2003
09240312A Sm (Mn, 100
9.37 5213
SIR of 1 Channel ES215.00 5.13e4 Area
9.34 10467
SIR of 1 Channel ES215.00 9.09e4 Area
A
0 09240315A Sm (Mn, 100
B
0 8.00
9.00
A
IN-KQ960 Standard
10.00
11.00
B
Time
IN-KQ960 Standard
0.050 µg/mL Standard
0.10 µg/mL Standard
Peak Area: 5213
Peak Area: 10467
Analysis Date:
Analysis Date:
24 Sep 2003
24 Sep 2003
Set No.: 2
Set No.: 2
32
DuPont-13753
FIGURE 5
C YMOXANIL - EXAMPLE CHROMATOGRAMS OF CONTROL AND FORTIFIED SPINACH SAMPLES
Control Spinach
19:28:15
29-Sep-2003
09260304B Sm (Mn, SIR of 1 Channel ES1 4 . 3 2 1 4 . 7 4 13.73 197.00 13.28 15.08 14.07 100 15.68 16.15 16.56 1.09e4 A 0 09260306B Sm (Mn, 100
14.80 1175
SIR of 1 Channel ES197.00 1.79e4 Area
14.81 11406
SIR of 1 Channel ES197.00 1.05e5 Area
B 0 09260309B Sm (Mn, 100 C 0 13.00
14.00
A
Control
15.00
B
Cymoxanil LOQ Fort.
16.00
Time
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:
Analysis Date:
Analysis Date:
29 Sep 2003
29 Sep 2003
29 Sep 2003
Set No.: 1
Set No.: 1
Set No.: 1
33
DuPont-13753
FIGURE 6
IN-KQ960 - EXAMPLE CHROMATOGRAMS OF CONTROL AND FORTIFIED SPINACH SAMPLES
Control Spinach
15:57:49
09240304A Sm (Mn, 3x1) 100
8.11
8.80
8.87 8.93
24-Sep-2003 SIR of 1 Channel ES215.00 10.03 10.52 10.61 10.94 1.11e4
A 0 09240306A Sm (Mn, 3x1) 100
9.38 924
SIR of 1 Channel ES215.00 1.66e4 Area
9.38 9608
SIR of 1 Channel ES215.00 9.16e4 Area
B 0 09240310A Sm (Mn, 3x1) 100 C 0 8.00
A
Control
9.00
B
IN-KQ960 LOQ Fort.
10.00
11.00
Time
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:
Analysis Date:
Analysis Date:
24 Sep 2003
24 Sep 2003
24 Sep 2003
Set No.: 2
Set No.: 2
Set No.: 2
34
DuPont-13753
FIGURE 7
SIGNAL-TO-NOISE RATIOS
A) Cymoxanil
Spinach LOQ 1
20:24:35
29-Sep-2003
09260306B
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
16:52:17
24-Sep-2003
09240306A
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
C YMOXANIL LC/MS/MS CONFIRMATION
20-Nov-2003 11200302 100
14.78 669
MRM of 2 Channels ES 197.00 > 42.00 6.76e3 Area
14.79 474
MRM of 2 Channels ES 197.00 > 42.00 4.64e3 Area
A
0 11200310 100
B
0 13.00
14.00
A 0.10 ppm Cymoxanil Standard
15.00
16.00
Time
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
9.19 97
MRM of 1 Channel ES214.80 > 140.00 1.30e3 Area
A
0 11140310 100
B
0 7.00
8.00
9.00
A
10.00
0.10 ppm IN-KQ960 Standard
11.00
12.00
Time
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 -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:
Volts Volts
Volts Volts Volts
2.0 0.0 15.0 15.0 250 40 0 700
Volts Volts
Volts Volts Volts Volts
2.2e-5 1.8e-3
Acquisition Threshold SIR or MRM Data Baseline level: General Ion count threshold:
mBar mBar
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 entr ies 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 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 -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:
15.0 15.0 250 40 0 700
Volts Volts Volt Volts
Pressures Analyser Vacuum: Gas Cell:
2.2e-5 1.8e-3
Acquisition Threshold SIR or MRM Data Baseline level:
1.0
mBar mBar
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 entr ies 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 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
25.0 200.0 200 0.00 27.00 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 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
44
