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
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PAGE RESERVED

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

JPM/grs

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GOOD LABORATORY PRACTICE STATEMENT

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

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

Applicant/Sponsor

DuPont Representative

Date

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CERTIFICATION

ANALYTICAL METHOD FOR THE DETERMINATION OF CYMOXANIL AND

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

Date Study Initiated:

September 23, 2003 (first set of validation samples prepared)

Date Study Completed:

January 22, 2004

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

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LIST OF ABBREVIATIONS AND SYMBOLS

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

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TABLE OF CONTENTS
Title Page .......................................................................................................................1
Page Reserved ................................................................................................................2
Good Laboratory Practice Statement .............................................................................3
Certification ...................................................................................................................4
List of Abbreviations and Symbols................................................................................5
Table of Contents...........................................................................................................6
1.0 Abstract .................................................................................................................8
2.0 Introduction ...........................................................................................................8
3.0 Materials................................................................................................................9
3.1 Equipment ......................................................................................................10
3.2 Reagents and Standards..................................................................................11
3.2.1 Reagents ................................................................................................11
3.2.2 Reference Analytical Standards.............................................................11
3.3 Safety and Health............................................................................................12
4.0 Methods...............................................................................................................12
4.1 Principle of the Analytical Method ................................................................12
4.2 Analytical Procedure ......................................................................................13
4.2.1 Glassware & Equipment Cleaning Procedures......................................13
4.2.2 Preparation & Stability of Reagent Solutions........................................13
4.2.3 Stock Standard Preparation and Stability ..............................................13
4.2.4 Fortification Standard Preparation and Stability ...................................13
4.2.5 Chromatographic Standard Preparation and Stability ...........................14
4.2.6 Source (& Characterization) of Samples ...............................................14
4.2.7 Storage & Preparation of Samples ........................................................14
4.2.8 Sample Fortification Procedure.............................................................14
4.2.9 Analyte Extraction Procedure................................................................15
4.2.10 Cymoxanil Purification Procedure ........................................................15
4.2.11 IN-KQ960 Purification Procedure.........................................................16
4.3 Instrumentation...............................................................................................16
4.3.1 Chromatography ....................................................................................16
4.3.2 LC/MS Analysis ....................................................................................17
4.3.3 Calibration Procedure and Sample Analysis .........................................18
4.4 Calculations ....................................................................................................18
4.4.1 Methods .................................................................................................18
4.4.2 Example.................................................................................................19
5.0 Results and Discussion........................................................................................20
5.1 Method Validation Results.............................................................................20
5.1.1 Detector Response .................................................................................20

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5.1.2 Control Samples ....................................................................................20

5.1.3 Recoveries (Accuracy & Precision).......................................................20
5.1.4 Extraction Efficiency.............................................................................21
5.1.5 Limit of Quantitation and Limit of Detection .......................................21
5.2 Timing ............................................................................................................21
5.3 Modifications or Special Precautions.............................................................21
5.4 Method Ruggedness .......................................................................................21
5.4.1 Stability..................................................................................................21
5.4.2 Specificity/Potential Interference ..........................................................22
5.4.3 Confirmatory Method ............................................................................22
6.0 Conclusions .........................................................................................................22
7.0 Retention of Records...........................................................................................22
8.0 References ...........................................................................................................22

TABLE
Table 1 Summary of Cymoxanil and IN-KQ960 Fortification (Recovery)
Data in Spinach ....................................................................................23

FIGURES
Figure 1 Flow Diagram of Analytical Method....................................................24
Figure 2 Full Scan Spectrum for Cymoxanil and IN-KQ960 .............................26
Figure 3 Cymoxanil Representative Curve and Standards .................................27
Figure 4 IN-KQ960 Representative Curve and Standards..................................30
Figure 5 Cymoxanil - Example Chromatograms of Control and Fortified
Spinach Samples...................................................................................33
Figure 6 IN-KQ960 - Example Chromatograms of Control and Fortified
Spinach Samples...................................................................................34
Figure 7 Signal-to-Noise Ratios .........................................................................35
Figure 8 Cymoxanil LC/MS/MS Confirmation..................................................36
Figure 9 IN-KQ960 LC/MS/MS Confirmation ..................................................37

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

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ANALYTICAL METHOD FOR THE DETERMINATION OF CYMOXANIL AND

IN-KQ960 IN SPINACH (LEAFY VEGETABLES) USING LC/MS

Joseph P. McClory and Robert M. Henze

1.0 ABSTRACT
The purpose of this study was to develop an analytical method for the detection,
quantitative analysis, and confirmation of cymoxanil and IN-KQ960 in spinach.
Cymoxanil and its metabolite, IN-KQ960, were extracted from samples of spinach
with a mixture of acetonitrile and water. This solvent mixture has been shown to be
effective at extracting cymoxanil from plant matrices.
For cymoxanil, NaCl was added to an aliquot to separate the aqueous phase from the
organic phase. The aqueous phase was discarded, and the acetonitrile layer
containing cymoxanil was passed through a SAX SPE column. The extract is then
further cleaned up using a hexane liquid/liquid extraction followed by Envi Carb SPE
column. Cymoxanil is not retained on either of these columns.
IN-KQ960 does not partition quantitatively when salted out, so a separate aliquot of
extract is used for the analysis of the metabolite. The extract is cleaned up using a
hexane liquid/liquid extraction followed by passing through SAX and Envi Carb SPE
columns. IN-KQ960 is not retained on either of these columns
The LOQ by LC/MS analysis was determined to be 0.050 µg/g (ppm) for both
cymoxanil and IN-KQ960. During method validation, acceptable recoveries were
generated for spinach samples fortified at the LOQ through the highest levels
anticipated in field treated samples as indicated in the following table:

Average Recovery

LEVEL CYMOXANIL IN-KQ960

(PPM) 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 from 10 freshly fortified
spinach samples was 83% ± 4.8% (RSD). Unfortified control samples showed no
quantifiable residues of cymoxanil and IN-KQ960.

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

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

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

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3.1 Equipment

Equipment Description Product ID Supplier

Freezer Labline Frigid-Cab Labline Instruments, Inc. (Melrose Park, IL)
Refrigerator 6FAR Marvel Industries, Inc. (Richmond, IN)
Commercial Food Processor Model 31FP93 Waring Products (New Hartford, CT)
AE163 Dual Range Balance; PM460 Toploading
Analytical Balance Balance; PM400 Toploading Balance; AE163 Dual Mettler Instrument Corp. (Hightstown, NJ)
Range Balance
N-Evap Model 111 with stainless steel luer fit
Analytical Evaporator Organomation Assoc. (South Berlin, MA)
needles
Tissumizer homogenizer Model SDT-20
Homogenizer equipped with Model SDT-182EN shaft (Teflon Tekmar Company (Cincinnati, OH)
bearing)
Bransonic 52 or 2200 Ultrasonic Cleaner,
Sonication Branson Ultrasonics Corp. (Danbury, CT)
0.75 gal. capacity
Vortex Mixer Vortex Genie K-550-G or Vortex-2 Genie VWR, Inc. (West Chester, PA)
Gelman Acrodisc 13 CR, 0.2-µm PTFE 13 mm
Filtration VWR (Bridgeport, NJ)
dia. membrane syringe filter, Cat. No. 4423
Supelclean ENVI-Carb SPE cartridge,
Solid Phase Extraction 0.5 g/6 mL, Cat. No. 57094; Visiprep DL SPE Supelco (Bellefonte, PA)
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.
Solid Phase Extraction Varian, Inc. (Palo Alto, CA)
No: 12131001; union adapter for 60 mL columns,
Cat. No. 12131004; Reservoir Adapters, Cat.
No. 12131003;
Centrifuge Sorvall Centrifuge, Model RC 3B Sorvall Instruments (Wilmington, DE)
Centrifuge Sorvall Centrifuge, Model GLC-2B(bench top) Sorvall Instruments (Wilmington, DE)
N-Evap Model 111(with stainless steel luer fit
Analytical Evaporator Organomation Assoc. (South Berlin, MA.)
needles)
Beckman φ 10 pH meter with combination pH
pH Meter Beckman Instruments, Inc. (Fullerton, CA)
electrode
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,
Labware 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
Labware Electronic 1000-µL and 10-mL Pipettors Rainin (Walnut Creek, CA)
Mechanical, positive displacement, 25-µL, 50-µL
Labware Gilson Inc. (Middletown, WI)
and 250-µL Pipettors
Falcon 2098 (50 mL), 2096 (15 mL)
Polypropylene Centrifuge Tubes; 3-mL Disposable
Labware Becton Dickinson (Franklin Lakes, NJ)
Syringe, Cat. No. BD309585; 60-mL Disposable
Syringe, Cat. No. BD309663

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HPLC/MS System
HP1100: G1322A degasser, G1312A binary pump,
G1311A quaternary pump; G1313A autosampler;
HPLC Agilent Technologies, Inc. (Palo Alto, CA)
G1316A column unit; G1314A variable wavelength
detector
Target DP Amber Kit, T/S/T Septa, 100 PK, Cat.
Autosampler Vials Agilent Technologies, Inc. (Palo Alto, CA)
No. 5182-0556
Eclipse XDB-C8; 4.6 mm × 150 mm, 5 µm
HPLC Column Agilent Technologies, Inc. (Palo Alto, CA)
particle size diameter
Valco zero dead-volume tee (split-flow to MS), Cat.
Splitter tee Valco Instruments, Inc. (Houston, TX)
No. ZT1C
Valco 6 Port Electrically Actuated Valve, Cat.
Switching Valve Valco Instruments, Inc. (Houston, TX)
No. 1384
MicroMass Quattro II triple quadrupole mass
spectrometer using an electrospray (ESI) or
Triple Quadrupole MS Waters Corporation (Milford, MA)
atmospheric chemical ionization (ACPI) interface
and MassLynx NT version 3.1 software

3.2 Reagents and Standards

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

Reagents Product Description Product ID Supplier

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

Methanol OmniSolv, 4L MX0488-1 EM Science (Gibbstown, NJ)
Hexane OmniSolv, 4L HX0296-1 EM Science (Gibbstown, NJ)
Acetonitrile OmniSolv, 4L AX0142-1 EM Science (Gibbstown, NJ)
Water OmniSolv, 4L WX0004-1 EM Science (Gibbstown, NJ)
Methylene Chloride OmniSolv, 1L DX0831-6 EM Science (Gibbstown, NJ)

3.2.2 Reference Analytical Standards

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

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N O DuPont Code: DPX-T3217

O
CAS Chemical Name:
N
H N 2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyimino)acetamide
O N H CH3
H3C CAS Registry Number: 57966-95-7
IUPAC Chemical Name:
1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea
Cymoxanil Molecular weight = 198.2 g/mole

O DuPont Code: IN-KQ960

C2H5
HN N CAS Chemical Name:
3-Ethyl-4-(methoxyamino)-2,5-dioxo-4-imidazolidinecarboxamide
CONH2
O CAS Registry Number: none
NHOCH3
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 METHODS

4.1 Principle of the Analytical Method

Samples are extracted using an acetonitrile/water mixture as described in DuPont
Report No. AMR 3705-95, Revision No. 2, “Analytical Method for the Determination
of DPX-JE874 and Cymoxanil Residues in Various Matrices” (EPA MRID
No. 44579102, Reference 2). Changes incorporated in the current study include the
addition of IN-KQ960 as an analyte. Also, LC/MS is used for the analysis of both
cymoxanil and IN-KQ960. Based on the sensitivity and selectivity of LC/MS, sample
cleanup procedures were simplified.
For cymoxanil, NaCl is added to an aliquot to separate the aqueous phase from the
organic phase. The aqueous phase is discarded, and the acetonitrile layer containing
cymoxanil is passed through a SAX SPE column. The extract is then further cleaned
up using a hexane liquid/liquid extraction followed by Envi Carb SPE column.
Cymoxanil is not retained on either of these columns.
IN-KQ960 does not partition quantitatively when salted out, so a separate aliquot of
extract is used for the analysis of the metabolite. The extract is cleaned up using a
hexane liquid/liquid extraction followed by passing through SAX and Envi Carb SPE
columns. IN-KQ960 is not retained on either of these columns.

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4.2 Analytical Procedure

4.2.1 Glassware & Equipment Cleaning Procedures

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

4.2.2 Preparation & Stability of Reagent Solutions

90/10 methylene chloride/methanol (v/v)- 100 mL of a 90/10 methylene
chloride/methanol (v/v) is prepared on the day of analysis by adding 90 mL of
methylene chloride to 10 mL of methanol. 15 mL of this solution is required per
sample; if analyzing more than six samples adjust volume of solution prepared
accordingly.
0.02% formic Acid (v/v)- 2.0 L of the solution is prepared by adding 0.4 mL of
formic acid to 2.0 liter of de-ionized water. Solution is stored at room temperature
and is stable for 1 month.

4.2.3 Stock Standard Preparation and Stability

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

4.2.4 Fortification Standard Preparation and Stability

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

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4.2.5 Chromatographic Standard Preparation and Stability

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

Desired Standard Volume of 10.0 µg/mL Intermediate Volume of 1.0 µg/mL Intermediate
Concentration (µg/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

4.2.6 Source (& Characterization) of Samples

Spinach samples were purchased fresh from the local supermarket.

4.2.7 Storage & Preparation of Samples

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

4.2.8 Sample Fortification Procedure

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

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4.2.9 Analyte Extraction Procedure

1. Weigh out 20 grams of Spinach into a 250-mL centrifuge bottle.
2. For fresh fortification samples, spike sample at the appropriate level and let sit for
5 minutes for the solvent to evaporate.
3. Add 60 mL of water and 120 mL of acetonitrile to the sample.
4. Blend with a tissuemizer for 5 minutes on medium speed. Centrifuge for
10 minutes at 3000 rpm.
5. Remove an 8-mL aliquot, transfer into a 15-mL centrifuge tube, and take this
aliquot through the purification procedure.

4.2.10 Cymoxanil Purification Procedure

1. Take the 8-mL aliquot from the extraction and add 4 grams of sodium chloride.
Shake vigorously for 1 minute and then centrifuge at 3000 rpm for 5 minutes.
2. Remove the upper layer (acetonitrile) and put into a new 15-mL centrifuge tube.
Add 3 mL of acetonitrile to the tube containing the NaCl layer from Step 1.
Shake vigorously for 1 minute and then centrifuge at 3000 rpm for 3 minutes.
Remove the upper layer and add this to the tube containing the acetonitrile from
this step.
3. Condition a 1-gram/6 mL SAX cartridge using 5 mL of acetonitrile, discard
conditioning solution.
4. Add the sample to the SAX cartridge, collecting the solution in a 15-mL
centrifuge tube. After the sample has gone through, add 3 mL of acetonitrile to
the tube from Step 3, vortex, and also pass that through the cartridge.
5. Remove tube from manifold, cap, vortex, and put on the N-Evap to reduce the
volume to approximately 9 mL.
6. Take sample (from Step 5) and add 5 mL of hexane. Vortex and centrifuge at
3000 rpm, then remove upper layer (hexane) and discard. Repeat this step again.
7. Condition a 1-gram/6 mL Envir- Carb carbon column using 5 mL of a
90% methylene chloride/10% acetonitrile and discard solution.
8. Add the sample (from Step 6) to the column and collect this elution in another
15-mL centrifuge tube. Rinse tube (from Step 6) with 4 mL of the
(90% methylene chloride/10% acetonitrile) solution, vortex and pass through the
Enviro Carb column and collect also.
9. Remove sample from the manifold, cap vortex, remove the cap and put on the
N-Evap to be evaporated to about 0.75 mL at 38 to 40°C. (After every 3 to 4 mL
is evaporated, remove sample from N-Evap, cap, vortex, and return to keep the
analyte in the solution.)
10. Dilute to a final volume of 4mL with 0.02% formic acid, cap, vortex, and sonicate
for 2 minutes. Filter through a syringe filter and analyze by LC/MS.

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4.2.11 IN-KQ960 Purification Procedure

1. Take the 8-mL aliquot from the extraction and add 5 mL of hexane. Shake
vigorously for 1 minute and then centrifuge at 3000 rpm for 5 minutes. Remove
the upper layer (hexane) and discard.
2. Repeat Step1. Discard top hexane layer. Save lower layer (water and
acetonitrile).
3. Condition a 1-gram/6 mL SAX cartridge using 5 mL of acetonitrile. Discard this
conditioning solution.
4. Add the sample from Step 2 to the cartridge, collecting the elution in a 15- mL
centrifuge tube. After the sample has gone through add an additional 3 mL of
acetonitrile to the tube from Step 2, vortex, and also pass that through the
cartridge.
5. Condition a 1-gram/6 mL Envir-Carb carbon column using 5 mL of
90% methylene chloride/10% acetonitrile. Discard solution.
6. Add the sample (from Step 4) to the column and collect this elution in another
15-mL centrifuge tube. Rinse tube (from Step 4) with 5 mL of the
(90% methylene chloride/10% acetonitrile) solution, vortex, and pass through the
Carbon column and collect also.
7. Remove sample from the manifold, cap vortex, remove the cap and put on the
N-Evap to be evaporated to about 0.75 mL at 38 to 40°C. (After every 3 to 4 mL
is evaporated, remove sample from N-Evap, cap, vortex, and return to keep the
analyte in the solution.)
8. Dilute to a final volume of 4 mL with 0.02% formic acid, cap, vortex, and
sonicate for 2 minutes.
9. Filter through a syringe filter and analyze by LC/MS.

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

4.3 Instrumentation

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

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System: Hewlett-Packard HP1100 HPLC

Column: 2.1 mm i.d. × 10 cm, Agilent Eclipse XDB-C8,
3 µm diameter packing
Column Temperature: 30°C
Auto-sampler Temperature 4°C
Injection Volume: 25-30 µL
Flow Rate: 1.0 ml/min
Conditions: A: 0.02% Formic Acid
B: Methanol
Time %A %B Time %A %B
------Cymoxanil---- ------IN-KQ960---

0.0 95 5 0.0 98.0 2.0

17.0 50 50 2.0 98.0 2.0
19.9 10 90 15.0 80 20.0
22.0 95 5 16.0 1.0 99.0
27.00 95 5 18.0 1.0 99.0
19.0 70.0 30.0
22.0 98.0 2.0
26.0 98.0 2.0
IN-KQ960 Retention Time: ~ 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 to and following elution of the compounds of interest. The use of
this valve reduced source contamination and enabled additional samples to be
analyzed before the ion source required cleaning. The valve switching times are
given in the following table.

TIME (MINUTES) COLUMN ELUATE FLOW

0.00-8.0 Waste
8.0-17.0 MS source
17-27 Waste

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

4.3.2 LC/MS Analysis

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

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 DuPont-13753

Micromass Quattro LC ESI-LC/MS Mass Spectrometer Conditions

Cone Source Dwell

Analytes Ions Monitored Voltage Temp. (Seconds)
Cymoxanil 197.0 ± 0.1 AMU 17V 125°C 0.02
IN-KQ960 215.0 ± 0.1 AMU 15V 125°C 0.02
Electrospray Voltage: 3.50 kV
Detector Voltage: 700 V
Nebulizing Gas Flow: 15 L/h
Drying Gas Flow: 300 L/h

A complete list of the experimental parameters is given in Appendix 1. A typical

LC/MS full scan spectrum is shown Figure 2.
The instrument was operated in MS (SIR) negative ion mode for quantitative analysis.
Peak area was used for quantitation. For confirmation MS/MS was used. The
relative ratio of the fragment ions was evaluated to confirm the presence of an analyte
in an unknown sample.

4.3.3 Calibration Procedure and Sample Analysis

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

4.4 Calculations

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

Concentration of analyte (µg/mL)

RFstd =
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.

(RFstd1 + RFstd 2 + RFstd3 + .......RFstdn )

RFave =
Total Number of Standards Injected

The concentration (µg/g or ppm) of analyte found in each sample was calculated as
follows:
[ Peak Area x RFave] x [ Final Vol. (mL) x mL solv ]
µg/g analyte Found =
Sample Wt. (g) x Aliquot Taken (mL)
Where:
Total Extract Volume (mL solv) = 180 mL
Final Extract Volume (Final Vol.) = 5.0 mL
Aliquot Taken = 4.0 mL
Sample Weight = 20.0 grams

The percent recovery found was calculated as follows:

(µg/g Found)
% Recovery = 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:
0.005 ( µ g/mL)
RFstd = = 0.00638 µ g/mL
784

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: = 1175

mL Solvent: = 180 mL
mL Aliquot 1: = 8.0 mL
Final Volume: = 5.0 mL

19
 DuPont-13753

Sample Weight: = 20.0 grams

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

ppm Cymoxanil = = 0.0439 µg/g
8.0mL × 20.0 g
0.0439 µg/g
% Recovery = x 100% = 88%
0.050 µg/g

5.0 RESULTS AND DISCUSSION

5.1 Method Validation Results

5.1.1 Detector Response

Standard calibration solutions used for quantitative analysis ranged from 5.0 to
100.0 ng/mL for both cymoxanil and IN-KQ960. Typical experimental conditions for
each analyte are provided in Appendix 1. Typical LC/MS chromatograms for
standards analyzed during method validation are provided in Figure 3 and Figure 4.
The response of the MS detector was linear over the range of standards analyzed, as
evidenced by relative standard deviation of the response factors consistently being
less than 15%.
Control samples fortified at 0.050 µg/g to 0.50 µg/g were successfully extracted,
cleaned up, and analyzed using this method. Representative chromatograms of
fortified and unfortified samples are shown in Figure 5 for cymoxanil and Figure 6 for
IN-KQ960.

5.1.2 Control Samples

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

5.1.3 Recoveries (Accuracy & Precision)

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

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 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.

5.1.5 Limit of Quantitation and Limit of Detection

The LOQ by LC/MS analysis was determined to be 0.050 µg/g for cymoxanil and
IN-KQ960. This quantitation limit is defined as the lowest fortification level
evaluated at which acceptable average recoveries (70-120%, RSD <20%) were
achieved. This quantitation limit also reflects the fortification level at which analyte
peaks were consistently generated at a level approximately 10-20 times the signal at
the retention time of each analyte in an untreated control sample. An example of the
signal-to-noise calculation is provided in Figure 7.
The limit of detection is estimated to be 0.02 µg/g, which is one-third the value of the
corresponding LOQ value.

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

5.3 Modifications or Special Precautions

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

5.4 Method Ruggedness

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

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 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.

5.4.3 Confirmatory Method

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

6.0 CONCLUSIONS
This method for determination of cymoxanil and IN-KQ960 residues extracted from
spinach (leafy vegetables) meets U.S. EPA and EU guidelines.
This LC/MS method with mass selective detection is free of interference above the
LOQ of 0.050 ppm at the retention times corresponding to cymoxanil and IN-KQ960
in unfortified samples. This method generated acceptable recoveries over
concentration levels expected in the samples tested.

7.0 RETENTION OF RECORDS

Originals or exact copies of all raw data and pertinent information, and the final
report will be retained at:
E.I. du Pont de Nemours and Company
DuPont Crop Protection
Global Technology Division
Stine-Haskell Research Center
Newark, Delaware 19714-0030

8.0 REFERENCES
1. Fox, G. C., “Metabolism of [2-14C]Cymoxanil in Lettuce”; DuPont Report
No. AMR 4375-97, E.I. du Pont de Nemours and Company, Wilmington,
Delaware; MRID No. 44944605.
2. Nathan, E.C. III, “Analytical Method for the Determination of DPX-JE874 and
Cymoxanil Residues in Various Matrices”; DuPont Report No. AMR 3705-95,
Rev. 2, E.I. du Pont de Nemours and Company, Wilmington, Delaware; MRID
No. 44579102.

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TABLE 1 SUMMARY OF CYMOXANIL AND IN-KQ960 FORTIFICATION

(RECOVERY) DATA IN SPINACH

Cymoxanil IN-KQ960
Fortification
Set No. Level (ppm)1 PK Area ppm % Recovery PK Area ppm % Recovery
2
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
Mean % Recovery ± SD (n = 4) = 92 ± 3.9 83 ± 3.0
RSD = 4.3 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) = 88 ± 2.3 83 ± 5.2
RSD = 2.6 6.2
Overall Mean % Recovery ± SD (n = 2) = 90 ± 3.9 83 ± 4.0
RSD = 4.3 4.8

1 Limit of quantitation (LOQ) for determination of both cymoxanil and IN-KQ960 in spinach was 0.050 ppm. Residue values
carried to an excessive number of significant figures were used to calculate % Recovery. After calculation, % Recovery
values were rounded to the nearest whole number and reported.
2 Additional data necessary to calculate % recoveries (see calculation on page 19)
sample wt. Extract Vol. Aliquot Final Vol. Res. Factor(MV-1) Res. Factor(MV-2)
Cymoxanil 20.0g 180ml 8.0ml 5.0ml 0.00664 0.00668
IN-KQ960 20.0g 180ml 8.0ml 4.0ml 0.00962 0.01011

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FIGURE 1 FLOW DIAGRAM OF ANALYTICAL METHOD

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

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 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) Scan ES-
196 2.64e5
100

197
%

41.5 242
166
0 m/z
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

B) IN-KQ960

10.0ug/ml std.
11130307 117 (9.235) Cm (106:119-(2:58+166:254)x4.000) 1: Scan ES-
215 1.11e4
100

140 216 261

0 m/z
50 100 150 200 250 300 350 400

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 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 Conc
0.0 0.1

27
 DuPont-13753

FIGURE 3 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS

(CONTINUED)

18:03:48 29-Sep-2003
09260301B Sm (Mn, 3x1) SIR of 1 Channel ES-
14.79 197.00
100
758 1.52e4
Area
A

0
09260302B Sm (Mn, 3x1) SIR of 1 Channel ES-
14.80 197.00
100
1138 1.82e4
Area
B

0
09260305B Sm (Mn, 3x1) SIR of 1 Channel ES-
14.81 197.00
100
1469 2.16e4
Area
C

0 Time
13.00 14.00 15.00 16.00

A Cymoxanil B Cymoxanil C Cymoxanil

Standard Standard 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 CYMOXANIL REPRESENTATIVE CURVE AND STANDARDS

(CONTINUED)

23:13:45 29-Sep-2003
09260312B Sm (Mn, 3x1) SIR of 1 Channel ES-
14.81 197.00
100
7448 7.06e4
Area

0
09260315B Sm (Mn, 3x1) SIR of 1 Channel ES-
14.79 197.00
100
14974 1.34e5
Area

0 Time
13.00 14.00 15.00 16.00

A Cymoxanil B Cymoxanil
Standard 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

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 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 Conc
0.0 0.1

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 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) SIR of 1 Channel ES-
9.43 215.00
100
537 1.49e4
Area
A

0
09240302A Sm (Mn, 3x1) SIR of 1 Channel ES-
9.41 215.00
100
691 1.57e4
Area
B

0
09240305A Sm (Mn, 3x1) SIR of 1 Channel ES-
9.39 215.00
100
963 1.66e4
Area
C

0 Time
8.00 9.00 10.00 11.00

A IN-KQ960 B IN-KQ960 C IN-KQ960

Standard Standard 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

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 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, SIR of 1 Channel ES-
9.37 215.00
100
5213 5.13e4
Area

0
09240315A Sm (Mn, SIR of 1 Channel ES-
9.34 215.00
100
10467 9.09e4
Area

0 Time
8.00 9.00 10.00 11.00

A IN-KQ960 B IN-KQ960
Standard 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

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 DuPont-13753

FIGURE 5 CYMOXANIL - EXAMPLE CHROMATOGRAMS OF CONTROL AND

FORTIFIED SPINACH SAMPLES

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

09260304B Sm (Mn, SIR of 1 Channel ES-
100 13.28 13.73 14.07 14.32 14.74 15.08 197.00
15.68 16.15 16.56 1.09e4

0
09260306B Sm (Mn, SIR of 1 Channel ES-
14.80 197.00
100
1175 1.79e4
Area
B

0
09260309B Sm (Mn, SIR of 1 Channel ES-
14.81 197.00
100
11406 1.05e5
Area
C

0 Time
13.00 14.00 15.00 16.00

A Control B Cymoxanil C Cymoxanil

LOQ Fort. 10XLOQ Fort.
Peak Area- Peak Area: 1175 Peak Area: 11406
<0.0.05 ppm 0.044 ppm 4.30 ppm
(89% Recovery) (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 24-Sep-2003

09240304A Sm (Mn, 3x1) SIR of 1 Channel ES-
100
8.87 8.93 10.03 10.52 10.61 10.94 215.00
8.11 8.80 1.11e4

0
09240306A Sm (Mn, 3x1) SIR of 1 Channel ES-
9.38 215.00
100
924 1.66e4
Area
B

0
09240310A Sm (Mn, 3x1) SIR of 1 Channel ES-
9.38 215.00
100
9608 9.16e4
Area
C

0 Time
8.00 9.00 10.00 11.00

A Control B IN-KQ960 C IN-KQ960

LOQ Fort. 10XLOQ Fort.
Peak Area- Peak Area: 924 Peak Area: 9608
<0.0.05 ppm 0.044 ppm 4.14 ppm
(88% Recovery) (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 ES-
197.00
2.00e4
45 Area
100

S/N=45/8=5.6

27 Time
13.00 14.00 15.00 16.00

B) IN-KQ960

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

09240306A SIR of 1 Channel ES-
215.00
2.00e4
Area
30
100
S/N=30/6=5

% 6

2 Time
8.00 9.00 10.00 11.00

35
 DuPont-13753

FIGURE 8 CYMOXANIL LC/MS/MS CONFIRMATION

20-Nov-2003
11200302 MRM of 2 Channels ES
14.78 197.00 > 42.00
100
669 6.76e3
Area

0
11200310 MRM of 2 Channels ES
14.79 197.00 > 42.00
100
474 4.64e3
Area

0 Time
13.00 14.00 15.00 16.00

A 0.10 ppm B Cymoxanil

Cymoxanil 10 X LOQ
Standard Fort. Spinach

36
 DuPont-13753

FIGURE 9 IN-KQ960 LC/MS/MS CONFIRMATION

14-Nov-2003
11140302 MRM of 1 Channel ES-
9.21 214.80 > 140.00
100
99 1.42e3
Area

0
11140310 MRM of 1 Channel ES-
9.19 214.80 > 140.00
100
97 1.30e3
Area

0 Time
7.00 8.00 9.00 10.00 11.00 12.00

A 0.10 ppm B IN-KQ960

IN-KQ960 10 X LOQ
Standard 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: ES-
Source Page (ESI)
Capillary: 3.50 kVolts
HV Lens: 0.87 kVolts
Cone: 15 Volts
Skimmer Offset: 5 Volts
Skimmer: 1.6 Volts
RF Lens: 0.3 Volts
Source Temp: 125 øC
MS1
Ion Energy: 2.0 Volts
Ion Energy Ramp: 0.0 Volts
LM Resolution: 15.0
HM Resolution: 15.0
Lens 5: 100 Volts
Lens 6: 5 Volts
Multiplier 1: 700 Volts
MS2
Ion Energy: 2.0 Volts
Ion Energy Ramp: 0.0 Volts
LM Resolution: 15.0
HM Resolution: 15.0
Lens 7: 250 Volts
Lens 8: 40 Volts
Lens 9: 0 Volts
Multiplier: 700 Volts
Pressures
Analyser Vacuum: 2.2e-5 mBar
Gas Cell: 1.8e-3 mBar
Acquisition Threshold
SIR or MRM Data
Baseline level: 1.0
General
Ion count threshold: 0

39
 DuPont-13753

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

HP1100 LC Pump Initial Conditions

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

HP1100LC Pump Gradient Timetable

The gradient Timetable contains 5 entries which are :
Time A% B% C% D% Flow Pressure
0.00 95.0 5.0 0.0 0.0 1.000 400
17.00 50.0 50.0 0.0 0.0 1.000 400
19.99 10.0 90.0 0.0 0.0 1.000 400
22.00 95.0 5.0 0.0 0.0 1.000 400
27.00 95.0 5.0 0.0 0.0 1.000 400
HP1100 LC Pump External Event Timetable
The Timetable contains 4 entries which are :
Time Column Switch Contact 1 Contact 2 Contact 3 Contact 4
Initial On Off Off Off Off
0.00 On Off Off On Off
8.00 On Off Off Off On
17.00 On On Off Off Off
HP1100 Autosampler Initial Conditions
Injection Volume(µl) 25.0
Draw Speed 200.0
Eject Speed (µl/min) 200
Draw Position (mm) 0.00
Stop Time (mins) 27.00

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: ES-
Data type: SIR or MRM data
Function type: SIR of 1 channel
Chan Mass Dwell(secs) Cone Volt.
1 : 197.00 0.02 17.0

IN-KQ960
Acquisition Experiment Report
File: g:\je874.pro\data\09260309b
Header
Acquired File Name: 09260309B
Acquired Date: 29-Sep-2003
Acquired Time: 21:49:08
Job code: 092603CymoxanilValidationSet1
Task code:
User Name: Administrator
Laboratory Name: Lab
Instrument: Inst
Conditions:
Submitter:
SampleID: Spinach 10X LOQ 1
Bottle Number: 18
Description: Spinach 10X LOQ 1
Instrument Calibration
Parameters
MS1 Static:
Mass 85 Da to 596 Da.
Resolution : 15.0/15.0
Ion Energy : 0.8
Reference File : peghnh4
Acquisition File : STATMS1
MS1 Scanning:
Mass 80 Da to 600 Da.
Resolution : 15.0/15.0
Ion Energy : 0.8
Reference File : peghnh4
Acquisition File : SCNMS1
MS1 Scan Speed:
Scan 64 to 473 amu/sec.

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: ES-
Source Page (ESI)
Capillary: 3.50 kVolts
HV Lens: 0.87 kVolts
Cone: 15 Volts
Skimmer Offset: 5 Volts
Skimmer: 1.6 Volts
RF Lens: 0.3 Volts
Source Temp: 125 øC
MS1
Ion Energy: 2.0 Volts
Ion Energy Ramp: 0.0 Volts
LM Resolution: 15.0
HM Resolution: 15.0
Lens 5: 100 Volts
Lens 6: 5 Volts
Multiplier 1: 700 Volts
MS2
Ion Energy: 2.0 Volts
Ion Energy Ramp: 0.0 Volts

42
 DuPont-13753

LM Resolution: 15.0
HM Resolution: 15.0
Lens 7: 250 Volts
Lens 8: 40 Volts
Lens 9: 0 Volt
Multiplier: 700 Volts
Pressures
Analyser Vacuum: 2.2e-5 mBar
Gas Cell: 1.8e-3 mBar
Acquisition Threshold
SIR or MRM Data
Baseline level: 1.0

General
Ion count threshold: 0
Prescan Statistics
Zero Level: 25
ADC zero: 82.54
ADC standard deviation: 1.14
Acquisition Threshold MS2
SIR or MRM Data
Baseline level: 1.0
General
Ion count threshold: 0
Prescan Statistics
Zero Level: 24
ADC zero: 70.48
ADC standard deviation: 1.20
ACE Experimental Record
--------------------- Run method parameters ----------------
HP1100 LC Pump Initial Conditions
Solvents
A% 95.0
B% 5.0
C% 0.0
D% 0.0
Flow (ml/min) 1.000
Stop Time (mins) 27.0
Min Pressure (bar) 0
Max Pressure (bar) 400
Oven Temperature Left(°C) 40.0
Oven Temperature Right(°C) 40.0

43
 DuPont-13753

HP1100 LC Pump Gradient Timetable

The gradient Timetable contains 5 entries which are :

Time A% B% C% D% Flow Pressure

0.00 95.0 5.0 0.0 0.0 1.000 400
17.00 50.0 50.0 0.0 0.0 1.000 400
19.99 10.0 90.0 0.0 0.0 1.000 400
22.00 95.0 5.0 0.0 0.0 1.000 400
27.00 95.0 5.0 0.0 0.0 1.000 400
HP1100 LC Pump External Event Timetable
The Timetable contains 4 entries which are :
Time Column Switch Contact 1 Contact 2 Contact 3 Contact 4
Initial On Off Off Off Off
0.00 On Off Off On Off
8.00 On Off Off Off On
17.00 On On Off Off Off
HP1100 Autosampler Initial Conditions
Injection Volume(µl) 25.0
Draw Speed 200.0
Eject Speed (µl/min) 200
Draw Position (mm) 0.00
Stop Time (mins) 27.00
Vial Number 18
---------------------------- oOo -----------------------------
End of experimental record.
Solvent Delay
None
Function 1
Scans in function: 10379
Cycle time (secs): 0.050
Inter Channel delay (secs): 0.00
Retention window (mins): 8.000 to 17.000
Ionization mode: ES-
Data type: SIR or MRM data
Function type: SIR of 1 channel
Chan Mass Dwell(secs) Cone Volt.
1 : 197.00 0.02 17.0

44