Fluensulfone ... - IR-4 Project

TITLE 

Method Validation of an Analytical Method for the Determination of MCW-2 and its 

Metabolites in Plant Matrices 

DATA REQUIREMENT 

OPPTS 860.1340 

SANCO/825/00 rev. 7 (17/3/04) 

AUTHOR 

Janine E. Marin, Ph.D. 

STUDY COMPLETION DATE 

June 14, 2010 

Amended Report Date: July __, 2010 

PERFORMING LABORATORY 

PTRL West, Inc. 

625-B Alfred Nobel Drive 

Hercules, California 94547 

SUBMITTER/SPONSOR 

Makhteshim-Agan of North America, Inc. 

4515 Falls of Neuse Road, Suite 300 

Raleigh, NC 27609 

LABORATORY PROJECT IDENTIFICATION 

PTRL West Project No. 1977W 

1977W Report 2 

Page 1 of 138


Report 2 Page 2 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIM 

No claim of confidentiality is made for any information contained in this study on the 

basis of its falling within the scope of FIFRA § 10(d)(1)(A), (B), or (C). Nor is any 

supplemental claim of confidentiality made for any information in this study on the basis 

of FIFRA § 10 (a) or (b). This document, however, is proprietary to Makhteshim-Agan of 

North America, Inc. and is considered to be confidential and trade-secret information in 

all other countries and for all purposes other than those enunciated in FIFRA § 3 & 10. 

Information contained in this study should not be reproduced, stored in a retrieval system 

or transmitted in any form or by any means by persons other than EPA without the 

express written consent of Makhteshim-Agan of North America, Inc. except as required 

to carry out the requirements of FIFRA. 

Company: Makhteshim-Agan of North America, Inc. 

Company Representative: 

Title: 

Signature: 

Date:



CERTIFICATION OF GOOD LABORATORY PRACTICE 

PTRL West, Inc. Project No. 1977W, “Method Validation of an Analytical Method for 

the Determination of MCW-2 and its Metabolites in Plant Matrices” has been performed 

in compliance with current US EPA FIFRA Good Laboratory Practice Standards (FIFRA 

Guidelines 40 CFR §160 by PTRL West, Inc., with no exceptions. 

Submitted by: 



Study Director 

Kris Venkatesh, Ph.D. 

Makhteshim-Agan of North America, Inc. 

Sponsor Representative 

Date 

Date 

Submitter Date



PTRL WEST, INC. QUALITY ASSURANCE UNIT STATEMENT

STUDY PERSONNEL 



The individuals listed below are the study personnel involved in the collection and/or 

interpretation of data generated in the course of the study. 

Name Position 

Janine E. Marin, Ph.D. Study Director 

Curtis Hatton, B.S. Senior Research Chemist 

Polina Kosovskaya, M.S. Research Chemist 

Sonia Ho, B.S. Associate Research Chemist 

Craig Warling, B.S. Assistant Research Chemist 

Rose Robinson Senior Laboratory Technician 

Sean Lucas Laboratory Technician 




Luis O. Ruzo, Ph.D. 


Director 

ARCHIVE STATEMENT: 

Study Completion Date 

Date 

The original project-specific data files are temporarily located in the archives of PTRL 

West, Inc., Hercules, California, 94547. The above materials may be transferred to a 

location specified by the Sponsor upon their authorization. Facility records, such as 

Compound Control logs, equipment use, standardization/calibration, and maintenance 

logs, personnel records, SOPs, etc. will be archived at PTRL West, Inc. A copy of the 

final analytical report will be maintained in the archives of PTRL West, Inc. 

Specimens will be retained by PTRL West, Inc. Following Sponsor's authorization, 

specimens may be sent to the Sponsor or disposed of, in accordance with PTRL West, 

Inc. standard operating procedures, after quality assurance verification.

TABLE OF CONTENTS 



TITLE ..................................................................................................................................1 

STATEMENT OF NO DATA CONFIDENTIALITY CLAIM ..........................................2 

CERTIFICATION OF GOOD LABORATORY PRACTICE ............................................3 

PTRL WEST, INC. QUALITY ASSURANCE UNIT STATEMENT ...............................4 

STUDY PERSONNEL ........................................................................................................5 

ARCHIVE STATEMENT: ..................................................................................................5 

TABLE OF CONTENTS .....................................................................................................6 

LIST OF TABLES ...............................................................................................................7 

LIST OF FIGURES .............................................................................................................7 

LIST OF APPENDICES ....................................................................................................10 

SUMMARY .......................................................................................................................12 

INTRODUCTION .............................................................................................................12 

MATERIALS AND METHODS .......................................................................................13 

Reference Substances ..................................................................................................13 

Reagents and Solvents .................................................................................................13 

Glassware and Miscellaneous Equipment ...................................................................13 

ANALYTICAL PROCEDURES .................................................................................14 

Preparation of Samples ..............................................................................................14 

Preparation of Stock Standards ..................................................................................14 

Preparation of Fortification Standards .......................................................................14 

Sample Fortification ...................................................................................................15 

Preparation of Calibrants ...........................................................................................15 

EXTRACTION METHOD ..........................................................................................16 

LC-MS/MS CHROMATOGRAPHY ..........................................................................18 

Statistical Methods .......................................................................................................21 

Limit of Quantitation ...................................................................................................21



METHODS OF CALCULATION ...............................................................................22 

Preparation of Stock Standards ..................................................................................22 

Residue in Matrices ....................................................................................................22 

Time Required for Analysis .........................................................................................24 

RESULTS AND DISCUSSION ........................................................................................24 

CONCLUSIONS ................................................................................................................26 

LIST OF TABLES 

Table I. Summary of Method Validation Recoveries of MCW-2, Butene Sulfonic 

Acid and Thiazole Sulfonic Acid in Tomato. .............................................. 27 

Table II. Summary of Method Validation Recoveries of MCW-2, Butene Sulfonic 

Acid and Thiazole Sulfonic Acid in Pepper. ............................................... 30 

Table III. Summary of Method Validation Recoveries of MCW-2, Butene Sulfonic 

Acid and Thiazole Sulfonic Acid in Cucumber. .......................................... 33 

Table IV. Summary of Method Validation Recoveries of MCW-2, Butene Sulfonic 

Acid and Thiazole Sulfonic Acid in Melon. ................................................ 36 

LIST OF FIGURES 

Figure 1. Representative Chromatogram of Solvent Blank.Error! Bookmark not 

defined. 

Figure 2. Representative Chromatogram of MCW-2 at 0.0005 µg/mL. ............... Error! 

Bookmark not defined. 

Figure 3. Representative Chromatogram of MCW-2 at 0.00075 µg/mL. ............. Error! 


Figure 4. Representative Chromatogram of MCW-2 at 0.001 µg/mL. ................. Error! 

Bookmark not defined.



Figure 5. Representative Chromatogram of MCW-2 at 0.0025 µg/mL. ............... Error! 










Figure 10. 

defined. 

Representative MCW-2 Calibration Curve.Error! Bookmark not 

Figure 11. Representative Chromatogram of M-3627 at 0.0005 µg/mL. ........... Error! 


Figure 12. Representative Chromatogram of M-3627 at 0.00075 µg/mL. ......... Error! 


Figure 13. Representative Chromatogram of M-3627 at 0.001 µg/mL. ............. Error! 









Bookmark not defined.





Figure 19. 

defined. 

Representative Calibration Curve for M-3627.Error! Bookmark not 



Figure 21. Representative Chromatogram of M-3625 at 0.00075 µg/mL. ......... Error! 














Figure 28. 

defined. 

Representative Calibration Curve for M-3625.Error! Bookmark not 

Figure 29. Representative Chromatogram of Tomato Control.Error! Bookmark not 

defined. 

Figure 30. Representative Chromatogram of Fortified Control Tomato at 0.01 ppm. 

Error! Bookmark not defined.



Figure 31. Representative Chromatogram of Fortified Control Tomato at 0.10 ppm. 

Error! Bookmark not defined. 

Figure 32. Representative Chromatogram of Pepper Control.Error! Bookmark not 

defined. 

Figure 33. Representative Chromatogram of Fortified Control Pepper at 0.01 ppm. 


Figure 34. Representative Chromatogram of Fortified Control Pepper at 0.10 ppm. 


Figure 35. Representative Chromatogram of Cucumber Control.Error! Bookmark 

not defined. 

Figure 36. Representative Chromatogram of Fortified Control Cucumber at 0.01 

ppm. ...........................................................Error! Bookmark not defined. 

Figure 37. Representative Chromatogram of Fortified Control Cucumber at 0.10 

ppm. ...........................................................Error! Bookmark not defined. 

Figure 38. Representative Chromatogram of Melon Control.Error! Bookmark not 

defined. 

Figure 39. Representative Chromatogram of Fortified Control Melon at 0.01 ppm. 


Figure 40. Representative Chromatogram of Fortified Control Melon at 0.10 ppm. 


LIST OF APPENDICES 

Appendix A. Study Protocol and Protocol Deviation. Error! Bookmark not defined. 

Appendix B. Certificates of Analysis. .........................Error! Bookmark not defined. 

Appendix C. Method Validation Spreadsheets. ......................................................... 39



Appendix D. Final Report Amendment. ..................................................................... 40

SUMMARY 



A method for quantitation of MCW-2 and its metabolites in tomato, pepper, cucumber 

and melon was successfully validated. Control matrices were fortified at 0.01 mg/kg (the 

LOQ) and 0.10 mg/kg (10x LOQ). MCW-2 and its metabolites butene sulfonic acid (M- 

3627) and thiazole sulfonic acid (M-3625) were extracted from fortified tomatoes, 

pepper, cucumber and melon with acetonitrile:water. The extract was subjected to further 

cleanup involving an SPE cartridge cleanup. The final samples were analyzed by LC- 

MS/MS for quantitation of MCW-2, butene sulfonic acid and thiazole sulfonic acid. The 

limit of quantitation for all three analytes in all matrices is 0.01 ppm. The limit of 

detection was 0.45 ng/mL in solution. 

INTRODUCTION 

Makhteshim-Agan of North America, Inc. contracted with PTRL West, Inc. to conduct 

method development and validation of an analytical method for determination of MCW-2 

and its metabolites commonly named butene sulfonic acid and thiazole sulfonic acid in 

tomato, pepper, cucumber and melon. 

The study is designed to comply with OPPTS 860.1340 and SANCO/825/00 rev.7 dated 

17/03/04 guidelines. The study was initiated on January 15, 2010. This study was 

conducted from January 19 to May 13, 2010. The study was conducted at PTRL West, 

Inc., 625-B Alfred Nobel Drive, Hercules, CA 94547 under an approved protocol 

according to the US EPA FIFRA Good Laboratory Practice Standards, 40 CFR §160 

(Appendix A).

MATERIALS AND METHODS 

Reference Substances 



The reference substance of MCW-2 (lot number 326-115-01, PTRL Sample No. 1785W- 

003C) was supplied by LABService with a stated purity of 99.05%. The expiration date 

on the certificate of analysis is May 31, 2010 and it was stored refrigerated upon receipt. 

The reference standard of thiazole sulfonic acid sodium salt (M-3625, lot number 381- 

174-00, PTRL Sample No. 1788W-003) was supplied by Makhteshim Chemical Works 

with a stated purity of 90.9%. The expiration date on the certificate of analysis is 

December 31, 2011 and it was stored refrigerated upon receipt. The reference standard of 

butene sulfonic acid sodium salt (M-3627, lot number 215PAL2, PTRL Sample No. 

1785W-021) was supplied by PharmAgra Labs, Inc. with a stated purity of 99.1%. The 

expiration date on the certificate of analysis is September 10, 2010 and it was stored at 

room temperature. See Appendix B for the certificates of analysis. 

All stock and working solutions were stored at < 0°C in amber bottles with Teflon ® -lined 

screw-top caps. All solutions were prepared using pipettmen with diposable plastic tips, 

avoiding reuse of any glassware as carryover of the metabolites was suspected. All 

reference substances were concluded to be stable in solution throughout the study period 

based on comparison of chromatograms generated over the study period. 

Reagents and Solvents 

All solvents were HPLC grade unless noted: 

Acetonitrile (ACN) 

Formic Acid 

Water 

Glassware and Miscellaneous Equipment 

Balance 

Bottle, amber, with Teflon ® -lined cap, various sizes 

Bottle, plastic, 250 mL centrifuge 

Centrifuges, Mistral 3000E and Eppendorf 5415C 

Graduated Cylinder, various sizes 

Microfilterfuge tube, Rainin 0.45 UM Nylon-66

Pipettemen, assorted sizes with disposable tips 

C18 SPE Cartridges, BondElut 0.5 g/6 mL 

Vials, glass with Teflon ® -lined cap 

Volumetric pipette, various sizes 

ANALYTICAL PROCEDURES 

Preparation of Samples 



Untreated organic tomato, cucumber and pepper matrices were purchased from Nugget 

Market (Vacaville, CA). The untreated melon was purchased from Safeway (605 Parker 

Street, Rodeo, CA 94572). The control tomato, cucumber and pepper samples were 

processed using a Hobart Cutter-Mixer in the presence of dry ice. The melon sample was 

processed using a Hobart K-25 processor in the presence of dry ice. The processed 

samples were transferred to a zipper-locking bag and stored frozen, allowing the dry ice 

to sublime. The processed samples were stored frozen until removed for sub-sampling. 

Preparation of Stock Standards 

A stock standard of MCW-2 was prepared by dissolving 28.74 mg MCW-2 in 10 mL of 

acetonitrile. When adjusted for purity, the concentration of the MCW-2 stock standard 

was 2.85 mg/mL. A stock standard of butene sulfonic acid sodium salt was prepared by 

dissolving 22.87 mg M-3627 in 10 mL of acetonitrile:water (1:1, v/v). When adjusted for 

purity, the concentration of the butene sulfonic acid sodium salt stock solution was 2.27 

mg/mL. Similarly, a stock standard of thiazole sulfonic acid sodium salt was prepared by 

dissolving 21.17 mg of M-3625 in 10 mL of acetonitrile:water (1:1, v/v). When adjusted 

for purity, the concentration of the thiazole sulfonic acid stock solution was 1.92 mg/mL. 

An intermediate working solution was prepared for each analyte at 50 µg/mL by dilution 

with acetonitrile for MCW-2 and acetonitrile:water (1:1, v/v) for butene sulfonic acid 

sodium salt and thiazole sulfonic acid sodium salt. Due to suspected glassware carryover 

of the acid metabolites, reuse of these volumetric flasks was avoided. 

Preparation of Fortification Standards 

Mixed fortification solutions were prepared by dilution of the stock standards. A 10 

µg/mL mixed MCW-2, butene sulfonic acid sodium salt (M-3627) and thiazole sulfonic



acid sodium salt (M-3625) fortification solution was prepared by diluting 2 mL each of 

the respective 50 µg/mL working solutions to 10 mL in a 10 mL volumetric flask, where 

the solution was brought to the mark with acetonitrile:water (1:1). A 1.0 µg/mL mixed 

fortification solution was prepared by diluting 0.2 mL each of the respective 50 µg/mL 

working solutions to 10 mL in a 10 mL volumetric flask, where the solution was brought 

to the mark with acetonitrile:water (1:1). Due to suspected glassware carryover of the 

acid metabolites, reuse of these volumetric flasks was avoided. 

Sample Fortification 

Portions of control matrices (10 g) were fortified with MCW-2, butene sulfonic acid 

sodium salt (M-3627) and thiazole sulfonic acid sodium salt (M-3625) in replicates (5), 

as shown below, for determination of method recovery. 

Fortification Level 

(ppm) 

Volume of Mixed Fortification Solution 

0.01 0.10 mL of 1.0 µg/mL Mixed Fort. Soln. 

0.10 0.10 mL of 10 µg/mL Mixed Fort. Soln. 

Preparation of Calibrants 

All calibrant dilutions were prepared in amber bottles using appropriate pipettemen and 

disposable tips to dispense mixed standard solutions and solvents. An 5.0 µg/mL mixed 

stock standard solution was prepared by combining 1 mL each of the 50 µg/mL stock 

standards and diluting with 7 mL of acetonitrile:water (1:1, v/v) in a disposable amber 

glass bottle. The solution was sonicated prior to use. The following dilutions were 

prepared in disposable amber vials and diluted with acetonitrile:water (1:1, v/v).

Calibrant Conc. 

(µg/mL) 

Conc. Stock Std. 

(µg/mL) 



Volume (mL) Final Volume (mL) 

0.050 5.0 0.1 10 

0.025 5.0 0.05 10 

0.010 0.05 2.0 10 

0.005 0.05 1.0 10 

0.0025 0.025 1.0 10 

0.001 0.05 0.2 10 

0.00075 0.05 0.15 10 

0.0005 0.05 0.1 10 

Note: Actual concentrations of M-3625 and M-3627 were corrected by molecular weight 

conversion to account for the analysis of the free acid, rather than the sodium salt. The 

corrected concentrations for the M-3625 and M-3627 calibrants are presented below, 

where the molecular weight conversion factor is 0.896 for M-3627 and 0.901 for M- 

3625. 

Calibrant Conc. (µg/mL) Corrected Conc. 

M-3627 (µg/mL) 

Corrected Conc. 

M-3625 (µg/mL) 

0.050 0.045 0.045 

0.025 0.022 0.023 

0.010 0.0090 0.0090 

0.005 0.0045 0.0045 

0.0025 0.0022 0.0023 

0.001 0.00090 0.00090 

0.00075 0.00067 0.00068 

0.0005 0.00045 0.00045 

EXTRACTION METHOD 

1. Weigh out 10 g portions of processed matrix into 250 ml plastic bottles. 

2. Spike as necessary, using a pipetteman and disposable tips.

3. Add 50 ml of ACN:HPLC water (1:1, v/v) to each bottle. 



4. Tissuemize for approximately 5 minutes. Rinse probe with 10 mL of ACN:HPLC 

water (1:1, v/v). Total volume 60 mL. 

5. Place bottles onto wrist action shaker for 5 minutes 

6. Transfer 30 mL of extract into 50 mL centrifuge plastic tube. 

7. Centrifuge at 2,000 rpm for 10 minutes 

8. Transfer aliquot of the supernatant to a snap-top GC vial for the direct MCW-2 

analysis. Microfilterfuge the sample as needed. 

9. Transfer 6.0 mL of the supernatant to 10 mL plastic centrifuge tube. Concentrate 

sample to 3 mL in Turbo-vap at ~35ºC. 

SPE Clean-up 

10. Condition the Bond Elut 500mg, 6cc SPE cartridge with 5 mL of ACN, then 5 mL 

of water. 

11. Apply concentrated extract from step 9 and collect the eluate into a clean plastic 

graduated tube. 

12. Rinse the sample tube from Step 9 with 5 mL of water, apply to the same 

cartridge and collect the eluate into the same graduated tube. Record the volume. 

Total volume should be about 8 mL. Mix the sample by vortexing. 

13. Aliquot in snap-top vials and analyze on LC-MS/MS for butene sulfonic acid and 

thiazole sulfonic acid. 

Note: In an effort to correct for the moisture derived from the matrix, three representative 

samples from each matrix were extracted as described in steps 1 through 5 and the 

samples were vacuum filtered through a Büchner funnel fitted with Whatman GF/A filter 

paper. The resultant extract volumes were measured. The average volumes were 66 mL 

for tomato, cucumber and green pepper and 63 mL for melon.

LC-MS/MS CHROMATOGRAPHY 



SCIEX API4000 Components (HPLC/Turbo Ion Spray Mode) or equivalent: 

LC Pump Agilent 1100 Series Binary Pump, Model G1312A 

Autosampler Agilent 1100 Series Autosampler, Model G1329A 

Vacuum Degasser Agilent 1100 Series Vacuum Degasser, Model G1379A 

A. MCW-2 Analysis Method 

Mass Spectrometer Settings 

Scan Type: MRM 

Polarity: Positive 

Ion Source: APCI (+) 

CUR: 40.0 

GS1: 70.0 

GS2: 0.0 

TEMP: 500.0 

CAD: 9.0 

Period 1 settings: 

Q1 Mass (amu) Q3 Mass (amu) Dwell (msec) 

292 166 (quantitation ion) 200 


292 89 (confirmation ion) 200 



Column: Synergi 4µ Fusion-RP 80A (75 mm x 2.0 mm) fitted with a Fusion guard 

cartridge (AJO-7556) 

Column Temperature: 30°C

Injection Volume: 20 µL 

Solvent System: 

Solvent A = Water (0.05% formic acid) 

Solvent B = Acetonitrile (0.05% formic acid) 

Solvent Program: Minutes Flow Rate Solvent A Solvent B 

0 0.20 mL/min 95 5 

1.0 0.20 mL/min 95 5 

15.0 0.20 mL/min 0 100 

16.0 0.40 mL/min 0 100 

16.5 0.35 mL/min 95 5 

18.0 0.20 mL/min 95 5 

Retention Time: MCW-2 was ~12.6 minutes 



B. Butene Sulfonic Acid (M-3627) and Thiazole Sulfonic Acid (M-3625) 

Analysis Method 

Mass Spectrometer Settings 

Period 1 

Scan Type: MRM 

Polarity: Negative 

Ion Source: ESI (-) 

CUR: 35.0 

GS1: 40.0 

GS2: 40.0 

TEMP: 500.0 

CAD: 8.0

Period 1 settings (Butene sulfonic acid): 





Period 2 settings: 







Column: Synergi 2.5µ Fusion-RP 100A (100mm x 2.00 mm) fitted with a Fusion guard 

cartridge (AJO-7556) 

Column Temperature: 30°C 

Injection Volume: 20 µL 

Solvent System: 

Solvent A = Water (0.05% formic acid) 

Solvent B = Acetonitrile (0.05% formic acid) 

Solvent Program: Minutes Flow Rate Solvent A Solvent B 

0 0.25 mL/min 95 5 

1.0 0.25 mL/min 95 5 

9.0 0.25 mL/min 42 58 

9.5 0.30 mL/min 0 100 

10.5 0.35 mL/min 0 100 

11.5 0.30 mL/min 95 5 

13.5 0.25 mL/min 95 5 

14.0 0.25 mL/min 95 5

Retention Time: Butene sulfonic acid (M-3627) was ~6.5 minutes 

Thiazole sulfonic acid (M-3625) was ~7.7 minutes 



Separation of the analytes was achieved by HPLC. The analytes were identified by the 

coincidence of their retention times with the reference standards and MS characteristics, 

and quantitated by integration of the peak areas. 

A typical injection sequence for MCW-2 in a method validation set as analyzed by LC- 

MS/MS was: solvent blank, 0.5 ng/mL calibrant, 0.75 ng/mL calibrant, 1.0 ng/mL 

calibrant, 2.5 ng/mL calibrant, 5.0 ng/mL calibrant, 10 ng/mL calibrant, 25 ng/mL 

calibrant, 50 ng/mL calibrant, solvent blank, 0.5 ng/mL calibrant, 0.75 ng/mL calibrant, 

reagent blank, control sample, control sample, 1.0 ng/mL calibrant, fortified control, 

fortified control, fortified control, 2.5 ng/mL calibrant, fortified control, fortified control, 

5.0 ng/mL calibrant, fortified control, fortified control, fortified control, 10 ng/mL 

calibrant, fortified control, fortified control, 25 ng/mL calibrant, 50 ng/mL calibrant, QC 

standard. The initial injection of calibrants was used to generate the calibration curve and 

the interspersed calibrants were used as quality control samples to insure the stability of 

the analysis throughout the sequence. A similar injection sequence was used for butene 

sulfonic acid and thiazole sulfonic acid analysis. 

Statistical Methods 

The residue data included the following statistical calculations: means, standard 

deviations, relative standard deviations and linear regression analysis. 

Limit of Quantitation 

The limit of quantitation was assigned as the lowest fortification level of analyte 

validated by the residue method. The LOQ for MCW-2, butene sulfonic acid sodium salt 

(M-3627) and thiazole sulfonic acid sodium salt (M-3625) in tomato, pepper, cucumber 

and melon was 0.01 ppm. The limit of detection was assigned as the lowest calibrant used 

in the analysis or 0.5 ng/mL MCW-2 and 0.45 ng/mL butene sulfonic acid and thiazole 

sulfonic acid.

METHODS OF CALCULATION 

Preparation of Stock Standards 

(W) x (P) 

Volume of solvent (mL) = 

(FC) 

where W = Milligrams of neat standard 

P = Chemical purity of neat standard 

FC = Final Concentration (mg/mL) 

Residue in Matrices 



The MCW-2, butene sulfonic acid (M-3627) and thiazole sulfonic acid (M-3625) 

quantitation was conducted by peak area relative to an external calibration curve. 

Linear regression formula for analyte peak area, calibration curve y = mx + b 

where y = peak area 

x = µg/mL analyte injected 

m = Slope 

b = Calibration intercept 

For butene sulfonic acid and thiazole sulfonic acid, the linear regression analysis was 

prepared using Excel 2007, where the molecular weight conversion factor had been used 

to convert the concentrations of sodium salts to concentrations as free acids. The 

molecular weight of butene sulfonic acid sodium salt is 212.12 g/mole, while the free 

acid molecular weight is 190.14 g/mole. The molecular weight conversion factor was 

190.14 divided by 212.12 or 0.896 for butene sulfonic acid. The molecular weight of 

thiazole sulfonic acid sodium salt is 221.62 g/mole, while the free acid molecular weight 

is 199.64 g/mole. The molecular weight conversion factor for the thiazole sulfonic acid 

was 0.901. 

µg/mL butene sulfonic acid = 0.896 x µg/mL butene sulfonic acid sodium salt 

µg/mL thiazole sulfonic acid = 0.901 x µg/mL thiazole sulfonic acid sodium salt 

The linear regression generated by the LCMS program (Analyst 1.4.2) was used to 

calculate the concentration (ng/mL) of the MCW-2 present in the sample for tomato



method validation, where the remaining were conducted with linear regression as 

calculated with Excel. 

The residue on treated matrices was calculated as follows: 

ppm MCW-2 (mg/kg) = 

ng/mL MCW - 2 x Init. Extract vol. 

(mL) x 0.001g/ng 

Sample Wt. (g) 

ppm butene sulfonic acid or thiazole sulfonic acid = 

g/mL 

acid 

x Eluate vol. (mL) x dilution factor x Init. Extract Vol. (mL) 

Aliquot vol. 

(mL) x Sample Wt. (g) 

Percent recovery for each analyte was calculated as follows: 

% MCW-2 Recovery = 

% BSA or %TSA Recovery = 

Residue Detected (ppm) - Average ppm Control 

Fortification 

Level (ppm) 

Residue Detected (ppm) - Average ppm Control 

Fortification 

Level(ppm) 

x Mol. Wt. Conversion Factor 

x 100 

x 100 

Validity of the MCW-2, butene sulfonic acid and thiazole sulfonic acid residue analytical 

methods was established by acceptable average recovery (70-110%) from fortified 

untreated control samples. 

An example calculation for the MCW-2 recovery in the tomato method validation (0.01 

ppm) is shown below: 

MCW-2 ppm = 

1.57 ng/mL x 

66 mL x 

10 g 

Percent Recovery MCW-2 = 

0.001 

0. 010 

0. 

000 

0.01ppm 

= 0.010 ppm 

ppm 

x 100 = 100% 

An example calculation for the butene sulfonic acid recovery in cucumber method 

validation is shown below, where a similar calculation was conducted for thiazole 

sulfonic acid:

ppm butene sulfonic acid = 

0. 0009 g/mL 

x 8.0 mL x 1x 

66 mL 

6.0 mL x 10 g 

0. 0079 0. 

000 ppm 

Percent Recovery = 

= 88% 

0.01ppm 

x 0.896 

Time Required for Analysis 



= 0.0079 

Time required per sample set, where a sample set consists of twelve (12) matrix samples, 

8 standards and 1 reagent blank: 

Extraction and Clean-up take approximately 8 hours for one analyst 

LC-MS/MS analysis and data processing takes approximately 28 hours 

TOTAL = approximately 14 hours for one analysis (2 calendar days) 

RESULTS AND DISCUSSION 

Method validation was successfully conducted for MCW-2 and its metabolites M-3625 

(thiazole sulfonic acid, TSA) and M-3627 (butene sulfonic acid, BSA) in tomatoes, 

peppers, cucumbers and melons. The validations were conducted at 0.01 ppm and 0.10 

ppm. The method validation results are summarized in Tables I through IV for tomatoes, 

pepper, cucumbers and melons, respectively. The supporting data is provided in 

Appendix C. Due to the chemical nature of the analytes, MCW-2 has one quantitation ion 

and two confirmation ions. Both M-3625 and M-3627 have one quantitation ion and one 

confirmation ion. Representative calibrant chromatograms and calibration curves are 

presented in Figures 1 through 28. 

Method validation for MCW-2, M-3625 and M-3627 in tomatoes was conducted with 

two controls, five (5) replicates at 0.01 ppm and 5 replicates at 0.10 ppm. The average 

percent recovery for MCW-2 was 106% at 0.01 ppm and 98% at 0.10 ppm. The overall 

average percent recovery of MCW-2 in tomatoes was 102% with a standard deviation of 

13%. The average percent recovery of M-3625 was 83% at 0.01 ppm and 84% at 0.10 

ppm. The overall average percent recovery of M-3625 in tomatoes was 84% with a 

standard deviation of 9%. The average percent recovery of M-3627 was 95% at 0.01 ppm 

and 97% at 0.10 ppm. The overall average percent recovery of M-3627 in tomatoes was



96% with a standard deviation of 7%. The confirmation ions for MCW-2, M-3625 and 

M-3627 yielded acceptable recoveries at both fortification levels and the overall average 

recoveries were acceptable. Representative chromatograms of control and fortified 

tomato samples are presented in Figures 29 through 31. 

Method validation for MCW-2, M-3625 and M-3627 in pepper followed the same 

method as was used in the tomato method validation. The pepper validation included 

with two controls, five (5) replicates at 0.01 ppm and 5 replicates at 0.10 ppm. The 

average percent recovery for MCW-2 was 103% at 0.01 ppm and 106% at 0.10 ppm. The 

overall average percent recovery of MCW-2 in peppers was 105% with a standard 

deviation of 9%. The average percent recovery of M-3625 was 100% at 0.01 ppm and 

94% at 0.10 ppm. The overall average percent recovery of M-3625 in peppers was 97% 

with a standard deviation of 7%. The average percent recovery of M-3627 was 84% at 

0.01 ppm and 95% at 0.10 ppm. The overall average percent recovery of M-3627 in 

peppers was 90% with a standard deviation of 7%. The confirmation ions for MCW-2, 

M-3625 and M-3627 yielded acceptable recoveries at both fortification levels and the 

overall average recoveries were acceptable in peppers. Representative control and 

fortified pepper chromatograms are presented in Figures 32 through 34. 

Method validation was similarly carried out in cucumbers. The cucumber validation 

included two controls, five (5) replicates at 0.01 ppm and 5 replicates at 0.10 ppm. The 

average percent recovery for MCW-2 was 103% at 0.01 ppm and 89% at 0.10 ppm. The 

overall average percent recovery of MCW-2 in cucumber was 96% with a standard 

deviation of 8%. The average percent recovery of M-3625 was 103% at 0.01 ppm and 

97% at 0.10 ppm. The overall average percent recovery of M-3625 in cucumber was 

100% with a standard deviation of 6%. The average percent recovery of M-3627 was 

86% at 0.01 ppm and 97% at 0.10 ppm. The overall average percent recovery of M-3627 

in cucumber was 92% with a standard deviation of 7%. The confirmation ions for MCW- 

2, M-3625 and M-3627 yielded acceptable recoveries at both fortification levels and the 

overall average recoveries were acceptable in cucumber. Representative control and 

fortified cucumber chromatograms are presented in Figures 35 through 37. 

Method validation for MCW-2, M-3625 and M-3627 in melon included with two 

controls, five (5) replicates at 0.01 ppm and 5 replicates at 0.10 ppm. The average percent 

recovery for MCW-2 was 101% at 0.01 ppm and 96% at 0.10 ppm. The overall average 

percent recovery of MCW-2 in melon was 98% with a standard deviation of 5%. The



average percent recovery of M-3625 was 83% at 0.01 ppm and 79% at 0.10 ppm. The 

overall average percent recovery of M-3625 in melon was 81% with a standard deviation 

of 4%. The average percent recovery of M-3627 was 83% at 0.01 ppm and 76% at 0.10 

ppm. The overall average percent recovery of M-3627 in melon was 79% with a standard 

deviation of 6%. The confirmation ions for MCW-2, M-3625 and M-3627 yielded 

acceptable recoveries at both fortification levels and the overall average recoveries were 

acceptable in melon. Representative control and fortified melon chromatograms are 

presented in Figures 38 through 40. 

CONCLUSIONS 

An analytical method was developed at validated for the analysis of MCW-2 and its 

metabolites thiazole sulfonic acid (M-3625) and butene sulfonic acid (M-3627) in 

tomatoes, peppers, cucumbers and melons. The method was validated at 0.01 ppm and 

0.10 ppm, where no residues were detected in control matrices. The limit of quantitation 

for all three analytes in all matrices is 0.01 ppm. The limit of detection was 0.45 ng/mL 

in solution.



Table I. Summary of Method Validation Recoveries of MCW-2, Butene 

Sulfonic Acid and Thiazole Sulfonic Acid in Tomato. 

Quantitation ions 

Fortification Level Percent Recovery 

Sample (ppm) MCW-2 M-3625 M-3627 

Controls NA NA NA NA 

Fort 1A 0.01 100 78 97 

Fort 1B 0.01 110 69 88 

Fort 1C 0.01 100 78 98 

Fort 1D 0.01 130 97 97 

Fort 1E 0.01 90 95 96 

Average 106 83 95 

Standard Deviation 15 12 4 

% Rel. Std. Dev. 14 14 4 

Fort 2A 0.10 116 88 96 

Fort 2B 0.10 94 89 97 

Fort 2C 0.10 93 82 81 

Fort 2D 0.10 100 72 102 

Fort 2E 0.10 88 89 107 

Average 98 84 97 


% Rel. Std. Dev. 11 8 10 

Overall Average 102 84 96 

Overall Standard Deviation 13 9 7 

Overall Average %RSD 13 11 7 

NA =Not Applicable 

M-3625 = Thiazole sulfonic acid 

M-3627 = Butene sulfonic acid



Table I (cont.). Summary of Method Validation Recoveries of MCW-2, Butene 


Confirmation ions 




Fort 1A 0.01 100 87 97 

Fort 1B 0.01 120 88 98 

Fort 1C 0.01 110 88 98 

Fort 1D 0.01 120 107 97 

Fort 1E 0.01 100 114 96 

Average 110 97 97 


% Rel. Std. Dev. 9 13 1 

Fort 2A 0.10 112 85 96 

Fort 2B 0.10 94 83 99 

Fort 2C 0.10 100 77 85 

Fort 2D 0.10 98 77 97 

Fort 2E 0.10 88 87 104 



% Rel. Std. Dev. 9 6 7 









Table I (cont.) Summary of Method Validation Recoveries of MCW-2, Butene 


Confirmation ion /z 59 




Fort 1A 0.01 100 NA NA 

Fort 1B 0.01 120 NA NA 

Fort 1C 0.01 100 NA NA 

Fort 1D 0.01 130 NA NA 

Fort 1E 0.01 90 NA NA 

Average 108 NA NA 

Standard Deviation 16 NA NA 

% Rel. Std. Dev. 15 NA NA 

Fort 2A 0.10 110 NA NA 

Fort 2B 0.10 94 NA NA 

Fort 2C 0.10 99 NA NA 

Fort 2D 0.10 100 NA NA 





Overall Average 103 NA NA 

Overall Standard Deviation 13 NA NA 

Overall Average %RSD 13 NA NA 






Table II. Summary of Method Validation Recoveries of MCW-2, Butene 

Sulfonic Acid and Thiazole Sulfonic Acid in Pepper. 





Fort 1A 0.01 106 99 79 

Fort 1B 0.01 92 98 78 

Fort 1C 0.01 92 88 88 

Fort 1D 0.01 119 108 88 

Fort 1E 0.01 106 108 88 

Average 103 100 84 


% Rel. Std. Dev. 11 8 6 

Fort 2A 0.10 106 94 95 

Fort 2B 0.10 107 94 95 

Fort 2C 0.10 110 92 91 

Fort 2D 0.10 112 95 95 

Fort 2E 0.10 96 93 100 

Average 106 94 95 


% Rel. Std. Dev. 6 1 3 









Table II (cont.). Summary of Method Validation Recoveries of MCW-2, Butene 






Fort 1A 0.01 99 69 79 

Fort 1B 0.01 92 69 79 

Fort 1C 0.01 79 78 78 

Fort 1D 0.01 99 98 88 

Fort 1E 0.01 86 88 88 



% Rel. Std. Dev. 10 16 6 

Fort 2A 0.10 103 85 92 

Fort 2B 0.10 108 85 94 

Fort 2C 0.10 110 88 90 

Fort 2D 0.10 109 85 93 

Fort 2E 0.10 90 95 96 

Average 104 88 93 


% Rel. Std. Dev. 8 5 2 









Table II (cont.). Summary of Method Validation Recoveries of MCW-2, Butene 






Fort 1A 0.01 99 NA NA 



Fort 1D 0.01 106 NA NA 





Fort 2A 0.10 106 NA NA 

Fort 2B 0.10 110 NA NA 

Fort 2C 0.10 110 NA NA 

Fort 2D 0.10 110 NA NA 













Table III. Summary of Method Validation Recoveries of MCW-2, Butene 

Sulfonic Acid and Thiazole Sulfonic Acid in Cucumber. 





Fort 1A 0.01 99 108 88 

Fort 1B 0.01 106 110 93 

Fort 1C 0.01 106 95 86 

Fort 1D 0.01 99 108 78 

Fort 1E 0.01 106 95 86 

Average 103 103 86 


% Rel. Std. Dev. 4 8 6 

Fort 2A 0.10 88 98 98 

Fort 2B 0.10 90 96 100 

Fort 2C 0.10 91 101 98 

Fort 2D 0.10 88 97 91 

Fort 2E 0.10 87 95 99 



% Rel. Std. Dev. 2 2 4 









Table III (cont.). Summary of Method Validation Recoveries of MCW-2, Butene 






Fort 1A 0.01 92 108 118 

Fort 1B 0.01 99 101 110 

Fort 1C 0.01 106 95 105 

Fort 1D 0.01 99 98 108 

Fort 1E 0.01 99 95 105 

Average 99 99 109 


% Rel. Std. Dev. 5 5 5 

Fort 2A 0.10 90 92 95 

Fort 2B 0.10 94 91 91 

Fort 2C 0.10 92 98 96 

Fort 2D 0.10 87 94 97 

Fort 2E 0.10 87 95 95 



% Rel. Std. Dev. 3 3 2 









Table III (cont.). Summary of Method Validation Recoveries of MCW-2, Butene 









Fort 1D 0.01 79 NA NA 





















Table IV. Summary of Method Validation Recoveries of MCW-2, Butene 

Sulfonic Acid and Thiazole Sulfonic Acid in Melon. 





Fort 1A 0.01 101 83 84 

Fort 1B 0.01 107 88 88 

Fort 1C 0.01 95 80 89 

Fort 1D 0.01 101 80 80 

Fort 1E 0.01 101 83 74 

Average 101 83 83 


% Rel. Std. Dev. 4 4 7 

Fort 2A 0.10 90 74 69 

Fort 2B 0.10 94 75 74 

Fort 2C 0.10 100 81 79 

Fort 2D 0.10 98 81 77 

Fort 2E 0.10 96 82 79 



% Rel. Std. Dev. 4 5 5 









Table IV (cont.). Summary of Method Validation Recoveries of MCW-2, Butene 






Fort 1A 0.01 95 73 84 

Fort 1B 0.01 101 88 98 

Fort 1C 0.01 101 80 80 

Fort 1D 0.01 95 71 80 

Fort 1E 0.01 88 73 74 



% Rel. Std. Dev. 5 9 11 

Fort 2A 0.10 88 73 72 

Fort 2B 0.10 92 71 76 

Fort 2C 0.10 96 80 77 

Fort 2D 0.10 95 81 77 

Fort 2E 0.10 95 82 79 



% Rel. Std. Dev. 3 6 4 









Table IV (cont.). Summary of Method Validation Recoveries of MCW-2, Butene 






Fort 1A 0.01 107 NA NA 

Fort 1B 0.01 101 NA NA 

Fort 1C 0.01 107 NA NA 

Fort 1D 0.01 101 NA NA 

Fort 1E 0.01 101 NA NA 



















Report 2 Page 39

Appendix A. Final Report Amendment. 

FINAL REPORT AMENDMENT NO. 1 



A. Title: Method Validation of an Analytical Method for the Determination of 

MCW-2 and its Metabolites in Plant Matrices 

B. Report Modifications: 

Pages were updated to include the following changes/additions: 

The synonym for butene sulfonic acid was changed from MAN-2 to M-3627 

(pages 1, 12, 17and 21) 

The name of M-3625 was changed to thiazole sulfonic acid sodium salt and M- 

3627 to butene sulfonic acid sodium salt (pages 11, 12 and 19) when referring to 

the reference substances in the solid form. 

An additional table and text was added on page 14. 

The LOD was changed to 0.45 ng/mL for thiazole sulfonic acid and butene 

sulfonic acid 

The residue calculations were updated to account for spiking with butene sulfonic 

acid sodium salt and thiazole sulfonic acid sodium salt, while analyzing for 

butene sulfonic acid and thiazole sulfonic acid by LC-MS/MS. This update allows 

for correct calculation of the percent recovery and will allow for quantitation of 

butene sulfonic acid and thiazole sulfonic acid residues as ppm free acids (pages 

20 and 22, Appendix C). 

All the tables and text (pages 21 to 24) were updated with the recalculated percent 

recoveries. The representative calibration curve figures were updated (pages 19 

and 28). The spreadsheets for butene sulfonic acid and thiazole sulfonic acid were 

updated (Appendix C) 

C. Reason(s): 

Corrects the residue method calculation for fortification with sodium salts but 

residue analysis as free acids for butene sulfonic acid (M-3627) and thiazole 

sulfonic acid (M-3625).

D. Impact on Study: 

Prepared By: 

No adverse impact on the study is expected. 


Amended Study Completion Date 


Report 2 Page 41

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