Cover Sheet for ENVIRONMENTAL CHEMISTRY METHOD Pestcide Name: Azoxystrobin MRID #: 436781-91 Matrix: Soil Analysis: HPLC/UV This method is provided to you by the Environmental Protection Agency's (EPA) Environmental Chemistry Laboratory (ECL). This method is not an EPA method but one which was submitted to EPA by the pesticide manufacturer to support product registration. EPA recognizes that the methods may be of some utility to state, tribal, and local authorities, but makes no claim of validity by posting these methods. Although the Agency reviews all Environmental Chemistry Methods submitted in support of pesticide registration, the ECL evaluates only about 30% of the currently available methods. Most methods perform satisfactorily but some, particularly the older methods, have deficiencies. Moreover, the print quality of the methods varies considerably because the methods originate from different sources. Therefore, the methods offered represent the best available copies. If you have difficulties in downloading the method, or further questions concerning the methods, you may contact Elizabeth Flynt at 228-688-2410 or via e-mail at flyntelizabeth@epa.gov.Page 24 Report No: R1864B Recipient : CopyNo: eee EEE TEE STANDARD OPERATING PROCEDURE RAM 269/01 RESIDUE ANALYTICAL METHOD FOR THE ANALYSIS OF ICIASSD4, R230310, R234886, R401553 and R402173 IN SOIL. Issue Date: Review Date nnuatly Authors + R1 Johnson, OJ Tummon Issuing Section _: Residue Chemistry M Eari Authorised by : R Elliott Section Manager ied, Copies are available from the Environmental istration Centre. (Contact extn 4322), REFERENCE COPY Page 1 of 7810 40.1 10.2 " Page 25 Report No: RJ1864B CONTENTS SCOPE. ‘SUMMARY |... PROCEDURE Extraction 2.22.1 : Liquidtiquid partition. 1.11! Preparation of ICIASS04, R23031 Derivatization of R4O1553 . . Derivatization of R402173 .... |. : Preparation of ICIAS504, R230310, R234886, Quantitative HPLC-MS-MS Analysis NANoao HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ULTRA-VIOLET DETECTION (HPLC-UV) : High Performance Liquid Chromatography Conditions GAS CHROMATOGRAPHY WITH MASS SELECTIVE DETECTION (GC-MS) 10 GC-MS Conditions for Analysis of R401553 Derivative re . ~ 10 Gas-Liquid Chromatography Conditions . . . wae 10 Mass Spectrometry Conditions sees . teense eee 10 GC-MS Conditions for Analysis of R402173 Derivative rr » 1 Gas-Liquid Chromatography Conditions . 1 Mass Spectrometry Conditions. " HIGH PERFORMANCE LIQUID: CHROMATOGRAPHY WITH TRIPLE QUADRUPOLE MASS SPECTROMETRY DETECTION (HPLC-MS-MS) .......... 12 CALCULATION OF RESIDUE RESULTS ........ wee . 13, CONTROL AND RECOVERY EXPERIMENTS . . 13 LIMIT OF DETERMINATION ... i see 13, METHOD VALIDATION STUDIES .......... |. pee fae 14 Recovery Data oe 4 Linearity Statement . EXAMPLES OF TYPICAL CHROMATOGRAMS - see Appendix 4 See erate. TABLE 2 : Results of Soil Analysis by HPLC-UV .. 7 TABLE 3 : Results of Soil Analysis by GC-MS .. 18 TABLE 4 : Results of Soil Analysis by HPLC-MS-MS 19 REFERENCE COPY RAM 268/01 Page No 2Page 26 Report No: RJ1864B CONTENTS Pa APPENDIX 4 Typical High Performance Liquid Chromatograms for ICLASS04, R230310 and R234886 Residue Determination in Soil ...... APPENDIX 2 Typical GC-MS Chromatograms for R401853 Residue Determination in Soil... 36 APPENDIX 3 Typical GC-MS Chromatograms for R402173 Residue Determination in Soil .... 43 ‘APPENDIX 4 ‘Typical High Performance Liquid Chromatography - Triple Quadrupole Mass Spectrometry Chromatograms for ICIA5504, R230310, R234886, R401553 and R402173 Residue Determination in Soll. .. : p APPENDIX 5 MaterialsiSafety . Apparatus Reagents . sone REFERENCE COPY RAM 269101 Page No 3Page 27 Report No: RJ1864B SCOPE The analytical procedures described are suitable for the determination of residues of the fungicide IC1AS504 (Figure 1), its geometrical isomer R230310 (Figure 2), and its soll ‘metabolites R234886 (Figure 3), R401553 (Figure 4) and R402173 (Figure §). To date, in these laboratories, the method has been applied to a variety of soil samples and the limits of determination of the method are 0.02 mg kg” for ICIAS504, R230310 and 234886, and 0.01 mg kg’ for R4015S3 and R402173, NAN oA, a He? cH, 0 Figure 1: Methyl (E)-2-(2-6-(2-cyanophenoxy)pyrimidin-4-yloxy|phenyl}-3- methoxyacrylate (UPAC), ns CO, cN oO. cH, uc? © Figure 2: Methyl @)-2-{2-16-(2-cyanophenoxy)pytimidin-4-yloxy}phenyl}-3- methoxyacryiate (IUPAC). SES fo} Figure 3: {Opa 2evsnoPhenoxy pyrimicin-+-yloxyiphenyt-3-methoxyacryte acid UF REFERENCE COPY RAM 269/01 Page No 4Page 28 Report No: R1864B a ot CN Figure 4: 4-(2-cyanophenoxy)-6-hydroxypyrimidine Figure S$: 24{6-(2-cyanophenoxy)pyrimidin-4-yloxy}benzoic acid (IUPAC). SUMMARY ICIAS504. R230310, R234886, R401553 and R402173 residues in soil samples are Qxtracted in 75:25 methanol:1M hydrochloric acid. An aliquot of the extract is subjected to |iquid-fiquid partition with acidified sodium chloride solution and dichloromethane. ‘The combined dichloromethane extract is evaporated to dryness and taken up in a known volume of chloroform. An aliquot is blown to dryness and resuspended in mobile phase for quantitative determination of ICIASS04, R230310 and R234886 resi using high performance liquid chromatography with triple quadrupole mass spectrometry. REFERENCE COPY RAM 269/01 Page No 534 32 Page 28 Report No: RJ1864B PROCEDURE Extraction a) ») ©) ®) Thoroughly mix the sample and weigh a representative aliquot (20 g), into @ screw top Nalgene bottle (250 em’). Fortity @ minimum of two contrat samples with an accurately known amount of ICIASSO4, R230310, R234886, R401553 and R402173 as recovery checks. ‘Add 75:25 methanol:1M hyd 130 +/- 20 rpm). Centi then decant the supem: rochioric acid (50 cm’) and shake for 30 minutes (at ifuge at 3500 +/- 500 rpm for approximately 4 minutes and ratant into @ round bottom flask. If centrifugation fails to Produce a supematant without soil particulate material then the supematant can be filtered through @ Whatman No. 1-or No. § fiter paper into'the round bottom flask. ‘Add a further aliquot of 75:25 methano1M hydrochloric acid (50 cm) to each ‘Sample and shake for a further 15.minutes (at 130 +/- 20 rpm). Centrifuge at 3500 {$i $00 rom for approximately 4 minutes and then decant the supematant through a Whatman No. 1 or No. 5 fier paper into the same round bottom flask , to combine the two extracts from each sample. Rinse the debris and residiuum with further washes of the extraction solvent. Adjust to a suitable known volume (eg.100-120 cm?) with 75:25 methanol: 1M hydrochloric acid. Liquid-tiquid partition a) » 9 Prepare an ac chloride in ultra- fed 5% (wv) sodium chloride solution by dissolving 15 g of sodium ‘pure water (300 cm") and adding 1M hydrochforic acid (15 cm?), Take @ 2 g soil aliquot from 3.1 (e) and partition with an equivalent volume of dichloromethane and an equivalent volume of acidified 5% (wiv) sodium chloride ‘Solution in @ separatory funnel. Collect the dichloromethane layer into a round bottom flask. ‘Add a further equivalent volume of dichloromethane to the aqueous extract and repartition. Combine the dichloromethane layers in the round bottom flask. Evaporate the dichloromethane to dryness on a rotary evaporator at <40°C and redissolve in chloroform (2 em’) using uttrasonication, Note: Additional soil aliquots from an untreated ‘sample should be taken through the procedure to be used to generate sta indards in presence of matrix. REFERENCE COPY RAM 269/01 Page No 633 34 a5 Page 30 Report No: RJ1864B Preparation of ICIA5504, R230310 and R234886 Samples for HPLC-UV Analysis Remove an aliquot equivalont to 1 g from 3.2 (c). Evaporate to dryness using a stream of lean, dry ait and re-suspend in a suitable volume (1 em) of HPLC mobile phase (60:40, \water:acetonitrile + 0.4% (VN) glacial acetic acid). Great care should be taken when fedissolving the sample to ensure quantitative transfer into the HPLC vial for analysis, Derivatization of R401553 8) Transfer @ 0.20 cm? atquot (equivalent to 0.2 g of soil) of the resuspended solution from 3.2( c) to an appropriate GC vial, . >) Add to the alquct 0.7 cm* chloroform and 0.10 cm of the derivatizing reagent N- ‘methyLN-(tertbutyidimethylsily)trifuoroacetamide (MTBSTFA ). ©) Gap the vial and leave to stand, with occasional shaking, at room temperature for ‘one hour and then analyse for the derivative by GC-MS, ‘A 0.50 em aliquot (equivalent to 0.5 g of soll) may be derivatised by the same procedure in order to achiove the required sensitivity, if necessary. 401859 derivatized standards must be prepared in the presence of soll matrix prior to analysis, as different responses are achieved in the presence of matrix compared to when ‘matrix is absent. . Standards are prepared as follows: © Transfer a 0.20 cm’ aliquot (equivalent to 0.2 g of soi) of the resuspended Solution from 3.2( ¢) of a control soil extract to an appropriate GC vial. @ Add an appropriate amount of R401553 standard to produce the required final standard concentration. Gi) Add 0.7 cm? minus the volume of standard added in (i) above, of chloroform, (v) Add 0.10 cm? of MTBSTFA, Cap the vial and leave to stand, with occassional shaking, at room temperature for one hour. W) Analyse standards alongside samples on GC-MS, Derivatization of R402173 8) Transfer a 0.50 cm? aliquot (equivalent to 0.5 from 3.2( c) to a disposable tube. 5) Add tothe aliquot 1 cm? of an ethereal solution of diazomethane and leave to stand ‘at room temperature for 30 minutes, 9 of soil) of the resuspended solution All operations carried out using diazomethane must be cartied out in a fume cupboard, REFERENCE COPY RAM 269/01 Page No 736 Page 31 Report Ru18648 ©) —__Evaporate the tube contents to dryness using a siream of clean, dry air, Resuspend in chloroform (1 em) and analyse for the methylated derivative by GC- Ms. ‘402173 derivatized standards must be prepared in the presence of soil matrix prior to analysis, as different responses are achieved in the presence of matrix compared to when ‘matrix is absent. Standards are prepared as follows: © Transfer a 0.50 cm? aliquot (equivalent to 0.5 g of soi) of the resuspended Solution from 3.2( c) of a control soil extract to an appropriate GC vial (Add an appropriate amount of R402173 standard to produce the required final standard concentration. Gi) Add 1 cm? of an ethereal solution of diazomethane and leave to stand at ‘oom temperature for 30 minutes. (™) _Evaporate the tube contents to dryness using a stream of clean, dry air anc resuspend in chloroform (1 om). (¥) Analyse standards alongside samples on GC-MS. Preparation of ICIASS04, R230310, R234886, R401853 and R402173 Samples for ‘Quantitative HPLC-MS-MS Analysis 2) Transfer a 0.50 cm? aliquot (equivalent to 0.5 g of soil of the resuspended solution from 3.2( c) to an appropriate vial. 5) Evaporate to dryness using a stream of clean, dry air and re-suspend in a suitable yolume (1 cm") of HPLC mobile phase (50:50 water:acetonitile + 0.4% (v/v) glacial ‘acetic acid) prior to analysis by HPLC-MS-MS, ©) Alternatively, of the sampl mobile if samples have been prepared for analysis by HPLC-UV take 0.5 cm? ein mobile phase and transfer to an appropriate vial, Add 0.5 cm? of phase to give a final sample concentration of 0.5 g soll cm?. ‘Standards must be prepared in the presence of soil matrix prior to analysis, as different fesponses are achieved in the presence of matrix compared to when matrix is absent, Standards are prepared as follows: @ Transfer a 0.50 cm? aliquot (equivalent to 0.5 g of soil of the resuspended ‘solution from 3.2( c) of a control soll extraet to an appropriate vial. @) Add an approy priate amount of ICIASSO4, R230310, R234886, R401553 and 402173 star indard to produce the required final standard concentration. i) Evaporate to dryness and re-suspend in a suitable volume (1 em) of HPLC mobile phase (50:50 water:acetonitile + 0.4% (viv) glacial acetic acid). (™) Analyse standards alongside samples on HPLC-MS-MS. REFERENCE copy RAM 269/01 Page No 844 Page 32 Report No: RJ1864B, HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ULTRA-VIOLET DETECTION (HPLC-UV) The conditions for the analysis by HPLC-UV will depend upon the equipment available, The operating manuals for the instruments should always be consulted to ensure safe optimum Use. The following conditions have been found to be satisfactory using a Hewlett Packard 1050 series HPLC gradient pump fitted with a Hewlett Packard 1050 sories autosampler end 8 1050 series LC-UV detector: Several method variables may be modified i.e. mobile phase composition and flow rate to ensure resolution of the analytes from co-eluting peaks. High Performance Liquid Chromatography Conditions. © Column: ‘Spherisorb 5 ym ODS 2 (25 cm x 3.2 mm internal diameter) @ Mobile phase: Solvent A - Uttra-pure water + 0.4% (viv) glacial acetic acid ‘Solvent B - Acetonitrile + 0.4% (viv) glacial acetic acid Following an injection, the above two solvents are combined in the gradient system to produce the required linear changes in mobile phase composition, as shown in Table 1, After 40 minutes, the solvent is lef in the zoro-iime Composition for 10 minutes prior to re-injection. Table 1: Gradient HPLC Solvent Composition Profile Solvent A (%) Solvent B (2) Gi) Flow rate: 0.8 cm? min? (¥) Injection volume: 200 ) Detection: 255 1m Under these conditions the retention times of ICLASS04, R230310 and R234886 were approximately 47, 41 and 25 minutes, respectively. REFERENCE COPY RAM 268/01 Page No ®