APPENDⅨ/ PROTOCOL A Pesticide Analytical Manual Vol I Calculate retention time of chemical relative to carbofuran on the hplc Recovery of Chemical Through Cleanup Column Prepare fortification solution by diluting stock solution with methanol Initially determine that charcoal/ silanized Celite column has proper elu- tion characteristics as described in Section 401 Cl In duplicate, add 25 ug pesticide to newly prepared charcoal/ silanized Celite column Then elute as described in method. After collection of eluate(20 mL methylene chloride 125 mL toluene/acetonitrile) in round- bottom(r-b)flask, momentarily stop flow, remove bottom flask and re- place with second r-b flask Elute column with additional 100 mL toluene/ acetonitrile. Evaporate solvents in both flasks to dryness as described in method. Dissolve residue in first flask to appropriate volume with appro- priate solvent. Dissolve residue in second flask with 5 mL solvent. Deter mine percentage of total added pesticide eluted in each eluate. Continue recovery studies with food products only if combined recoveries from charcoal column are >50%. If 210% of pesticide elutes in second flask, collect separate additional 100 mL eluting solution in recovery stud es with food products Recovery Through Complete Method Select representative food sample. Analyze by Section 401 to ensure that there are no interferences with chemical being tested. Simultaneously, analyze reagent blank for further information on source of possible inter- ferences Fortify duplicate 150 g portions of chopped food product, while it is in homogenizer, at about 0.05 ppm; analyze as above Fortify duplicate 150 g samples at level near tolerance or, if no tolerance exists, at about 0.25 ppm; analyze as above REPORTING RESULTS Report all results on copy of Reporting Form A. An asterisk ()appears on form herever name of tested chemical should be entered APPENDIX I-6
APPENDIX II/PROTOCOL A Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) APPENDIX II-6 Form FDA 2905a (6/92) • Calculate retention time of chemical relative to carbofuran on the HPLC system. Recovery of Chemical Through Cleanup Column Prepare fortification solution by diluting stock solution with methanol. • Initially determine that charcoal/silanized Celite column has proper elution characteristics as described in Section 401 C1. • In duplicate, add 25 µg pesticide to newly prepared charcoal/silanized Celite column. Then elute as described in method. After collection of eluate (20 mL methylene chloride + 125 mL toluene/acetonitrile) in roundbottom (r-b) flask, momentarily stop flow, remove bottom flask and replace with second r-b flask. Elute column with additional 100 mL toluene/ acetonitrile. Evaporate solvents in both flasks to dryness as described in method. Dissolve residue in first flask to appropriate volume with appropriate solvent. Dissolve residue in second flask with 5 mL solvent. Determine percentage of total added pesticide eluted in each eluate. • Continue recovery studies with food products only if combined recoveries from charcoal column are >50%. If ≥10% of pesticide elutes in second flask, collect separate additional 100 mL eluting solution in recovery studies with food products. Recovery Through Complete Method • Select representative food sample. Analyze by Section 401 to ensure that there are no interferences with chemical being tested. Simultaneously, analyze reagent blank for further information on source of possible interferences. • Fortify duplicate 150 g portions of chopped food product, while it is in homogenizer, at about 0.05 ppm; analyze as above. • Fortify duplicate 150 g samples at level near tolerance or, if no tolerance exists, at about 0.25 ppm; analyze as above. REPORTING RESULTS Report all results on copy of Reporting Form A. An asterisk (*) appears on form wherever name of tested chemical should be entered
Pesticide Analytical Manual Vol. I APPENDⅨ/ REPORT A REPORTING FORM A: BEHAVIOR THROUGH SECTION 401 The following data resulted from testing the chemical s through pam i Section 401 El+ Cl+ DLl or DL2, according to Appendix Il, Protocol A Alternative names Reference Standard(source and number) Molecular Formula: Structure Comments: Results of hplc test The following HPlC system was used Analytical Column: Guard column Mobile phase For natural fluorescence DL2. fluoresces at excitation and emission wavelengths of respectively Peak characteristics DLI Peak sha Retention time (relative to carbofuran) ausing 50% FSD Results of Stability in Methanol Studies Short Term Study Long Term Study Ti Ime Peak Ht(mm) y Peak Ht(mm) APPENDIX II-7
Pesticide Analytical Manual Vol. I APPENDIX II/PROTOCOL A APPENDIX II–7 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) REPORTING FORM A: BEHAVIOR THROUGH SECTION 401 The following data resulted from testing the chemical * through PAM I Section 401 E1 + C1 + DL1 or DL2, according to Appendix II, Protocol A. Name: * Alternative Names: Reference Standard (source and number): Molecular Formula: Structure: Comments: Results of HPLC Tests The following HPLC system was used: Analytical Column: Guard Column: Mobile Phase: For natural fluorescence, DL2: * fluoresces at excitation and emission wavelengths of _________ and _________ nm, respectively. Peak Characteristics: DL1 DL2 Peak shape _______ _______ Retention time (relative to carbofuran) _______ _______ ng causing 50% FSD _______ _______ Linear range _______ _______ Results of Stability in Methanol Studies Short Term Study Long Term Study Time Peak Ht (mm) Day Peak Ht (mm) _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ _______ APPENDIX II/REPORT A
APPENDⅨX/ REPORT A Pesticide Analytical Manual Vol I Recovery Through Charcoal/Silanized Celite Column ug added to column: Percent recovered from charcoal/silanized Celite column Methylene chloride+ Additional 100 mL toluene/acetonitrile toluene/ acetonitrile Trial I Trial I Recovery Through Complete Method Food sample Does sample extract cause any interference with test chemical? Does reagent blank cause any interference with test chemical? Determinative step used for recovery test: Duplicate 150 g samples fortified at_Ppm and_ Percent a d: PF Trial l Trial 2 Trial l Trial 2 Additional Data on Crop Used as Samples Pesticide residues found Unidentified peaks(specify determinative step used and list peaks by retention time relative to appropriate chemical) Information submitted by: Address: APPENDIX I-8
APPENDIX II/PROTOCOL A Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) APPENDIX II-8 Form FDA 2905a (6/92) Recovery Through Charcoal/Silanized Celite Column µg * added to column: __________ Percent recovered from charcoal/silanized Celite column: Methylene chloride + Additional 100 mL toluene/acetonitrile toluene/acetonitrile Trial 1 Trial 2 Trial 1 Trial 2 _______ _______ _______ _______ Recovery Through Complete Method Food sample: Does sample extract cause any interference with test chemical? Does reagent blank cause any interference with test chemical? Determinative step used for recovery test: Duplicate 150 g samples fortified at __________ ppm and __________ ppm. Percent * recovered: _______ ppm _______ ppm Trial 1 Trial 2 Trial 1 Trial 2 _______ _______ _______ _______ Additional Data on Crop Used as Samples Pesticide residues found: Unidentified peaks (specify determinative step used and list peaks by retention time relative to appropriate chemical). Information submitted by: Address: Phone: ( ) Date: APPENDIX II/REPORT A
Pesticide Analytical Manual Vol. I APPENDⅨX‖/ PROTOCOL E PROTOCOL B: PROCEDURE FOR TESTING CHEMICALS THROUGH SECTION 402 BACKGROUND Method: Section 402 Chemical Type: Applicable to chemicals with acid or phenol structure. Chemicals are methylated before determination by glC, because esters and ethers are more easily chromatographed than acids and phenols Commodity Type: Applicable to wide variety of commodities, both fatty and nonfatty Different extraction steps are used depending on commodity PAM I Tables: Table 402-a DATA DEVELOPMENT Gas Chromatography Because the method being tested includes methylation of the analyte, glC chan acteristics of both the acid/ phenol and its methylation product are collected Perform the following operations Dissolve methyl ester/ether reference standard, if available, in 10% acetone/isooctane(v/v) to prepare stock standard solution. Dilute wit in Protocol C for chromatography of methyl ester/ ether reference standard. Report results on Reporting Form C Stop work if methyl ester/ether does not cause response on any detector when chromatographed on any appropriate column Dissolve acid/phenol reference standard in acetone to prepare stock standard solution. Dilute as needed with acetone. Also dilute with 50% methylene chloride/hexane to prepare solution suitable for testing re- covery through gel permeation chromatography(GPC).(Note: 2, 4,5-T will not dissolve in 50% methylene chloride/hexane, so solutions in that solvent mixture must be prepared by diluting acetone stock solution. Follow directions in Protocol C for chromatography of acid/phenol ref erence standard. Report results on separate copy of Reporting Form C Methylate 100 ug acid/phenol reference standard in acetone solution according to procedure described in Section 402 CIb Dilute as needed with hexane. If any other methylation procedure is used, describe pro- cedure on Reporting Form B Follow directions in Protocol C for chromatography of methylated ref- erence standard Report results on separate copy of Reporting Form C (Note: if ester/ether reference standard is available, these results should Form FDA 2905a(6/92) ppendix -9
Pesticide Analytical Manual Vol. I APPENDIX II/PROTOCOL B Appendix II–9 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) PROTOCOL B: PROCEDURE FOR TESTING CHEMICALS THROUGH SECTION 402 BACKGROUND Method: Section 402 Chemical Type: Applicable to chemicals with acid or phenol structure. Chemicals are methylated before determination by GLC, because esters and ethers are more easily chromatographed than acids and phenols. Commodity Type: Applicable to wide variety of commodities, both fatty and nonfatty. Different extraction steps are used depending on commodity. PAM I Tables: Table 402-a DATA DEVELOPMENT Gas Chromatography Because the method being tested includes methylation of the analyte, GLC characteristics of both the acid/phenol and its methylation product are collected. Perform the following operations: • Dissolve methyl ester/ether reference standard, if available, in 10% acetone/isooctane (v/v) to prepare stock standard solution. Dilute with isooctane. • Follow directions in Protocol C for chromatography of methyl ester/ ether reference standard. Report results on Reporting Form C. • Stop work if methyl ester/ether does not cause response on any detector when chromatographed on any appropriate column. • Dissolve acid/phenol reference standard in acetone to prepare stock standard solution. Dilute as needed with acetone. Also dilute with 50% methylene chloride/hexane to prepare solution suitable for testing recovery through gel permeation chromatography (GPC). (Note: 2,4,5-T will not dissolve in 50% methylene chloride/hexane, so solutions in that solvent mixture must be prepared by diluting acetone stock solution.) • Follow directions in Protocol C for chromatography of acid/phenol reference standard. Report results on separate copy of Reporting Form C. • Methylate 100 µg acid/phenol reference standard in acetone solution according to procedure described in Section 402 C1b. Dilute as needed with hexane. If any other methylation procedure is used, describe procedure on Reporting Form B. • Follow directions in Protocol C for chromatography of methylated reference standard. Report results on separate copy of Reporting Form C (Note: if ester/ether reference standard is available, these results should
APPENDⅨ/ PROTOCOL E Pesticide Analytical Manual Vol I verify retention times and response characteristics previously found. Note this on Reporting Form prepared for methyl ether/ester reference stan- dard test results Determine efficiency of methylation by direct comparison to methyl es- er/ether reference standard, if available. Report percentage conversion to methylated product on Reporting Form B If methyl ester/ether refer ence standard is not available, assume complete methylation of chemical for calculating amount of reference standard causing 50% FSD Stop work if acid/ phenol cannot be methylated or if there is no GlC response to ester/ether. Recovery Through GPC and Florisil Methyl Ester/Ether Reference Standard In duplicate, place 1-100 ug methyl ester/ether reference standard, in 1 10 mL hexane, on Florisil column prepared as described in Section 402 CIc.(Use Florisil that has been shown to permit elution of both hep- tachlor epoxide and endrin by eluant 2 [Section 204].) Elute columns with 35 mL eluant 1, 60 mL eluant 2, and 100 mL ethyl ether, and determine percentage recovered in each eluate If total recovery is <g0%, report results on Reporting Form B and termi- nate work Acid/Phenol Reference Standards Place 100 ug acid/ phenol reference standard, dissolved in 50% methylene chloride/hexane, on calibrated GPC column and elute as directed in ection 402 cla Methylate collected fraction according to Clb Determine percentage recovery by comparison to methyl ester/ether ref erence standard, if available, or to reference standard previously methy lated in laboratory If recoveries are <30%, report results on Reporting Form B and terminate work Recovery Through Complete Method Select one representative fatty and one nonfatty food. Analyze by Section 402 using extraction module appropriate to commodity, to ensure that there are no interferences with chemical being tested. Simultaneously, nalyze reagent blank for further information on source of possible inter- ences Fortify duplicate portions of food samples with 1-2 mL acetone solution of acid/phenol reference standard at tolerance level or, if no tolerance ex- ists, at 0.05 ppm APPENDIX‖10 Form
Transmittal No. 94-1 (1/94) APPENDIX II-10 Form FDA 2905a (6/92) APPENDIX II/PROTOCOL B Pesticide Analytical Manual Vol. I verify retention times and response characteristics previously found. Note this on Reporting Form prepared for methyl ether/ester reference standard test results.) • Determine efficiency of methylation by direct comparison to methyl ester/ether reference standard, if available. Report percentage conversion to methylated product on Reporting Form B. If methyl ester/ether reference standard is not available, assume complete methylation of chemical for calculating amount of reference standard causing 50% FSD. Stop work if acid/phenol cannot be methylated or if there is no GLC response to ester/ether. Recovery Through GPC and Florisil Methyl Ester/Ether Reference Standards • In duplicate, place 1-100 µg methyl ester/ether reference standard, in 1- 10 mL hexane, on Florisil column prepared as described in Section 402 C1c. (Use Florisil that has been shown to permit elution of both heptachlor epoxide and endrin by eluant 2 [Section 204].) • Elute columns with 35 mL eluant 1, 60 mL eluant 2, and 100 mL ethyl ether, and determine percentage recovered in each eluate. • If total recovery is <30%, report results on Reporting Form B and terminate work. Acid/Phenol Reference Standards • Place 100 µg acid/phenol reference standard, dissolved in 50% methylene chloride/hexane, on calibrated GPC column and elute as directed in Section 402 C1a. • Methylate collected fraction according to C1b. • Determine percentage recovery by comparison to methyl ester/ether reference standard, if available, or to reference standard previously methylated in laboratory. • If recoveries are <30%, report results on Reporting Form B and terminate work. Recovery Through Complete Method • Select one representative fatty and one nonfatty food. Analyze by Section 402, using extraction module appropriate to commodity, to ensure that there are no interferences with chemical being tested. Simultaneously, analyze reagent blank for further information on source of possible interferences. • Fortify duplicate portions of food samples with 1-2 mL acetone solution of acid/phenol reference standard at tolerance level or, if no tolerance exists, at 0.05 ppm