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-6APPENDIX 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 elu￾tion 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 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 ≥10% of pesticide elutes in second flask, collect separate additional 100 mL eluting solution in recovery stud￾ies 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 wherever name of tested chemical should be entered