Pesticide Analytical Manual Vol. I APPENDⅨ APPENDIX II: PROTOCOLS AND REPORTING FORMS FOR TESTING CHEMICALS THROUGH PAMI MULTIRESIDUE METHODS INTRODUCTION: MULTIRESIDUE METHOD TESTING Use of any multiresidue method(MRM)is supported by available information about how potential residues behave through the steps of the method To provide that support for PAM I MRMS, additional chemicals are continually tested through the method steps and the resulting data compiled in a single database. All PAM I tables in Chapters 3 and 4 and Appendix I are produced from that database This Appendix provides directions for performing such tests and forms for report- ing results The effort spent on the testing of MRMs and compilation of results is justified by the advantages such compilations offer the analytical chemist. When analytical behavior data for numerous chemicals through the method in use are known, the analyst is better equipped to identify residues that may be present in a sample of unknown treatment history. In situations where the likelihood of some particular residue is known, the data lists for several methods can be consulted to help choose which method should be used Regulatory agencies often must assess the incidence of residue occurrence. This effort is also assisted by compilations of method behavior data. The absence of many chemicals from the sample can be ascertained when it is known that those chemicals could have been detected had they been present It has been found advisable to define protocols for developing data on MRM behavior. In order to compile data into usable formats, it is imperative that all contributing laboratories perform the tests uniformly. The goal of this method- testing is not to find the optimum conditions for the one chemical currently being tested, but to be able to describe how the chemical will behave when determined by the precisely defined method This Appendix includes one protocol for determining GlC characteristics of chemi- cals and six protocols for testing their behavior through individual MRMs. Forms for reporting the results of testing by each protocol are also included. Each pro- tocol references the PAM I method(s) involved, the types of chemicals to which applies, and the PAM I table(s) in which previously collected data are published Some PAM I MRMs are applicable to a wide variety of residues, while others are targeted to those with specific chemical structures. A Decision Tree is included in this Appendix to direct the user to the most appropriate protocol(s) for each chemical being tested. Follow the Decision Tree in deciding which protocol(s)to Follow the steps of these protocols, in the order written Report data on a copy of the appropriate form and send it to: PAM I Editors, HFS-337, Food and Drug Administration, 200 C Street SW, Washington, DC 20204 ppendⅸ‖1
Pesticide Analytical Manual Vol. I APPENDIX II Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Appendix II–1 APPENDIX II: PROTOCOLS AND REPORTING FORMS FOR TESTING CHEMICALS THROUGH PAM I MULTIRESIDUE METHODS INTRODUCTION: MULTIRESIDUE METHOD TESTING Use of any multiresidue method (MRM) is supported by available information about how potential residues behave through the steps of the method. To provide that support for PAM I MRMs, additional chemicals are continually tested through the method steps and the resulting data compiled in a single database. All PAM I tables in Chapters 3 and 4 and Appendix I are produced from that database. This Appendix provides directions for performing such tests and forms for reporting results. The effort spent on the testing of MRMs and compilation of results is justified by the advantages such compilations offer the analytical chemist. When analytical behavior data for numerous chemicals through the method in use are known, the analyst is better equipped to identify residues that may be present in a sample of unknown treatment history. In situations where the likelihood of some particular residue is known, the data lists for several methods can be consulted to help choose which method should be used. Regulatory agencies often must assess the incidence of residue occurrence. This effort is also assisted by compilations of method behavior data. The absence of many chemicals from the sample can be ascertained when it is known that those chemicals could have been detected had they been present. It has been found advisable to define protocols for developing data on MRM behavior. In order to compile data into usable formats, it is imperative that all contributing laboratories perform the tests uniformly. The goal of this methodtesting is not to find the optimum conditions for the one chemical currently being tested, but to be able to describe how the chemical will behave when determined by the precisely defined method. This Appendix includes one protocol for determining GLC characteristics of chemicals and six protocols for testing their behavior through individual MRMs. Forms for reporting the results of testing by each protocol are also included. Each protocol references the PAM I method(s) involved, the types of chemicals to which it applies, and the PAM I table(s) in which previously collected data are published. Some PAM I MRMs are applicable to a wide variety of residues, while others are targeted to those with specific chemical structures. A Decision Tree is included in this Appendix to direct the user to the most appropriate protocol(s) for each chemical being tested. Follow the Decision Tree in deciding which protocol(s) to use. Follow the steps of these protocols, in the order written. Report data on a copy of the appropriate form and send it to: PAM I Editors, HFS-337, Food and Drug Administration, 200 C Street SW, Washington, DC 20204
APPENDIX‖l Pesticide Analytical Manual Vol I Decision Tree for MRM Testing Do data already exist (Index to Methods, Appendix I]? evelop only data nd t Does the compound have this structure or characteristic Yes N-methylcarbamate? Test per Protocol A Yes naturally fluorescent? Test per Protocol A. acid or phenol? Yes Test per Protocol B Yes substituted urea? Test per Protocol G Does it chromatograph on GLC systems? vel I Determine GLC characteristics per Protocol C, Level I Yes Discontinue tests Does it chromatograph on at least one Level system in a reasonable time [o3<rrt<5.0)? No Report rrts for Level I DG modules tested, then retest using appropriate Level ll DG module[s] Is the product a nonfatty (27 fat] food? Yesh Test per Protocol F for fatty food Test per Protocol D. Is the chemical recovered through Section 302 extraction? No Yesh Discontinue tests Test per protocol e for nonfatty food ppendⅸ|2 vo.941(1 2905a(6
Transmittal No. 94-1 (1/94) Appendix II–2 Form FDA 2905a (6/92) APPENDIX II Pesticide Analytical Manual Vol. I Decision Tree for MRM Testing Do data already exist (Index to Methods, Appendix I)? Develop only data not yet available. Yes No Does the compound have this structure or characteristic: Yes Yes Yes Yes Determine GLC characteristics per Protocol C, Level I. Does it chromatograph on GLC systems? No Discontinue tests. No Yes Does it chromatograph on at least one Level I system in a reasonable time (0.3<rrt<5.0)? No Yes Report rrts for Level I DG modules tested, then retest using appropriate Level II DG module(s). Is the product a nonfatty (2% fat) food? No Yes Test per Protocol F for fatty food. Test per Protocol D. Is the chemical recovered through Section 302 extraction? No Yes Discontinue tests. Test per Protocol E for nonfatty food. N-methylcarbamate? naturally fluorescent? acid or phenol? substituted urea? Test per Protocol A. Test per Protocol A. Test per Protocol B. Test per Protocol G
Pesticide Analytical Manual Vol. I APPENDⅨ SUGGESTONS FOR PRODUCING QUALITY DATA The following suggestions were developed in response to data received and to pecific questions that have been raised Decisions on what Protocols to follow As directed in the first step of the Decision Tree, review existing data on the chemical before performing experiments Examine PAM I Index to Methods for entries, then review the details in appropriate Chapter 8 or 4 table(s); review Appendix I for available GLC data. If these sources reveal gaps in the data, perform the tests necessary to provide missing data, but do not repeat experiments unless specifically asked to do so or unless current data reflect variability Develop glC data(Protocol C) with system(s) likely to detect the chemi- cal ie. of the dg modules listed. choose at least one whose detector selective to elements in the molecule. The electron capture detector may not be suitable to examine uncleaned extracts from Section 302 Protocol D), so DGl, 19, and 18 are often insufficient. If an electron capture detector is the only one that responds to the chemical, apply caution when injecting extracts from Section 302 Choose the GlC system that provides the best chromatography and sensitivity for examining solutions from recovery studies; it is not nec. essary to re-examine the extracts by multiple glc systems The Decision Tree provides basic criteria for making decisions about which methods to test Where situations exist that make testing or con- tinuation of testing illogical, suspend testing and report the reasons Examples If previous studies with radiolabelled chemical clearly show that the residue will partition into the(discarded) water layer of the method, nethod recovery tests need not be run; however, collection of GLC data should still be attempted If the only commodity of interest is fatty, do not attempt to perform tests on the product with methods designed for nonfatty foods. If the lodity is ly Section 304 El(Protocol F)or, if the hemical is an acid or phenol, Section 402 El(Protocol B) Suspend testing if the GlC tests(Protocol C) indicate that even the most sensitive GLC system is insensitive to the compound As a gen- eral rule, suspend testing if the minimum weight of compound that causes 10% full scale deflection(FSD) is equivalent to 210 times the tolerance for jection of the normal mg sample equivalent de- scribed in the method If the method being tested proves not amenable to analysis of a particular commodity, as evidenced by severe emulsions, failure to form distinct phases, etc, suspend testing ppendix l-3
Pesticide Analytical Manual Vol. I APPENDIX II Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Appendix II–3 SUGGESTIONS FOR PRODUCING QUALITY DATA The following suggestions were developed in response to data received and to specific questions that have been raised. Decisions on What Protocols to Follow • As directed in the first step of the Decision Tree, review existing data on the chemical before performing experiments. Examine PAM I Index to Methods for entries, then review the details in appropriate Chapter 3 or 4 table(s); review Appendix I for available GLC data. If these sources reveal gaps in the data, perform the tests necessary to provide missing data, but do not repeat experiments unless specifically asked to do so or unless current data reflect variability. • Develop GLC data (Protocol C) with system(s) likely to detect the chemical; i.e., of the DG modules listed, choose at least one whose detector is selective to elements in the molecule. The electron capture detector may not be suitable to examine uncleaned extracts from Section 302 (Protocol D), so DG1, 13, and 18 are often insufficient. If an electron capture detector is the only one that responds to the chemical, apply caution when injecting extracts from Section 302. • Choose the GLC system that provides the best chromatography and sensitivity for examining solutions from recovery studies; it is not necessary to re-examine the extracts by multiple GLC systems. • The Decision Tree provides basic criteria for making decisions about which methods to test. Where situations exist that make testing or continuation of testing illogical, suspend testing and report the reasons. Examples: – If previous studies with radiolabelled chemical clearly show that the residue will partition into the (discarded) water layer of the method, method recovery tests need not be run; however, collection of GLC data should still be attempted. – If the only commodity of interest is fatty, do not attempt to perform tests on the product with methods designed for nonfatty foods. If the commodity is meat, use only Section 304 E1 (Protocol F) or, if the chemical is an acid or phenol, Section 402 E1 (Protocol B). – Suspend testing if the GLC tests (Protocol C) indicate that even the most sensitive GLC system is insensitive to the compound. As a general rule, suspend testing if the minimum weight of compound that causes 10% full scale deflection (FSD) is equivalent to ≥10 times the tolerance for an injection of the normal mg sample equivalent described in the method. – If the method being tested proves not amenable to analysis of a particular commodity, as evidenced by severe emulsions, failure to form distinct phases, etc., suspend testing
APPENDIX‖l Pesticide Analytical Manual Vol I Proper Application of Methods Do not combine a GlC column that contains cyano groups with a nitro- gen-selective detector Adjust GlC column temperature to establish the correct relative retention time (rrt)of the marker chemical(s). Absolute retention times(min)are provided in DG modules to provide an indication of normal behavior, but the temperature should not be adjusted to achieve this Measure retention times on glc columns from the solvent front wher ever possible for both the compound being tested and the marker com- pound. If the instrumentation in use precludes measurement from the solvent front, state this fact in the report. When submitting chromatograms with reporting forms, label them clearly indicate how many nanograms(ng) or picograms(pg)are represented by the peak. Do not label a chromatogram of a standard in ppm For chro- matograms that result from recovery studies, indicate on the chromato- gram label how much sample weight equivalent(mg) was injected For accurate quantitation, detector response to the reference standard should be within +25% of the response to the analyte in the sample, based on observable(on-scale) peak heights. Usual GLC linearity does not sup- port quantitation that compares responses differing in size by more than that amount Injection to elute before the next injection is made. peaks from one Make sure that chromatograms do not overlap; allow when the chromatogram contains multiple peaks representing different components in the standard, report the rrt of each peak >5%0 FSD It is not necessary to evaporate the solvent from the fortification solution once it has been added to the sample, unless there is some reason to expect it to interfere with the analysis. It is preferable for the fortifying solution to be made from the same solvent used to extract the sample Use only the 60/100 mesh PR grade Florisil specified by Sections 303 and 304(Protocols E and F). Other grades of commercially available Florisil are likely to result in different elution patterns Florisil column in a polar solvent, which will affect elution patern. ch During tests of elution from Florisil, do not add reference material to Test Florisil elution by both ethyl ether/petroleum ether (Sections 303 Cl, 304 Cl and C3)and methylene chloride($0% C2, 304 C2 and C4) elution systems when following the steps of Protocols E and F. Even when florisil elution tests with reference standards indicate no elu tion with later eluants amine by GLC all the Florisil eluates of the recovery test from the fortified sample. Sometimes the presence of extract changes the Florisil elution pattern ppendⅸ|4 vo.941(1 2905a(6
Transmittal No. 94-1 (1/94) Appendix II–4 Form FDA 2905a (6/92) APPENDIX II Pesticide Analytical Manual Vol. I Proper Application of Methods • Do not combine a GLC column that contains cyano groups with a nitrogen-selective detector. • Adjust GLC column temperature to establish the correct relative retention time (rrt) of the marker chemical(s). Absolute retention times (min) are provided in DG modules to provide an indication of normal behavior, but the temperature should not be adjusted to achieve this. • Measure retention times on GLC columns from the solvent front wherever possible for both the compound being tested and the marker compound. If the instrumentation in use precludes measurement from the solvent front, state this fact in the report. • When submitting chromatograms with reporting forms, label them clearly: indicate how many nanograms (ng) or picograms (pg) are represented by the peak. Do not label a chromatogram of a standard in ppm. For chromatograms that result from recovery studies, indicate on the chromatogram label how much sample weight equivalent (mg) was injected. • For accurate quantitation, detector response to the reference standard should be within ±25% of the response to the analyte in the sample, based on observable (on-scale) peak heights. Usual GLC linearity does not support quantitation that compares responses differing in size by more than that amount. • Make sure that chromatograms do not overlap; allow peaks from one injection to elute before the next injection is made. • When the chromatogram contains multiple peaks representing different components in the standard, report the rrt of each peak >5% FSD. • It is not necessary to evaporate the solvent from the fortification solution once it has been added to the sample, unless there is some reason to expect it to interfere with the analysis. It is preferable for the fortifying solution to be made from the same solvent used to extract the sample. • Use only the 60/100 mesh PR grade Florisil specified by Sections 303 and 304 (Protocols E and F). Other grades of commercially available Florisil are likely to result in different elution patterns. • During tests of elution from Florisil, do not add reference material to the Florisil column in a polar solvent, which will affect elution pattern. • Test Florisil elution by both ethyl ether/petroleum ether (Sections 303 C1, 304 C1 and C3) and methylene chloride (303 C2, 304 C2 and C4) elution systems when following the steps of Protocols E and F. • Even when Florisil elution tests with reference standards indicate no elution with later eluants, examine by GLC all the Florisil eluates of the recovery test from the fortified sample. Sometimes the presence of extract changes the Florisil elution pattern
Pesticide Analytical Manual Vol. I APPENDIX I/PROTOCOL A PROTOCOL A: PROCEDURE FOR TESTING CHEMICALS THROUGH SECTION 401 BACKGROUND Methods: Section 401 El+cl+ dll or dl2 Chemical Type: Applicable to chemicals with N-methylcarbamate structure (DLl) and to some chemicals that are naturally fluorescent(DL2) soybeans and pe: Applicable to nonfatty foods and to certain fatty foods such as PAM I Tables: Tables 401-a. 401-b DATA DEVELOPMENT HPLC Analytical Behavior N-Methylcarbamates: Set up HPLC with post-column fluorescence labeling and fluorescence detector, as described in Section 401 DLl; check for proper opera- tion using system suitability test described Fluorescent Pesticides: Set up HPLC and fluorescence detector, as described in Section 401 Dl2 Develop information on test chemical(s)as follows Dilute with methanol for HPLC working standards e stock solutions Determine amount (ng) of chemical that causes detector response of 50% full scale deflection(FSD) on recorder or printer/plotter. Note peak shape of response to determine adequacy of chromatography Determine linear response range of detector to chemical Determine stability of chemical in methanol Short term. Prepare l ug/mL methanol solution of chemical. Use actinic glassware. Inject 5-10 uL injections of this solution into HPLC ystem over 8 hr to measure short term stability of chemical in solu- tion Report results as peak height(mm)response Long term. Using same solution as above, inject 10 uL into system once a day for 1-2 wk to measure long term stability of solution. Store solution on laboratory bench during day and in refrigerator over night. Report results as peak height(mm)response to test chemical, normalized to peak height for carbofuran standard injected on same APPENDIX‖-5
Pesticide Analytical Manual Vol. I APPENDIX II/PROTOCOL A APPENDIX II–5 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) PROTOCOL A: PROCEDURE FOR TESTING CHEMICALS THROUGH SECTION 401 BACKGROUND Methods: Section 401 E1 + C1 + DL1 or DL2 Chemical Type: Applicable to chemicals with N-methylcarbamate structure (DL1) and to some chemicals that are naturally fluorescent (DL2). Commodity Type: Applicable to nonfatty foods and to certain fatty foods such as soybeans and nuts. PAM I Tables: Tables 401-a, 401-b DATA DEVELOPMENT HPLC Analytical Behavior N-Methylcarbamates: Set up HPLC with post-column fluorescence labeling and fluorescence detector, as described in Section 401 DL1; check for proper operation using system suitability test described. Fluorescent Pesticides: Set up HPLC and fluorescence detector, as described in Section 401 DL2. Develop information on test chemical(s) as follows: • Dissolve reference standard in methanol to prepare stock solutions. Dilute with methanol for HPLC working standards. • Determine amount (ng) of chemical that causes detector response of 50% full scale deflection (FSD) on recorder or printer/plotter. Note peak shape of response to determine adequacy of chromatography. • Determine linear response range of detector to chemical. • Determine stability of chemical in methanol: – Short term. Prepare 1 µg/mL methanol solution of chemical. Use actinic glassware. Inject 5-10 µL injections of this solution into HPLC system over 8 hr to measure short term stability of chemical in solution. Report results as peak height (mm) response. – Long term. Using same solution as above, inject 10 µL into system once a day for 1-2 wk to measure long term stability of solution. Store solution on laboratory bench during day and in refrigerator overnight. Report results as peak height (mm) response to test chemical, normalized to peak height for carbofuran standard injected on same day
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