SECTION 401 Pesticide Analytical Manual Vol I Baseline resolution should be achieved between aldicarb sulfoxide and aldicarb sulfone and between carbaryl and carbofuran Directions See Figure 401-d for typical chromatogram of carbamate pesticides and metabolites filter methanol extract from cl r-b flask through filtration device. Collect filtrate in 10 mL centrifuge tube or other suitable container. About 4.5 mL filtrate will be collected. Exact volume of filtrate collected is not critical because sample concentration (g sample/mL methanol) is known If solution requires dilution, pipet aliquot into another container and dilute to volume. as needed Inject 10 uL methanol solution into HPLC system Tentatively identify residue peaks on basis of retention times. Measure peak area or height and determine residue amount by comparison to peak area or height obtained from known amount of appropriate refer ence standard (s). To ensure valid measurement of residue amount, sizes of peaks from sample residue and reference standard should match within +25%. Chromatograph reference standard (s) immediately after sample Figure 401-d HPLC Chromatogram of Carbamates and Metabolites 不 Retention(min Chromatographed at conditions described in DL1, with post-column derivatization. 1]aldicarb sulfoxide; 2]aldicarb sulfone; 3]oxamyl; 4)methor 3-hydroxycarbofuran; 6]methiocarb sulfoxide; 7] aldicarb; 8]carbofuran; 9]carbaryl; 10]methiocarb; 11)bufencarb 401-12 Form FDA 2905a (6/92]
SECTION 401 401–12 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I • Baseline resolution should be achieved between aldicarb sulfoxide and aldicarb sulfone and between carbaryl and carbofuran. Directions See Figure 401-d for typical chromatogram of carbamate pesticides and metabolites. • Filter methanol extract from C1 r-b flask through filtration device. • Collect filtrate in 10 mL centrifuge tube or other suitable container. About 4.5 mL filtrate will be collected. Exact volume of filtrate collected is not critical because sample concentration (g sample/mL methanol) is known. • If solution requires dilution, pipet aliquot into another container and dilute to volume, as needed. • Inject 10 µL methanol solution into HPLC system. • Tentatively identify residue peaks on basis of retention times. Measure peak area or height and determine residue amount by comparison to peak area or height obtained from known amount of appropriate reference standard(s). To ensure valid measurement of residue amount, sizes of peaks from sample residue and reference standard should match within ±25%. Chromatograph reference standard(s) immediately after sample. Chromatographed at conditions described in DL1, with post-column derivatization. 1) aldicarb sulfoxide; 2) aldicarb sulfone; 3) oxamyl; 4) methomyl; 5) 3-hydroxycarbofuran; 6) methiocarb sulfoxide; 7) aldicarb; 8) carbofuran; 9) carbaryl; 10) methiocarb; 11) bufencarb. Figure 401-d HPLC Chromatogram of Carbamates and Metabolites 1 2 3 8 10 11 11 9 7 6 5 4 0 5 10 15 20 25 30 Retention (min) 10% FSD
Pesticide Analytical Manual Vol. I SECTION 401 ALTERNAT/VE DL2 HPLC FLUORESCENCE DETECTION Referene Krause, RT(1983)/ Chromatogr. 255, 497-510 Principles Residues in methanol solution are separated on a C-8 reverse phase HPLC column using an acetonitrile/water gradient mobile phase. Naturally fluorescent residues eluting from the column are detected and measured by a fluorescence detector Directions Set up and operate an HPlC system in the same manner as dll, except set detector excitation and emission wavelengths at 288 and 330 nm turn off pumps or nitrogen flow that add sodium hydroxide and OPa- MERC reaction solutions to mobile phase Perform determination as in dll CONFIRMAT/ON Confirm tentative identification of naturally fluorescent residues by using Dl2 See Table 401-b for list of chemicals for which this confirmatory step is appropri- lte Use excitation and emission wavelengths that are optimum for residue being confirmed Confirm N-methylcarbamates that include phenolic structure by injecting final extract into HPLC with post-column hydrolysis-electrochemical detection method described in Krause, RT.(1988)/ Chromatogr. 442, 333-343. This method is based on selective detection of phenolic group of insecticides, rather than carbamate moiety. Intact carbamates are separated by reverse phase HPLC using gradient acetonitrile/water mobile phase as described above. Eluted carbamates are hydro- lyzed in-line with dilute sodium hydroxide at 100 C, and resulting phenols are detected with coulometric electrochemical detector. Technique has been tested with six carbamates(bufencarb, carbaryl, carbofuran, 3-hydroxycarbofuran isoprocarb, and methiocarb)and four crops(apples, cabbage, grapes, and toma- 401-13
Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 401–13 Pesticide Analytical Manual Vol. I SECTION 401 ALTERNATIVE: DL2 HPLC, FLUORESCENCE DETECTION Reference Krause, R.T. (1983) J. Chromatogr. 255, 497-510 Principles Residues in methanol solution are separated on a C-8 reverse phase HPLC column using an acetonitrile/water gradient mobile phase. Naturally fluorescent residues eluting from the column are detected and measured by a fluorescence detector. Directions • Set up and operate an HPLC system in the same manner as DL1, except: operate hydrolysis chamber at ambient temperature. set detector excitation and emission wavelengths at 288 and 330 nm, respectively. turn off pumps or nitrogen flow that add sodium hydroxide and OPAMERC reaction solutions to mobile phase. • Perform determination as in DL1. CONFIRMATION Confirm tentative identification of naturally fluorescent residues by using DL2. See Table 401-b for list of chemicals for which this confirmatory step is appropriate. Use excitation and emission wavelengths that are optimum for residue being confirmed. Confirm N-methylcarbamates that include phenolic structure by injecting final extract into HPLC with post-column hydrolysis-electrochemical detection method described in Krause, R.T. (1988) J. Chromatogr. 442, 333-343. This method is based on selective detection of phenolic group of insecticides, rather than carbamate moiety. Intact carbamates are separated by reverse phase HPLC using gradient acetonitrile/water mobile phase as described above. Eluted carbamates are hydrolyzed in-line with dilute sodium hydroxide at 100° C, and resulting phenols are detected with coulometric electrochemical detector. Technique has been tested with six carbamates (bufencarb, carbaryl, carbofuran, 3-hydroxycarbofuran, isoprocarb, and methiocarb) and four crops (apples, cabbage, grapes, and tomatoes)
SECTION 401 Pesticide Analytical Manual Vol I 401-14 Form FDA 2905a (6/92]
SECTION 401 401–14 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I
Pesticide Analytical Manual Vol. I SECTION 402 402: METHOD FOR ACIDS AND PHENOLS BASIC REFERENCE Hopper, M L et al (1992)/ AOAC Int. 75, 707-718 GENERAL PRINCIPLES Acidic and phenolic residues are extracted from commodity acidified with sulfuric acid by various techniques dictated by the type of commodity. The extract is cleaned up by gel permeation chromatography(GPC)Residues in the concentrated extract are methylated by ion pair alkylation and further cleaned up by Florisil column chromatography. The resulting methyl esters are determined by GlC. Certain residues can be determined only by element-selective GLC detectors APPLICABILITY Consult Guide to PAM I for additional information pertinent to the appropriate application of multiresidue methodology Method is applicable to a wide variety of fatty and nonfatty foods; several different extraction steps are available for the different food types. Cleanup by GPc is most practical when a large number of analyses are being performed. Method was originally designed for chlorophenoxy acids, but has been found applicable to a variety of acidic and phenolic residues. See Table 402-a, following method description, for pesticides and metabolites tested through the steps of this method REFERENCE STANDARDS Use reference standards of methyl esters/ ethers of acids or phenols, if available Otherwise, use standards of the acids/phenols methylated through Cla and cleaned up through Clb. Prepare stock solutions in acetone STEPS OF THE METHOD Extraction E) Recommended Use El (p 402-3) Extraction with solvents from animal tissues, dairy prod- ucts,fats,and shortenings E2(p. 402-7) Extraction with acidified methylene fruits, vegetables other chloride than legumes, and beverages E3(p. 402-9) Extraction with acidified methanol legumes E4(p. 402-10) Extraction with acidified water/ grains and cere E5(P. 402-11) Extraction with acidified methanol sugar and high sugar pro- E6(p. 402-18) Dissolution in methylene chloride/ vegetable oils E7 (P. 402-15) Extraction with acidified methylene water chloride Cleanup and methylation(C) Cl (p. 402-17)GPC cleanup, methylation, and products Florisil column cleanup 40a-1
Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 402–1 Pesticide Analytical Manual Vol. I SECTION 402 402: METHOD FOR ACIDS AND PHENOLS BASIC REFERENCE Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713 GENERAL PRINCIPLES Acidic and phenolic residues are extracted from commodity acidified with sulfuric acid by various techniques dictated by the type of commodity. The extract is cleaned up by gel permeation chromatography (GPC). Residues in the concentrated extract are methylated by ion pair alkylation and further cleaned up by Florisil column chromatography. The resulting methyl esters are determined by GLC. Certain residues can be determined only by element-selective GLC detectors. APPLICABILITY Consult Guide to PAM I for additional information pertinent to the appropriate application of multiresidue methodology. Method is applicable to a wide variety of fatty and nonfatty foods; several different extraction steps are available for the different food types. Cleanup by GPC is most practical when a large number of analyses are being performed. Method was originally designed for chlorophenoxy acids, but has been found applicable to a variety of acidic and phenolic residues. See Table 402-a, following method description, for pesticides and metabolites tested through the steps of this method. REFERENCE STANDARDS Use reference standards of methyl esters/ethers of acids or phenols, if available. Otherwise, use standards of the acids/phenols methylated through C1a and cleaned up through C1b. Prepare stock solutions in acetone. STEPS OF THE METHOD Extraction (E) Recommended Use E1 (p. 402-3) Extraction with solvents from animal tissues, dairy prodacidified, denatured products ucts, fats, and shortenings E2 (p. 402-7) Extraction with acidified methylene fruits, vegetables other chloride than legumes, and beverages E3 (p. 402-9) Extraction with acidified methanol legumes E4 (p. 402-10) Extraction with acidified water/ grains and cereals methanol E5 (p. 402-11) Extraction with acidified methanol sugar and high sugar processed foods E6 (p. 402-13) Dissolution in methylene chloride/ vegetable oils hexane E7 (p. 402-15) Extraction with acidified methylene water chloride Cleanup and methylation (C) C1 (p. 402-17) GPC cleanup, methylation, and all products Florisil column cleanup
SECTION 402 Pesticide Analytical Manual Vol I Determination (D) Recommended use DGl (P. 302-25)GLC, 100% methyl siloxane column halogenated acids and 200°, eC detector phenols DG3(P $02-29)GLC, 100% methyl siloxane column halogenated acids and 200° EICD-X phenols DG4(P. 302-31)GLC, 100% methyl siloxane column, acids and phenols con- 200°, EICD-N taining nitrogen Figure 402-a Method for acids and phenols fr animal tissues vegeta sugar and dairy products fats and gums, and rocessed shortenings beverages cereals water C1 DG1/3 residues residues with halogen nitrogen VALIDATION The following combination has undergone validation in a single laboratory, over many years, with repeated recoveries performed in conjunction with FDA's Total Study; these are recommended for use Validation report: Hopper, M.L. et aL.(1992)J. AOAC Int. 75, 707-713 Where slight differences occur between the method description in Section 402 and in the Hopper et al. 1992 publication, this section reflects standard operating procedure in the Total Diet Study 402-2 Transmittal No. 93-1(1/941 Form FDA 2905a(6/92
Transmittal No. 93-1 (1/94) 402–2 Form FDA 2905a (6/92) SECTION 402 Pesticide Analytical Manual Vol. I Determination (D) Recommended Use DG1 (p. 302-25) GLC, 100% methyl siloxane column, halogenated acids and 200°, EC detector phenols DG3 (p. 302-29) GLC, 100% methyl siloxane column, halogenated acids and 200°, ElCD-X phenols DG4 (p. 302-31) GLC, 100% methyl siloxane column, acids and phenols con- 200°, ElCD-N taining nitrogen VALIDATION The following combination has undergone validation in a single laboratory, over many years, with repeated recoveries performed in conjunction with FDA’s Total Diet Study; these are recommended for use: E1 + C1 + DG1 Validation report: Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713. Where slight differences occur between the method description in Section 402 and in the Hopper et al. 1992 publication, this section reflects standard operating procedure in the Total Diet Study. Figure 402-a Method for Acids and Phenols animal tissues, dairy products, fats and shortenings E1 fruits, vegetables other than legumes, and beverages E2 legumes E3 grains and cereals E4 sugar and high sugar processed foods E5 C1 DG1/3 residues with halogen DG4 residues with nitrogen vegetable oils E6 water E7