Pesticide Analytical Manual Vol. I Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Chapter 4 Multiclass Selective MRM ARMs Chapter 5 GLC Ch HPLC Table of contents page d 401: Method for N-Methylcarbamates Basic references 401-1 1/94 General Principles 401-1 1/94 Steps of the method 401-1 1/94 Extraction with methanol 401-3 E2 with methanol, reduced samp Liquid-Liquid Partitioning and Charcoal/Celite 401-5 1/94 Column clean DLI HPLC. Post-Column Derivatization 401-91/94 Flue Form FDA 2905a (6, 400-1
Pesticide Analytical Manual Vol. I 400–1 Transmittal No. 96-E1 (9/96) Form FDA 2905a (6/92) Table of Contents page date 401: Method for N-Methylcarbamates Basic References 401-1 1/94 General Principles 401-1 1/94 Applicability 401-1 1/94 Reference Standards 401-1 1/94 Steps of the method 401-1 1/94 Validation 401-2 1/94 E1 Extraction with methanol 401-3 1/94 E2 Extraction with methanol, reduced sample size 401-4 1/94 C1 Liquid-Liquid Partitioning and Charcoal/Celite 401-5 1/94 Column Cleanup DL1 HPLC, Post-Column Derivatization, 401-9 1/94 Fluorescence Detection Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC
Pesticide Analytical Manual Vol page HPLC. Fluorescence detection 131/94 Confirmation 1/94 102: Method for Acids and Phenols Basic reference 40211/94 General Principles 40211/94 Applicability 40211/94 Reference standards 402-1 1/9 Steps of the method 40211/94 Validation 402-2 1/94 Extraction with solvents from acidified 40231/94 denatured products Extraction with acidified methylene chloride 027 1/94 Extraction with acidified methanol 402-9 1/94 E4 Extraction with acidified water/ methanol 402101/94 Extraction with acidified methanol 402111/94 Dissolution in methylene chloride/hexane 402131/94 Extraction with acidified methylene chloride 02-151/94 GPC Cleanup, methylation, and Florisi 402-171/94 column cleanup Cla GPC Clean 402-171/94 CIb Methylation 02211/94 Clc Florisil column cleanup 402221/94 Determination 402251/94 Confirmation 402251/94 403 Method for Phenylurea Herbicides Basic reference 403-11/94 General Principles 40311/94 Applicability 40311/94 Reference standard 40311/94 Steps of the method 403-11/94 Validation 40321/94 Extraction with Methanol 40331/94 Liquid-Liquid Partitioning and Florisil 40351/94 Column Cleanup dL3 HPLC, Post-Column Photolysis and 40371/94 Derivatization fluorescence detectio DL4 HPLC, Different mobile phase 403101/94 403-111/94 404 Method for benzimidazoles c referenc 40411/94 General Principles 4002 Form FDA 2905a(6/92]
SECTION 400 Pesticide Analytical Manual Vol. I 400–2 Transmittal No. 96-E1 (9/96) Form FDA 2905a (6/92) page date DL2 HPLC, Fluorescence Detection 401-13 1/94 Confirmation 401-13 1/94 402: Method for Acids and Phenols Basic Reference 402-1 1/94 General Principles 402-1 1/94 Applicability 402-1 1/94 Reference Standards 402-1 1/94 Steps of the method 402-1 1/94 Validation 402-2 1/94 E1 Extraction with solvents from acidified, 402-3 1/94 denatured products E2 Extraction with acidified methylene chloride 402-7 1/94 E3 Extraction with acidified methanol 402-9 1/94 E4 Extraction with acidified water/methanol 402-10 1/94 E5 Extraction with acidified methanol 402-11 1/94 E6 Dissolution in methylene chloride/hexane 402-13 1/94 E7 Extraction with acidified methylene chloride 402-15 1/94 C1 GPC Cleanup, methylation, and Florisil 402-17 1/94 column cleanup C1a GPC Cleanup 402-17 1/94 C1b Methylation 402-21 1/94 C1c Florisil column cleanup 402-22 1/94 Determination 402-25 1/94 Confirmation 402-25 1/94 403: Method for Phenylurea Herbicides Basic Reference 403-1 1/94 General Principles 403-1 1/94 Applicability 403-1 1/94 Reference Standards 403-1 1/94 Steps of the method 403-1 1/94 Validation 403-2 1/94 E1 Extraction with Methanol 403-3 1/94 C1 Liquid-Liquid Partitioning and Florisil 403-5 1/94 Column Cleanup DL3 HPLC, Post-Column Photolysis and 403-7 1/94 Derivatization, Fluorescence Detection DL4 HPLC, Different mobile phase 403-10 1/94 Confirmation 403-11 1/94 404: Method for Benzimidazoles Basic Reference 404-1 1/94 General Principles 404-1 1/94
Pesticide Analytical Manual Vol. I date 40411/94 Reference standards 4041 /94 Steps of the method 40411/94 Validation 4043 1/94 Extraction with methanol transfer to 40451/94 Extraction with methanol, removal of oil with 404-7 1/94 hexane, transfer to methylene chloride Extraction with methanol, removal of oil with 404-8 1/94 thylene chloride DL5 HPLC, Ion Pair Mobile Phase, UV and 4049 1/94 Fluorescence detector DL6 HPLC, Concentrated Ion Pair Mobile Phase, 404-12 1/9 UV and Fluorescence Detector DL7 HPLC, Ion Pair Mobile Phase, Changes in 404141/94 Confirmation 404141/94 res 101-a Method for N-Methylcarbamates 401-2 1/94 401-b Vacuum Rotary Evaporator 401-31/94 HPLC System for Determination of 401-91/94 N-Methylcarbamates 401-d HPLC Chromatogram of carbamates 401-121/94 and metabolites 402-a Method for Acids and Phenols 1/94 402-b Delivery Tube apparatus 402-3 1/9 403-a Method for Phenylureas 403-2 1/94 403b HPLC System for Determination of 403-71/94 Phenylurea Herbicides 403-c HPLC Chromatograms of carrot extract 403-101/94 403-d Chromatographic Pattern Comparison 403-111/94 404a lethod for benzimidazoles 0421/94 404-b Chromatograms of Benzimidazole Compounds 4104-12 9/96 404-C Effect of Ion Pairing Solution Concentration 404-18 1/94 on Chromatography of MBC Tables 401-a: Recovery of Chemicals Through Method 401 401-a-1 9/96 401-b: Recovery of Chemicals Through Method 401 401-b-l 9/96 402-a: Recovery of Chemicals Through Method 402 402-a-l 9/96 (El-E7 +Cl+DGl or DG% or DG4) 403a: Recovery of Chemicals Through Method 403 403-a-1 9/96 (El +Cl+ dl3 and DL4) Form FDA 2905a (6/92 400-3
Pesticide Analytical Manual Vol. I 400–3 Transmittal No. 96-E1 (9/96) Form FDA 2905a (6/92) page date Applicability 404-1 1/94 Reference Standards 404-1 1/94 Steps of the Method 404-1 1/94 Validation 404-3 1/94 E1 Extraction with methanol, transfer to 404-5 1/94 methylene chloride E2 Extraction with methanol, removal of oil with 404-7 1/94 hexane, transfer to methylene chloride E3 Extraction with methanol, removal of oil with 404-8 1/94 methylene chloride, transfer to methylene chloride DL5 HPLC, Ion Pair Mobile Phase, UV and 404-9 1/94 Fluorescence Detector DL6 HPLC, Concentrated Ion Pair Mobile Phase, 404-12 1/94 UV and Fluorescence Detector DL7 HPLC, Ion Pair Mobile Phase, Changes in 404-14 1/94 Detector Settings Confirmation 404-14 1/94 Figures 401-a Method for N-Methylcarbamates 401-2 1/94 401-b Vacuum Rotary Evaporator 401-3 1/94 401-c HPLC System for Determination of 401-9 1/94 N-Methylcarbamates 401-d HPLC Chromatogram of Carbamates 401-12 1/94 and Metabolites 402-a Method for Acids and Phenols 402-2 1/94 402-b Delivery Tube Apparatus 402-3 1/94 403-a Method for Phenylureas 403-2 1/94 403-b HPLC System for Determination of 403-7 1/94 Phenylurea Herbicides 403-c HPLC Chromatograms of Carrot Extract 403-10 1/94 403-d Chromatographic Pattern Comparison 403-11 1/94 404-a Method for Benzimidazoles 404-2 1/94 404-b Chromatograms of Benzimidazole Compounds 404-12 9/96 404-c Effect of Ion Pairing Solution Concentration 404-13 1/94 on Chromatography of MBC Tables 401-a: Recovery of Chemicals Through Method 401 401-a–1 9/96 (E1-E2 + C1 + DL1) 401-b: Recovery of Chemicals Through Method 401 401-b–1 9/96 (E1-E2 + C1 + DL2) 402-a: Recovery of Chemicals Through Method 402 402-a–1 9/96 (E1-E7 + C1 + DG1 or DG3 or DG4) 403-a: Recovery of Chemicals Through Method 403 403-a–1 9/96 (E1 + C1 + DL3 and DL4)
Pesticide Analytical Manual Vol page 404-a: Recovery of Chemicals Through Method 404 404-a-1 9/96 (E1E3+DL5) 4004 Form FDA 2905a(6/92]
SECTION 400 Pesticide Analytical Manual Vol. I 400–4 Transmittal No. 96-E1 (9/96) Form FDA 2905a (6/92) page date 404-a: Recovery of Chemicals Through Method 404 404-a–1 9/96 (E1-E3 + DL5)
Pesticide Analytical Manual Vol. I SECTION 401 401: METHOD FOR N-METHYLCARBAMATES BASIC REFERENCES Krause, RT(1979). Chromatogr. 185, 615-624 Krause, RT(1980)). Assoc. Off. Anal. Chem. 63, 1114-1124 Krause, RT.(1985)/. Assoc. Of Anal. Chem. 68, 734-741 GENERAL PRINCIPLES N-methylcarbamate insecticide residues, including carbamate metabolites, are ex- tracted with methanol. The extract is cleaned up by partitioning and column chromatography on a charcoal/Celite column. Residues are selectively determined with an HPLC system consisting of a reverse phase (RP)column, post-column hydrolysis and derivatization, and detection of the resultant derivative with a fluo- rescence detector Variations in the determinative step may be used for additional residues not of the N-methylcarbamate structure APPLICABILITY Consult Guide to PAM I for additional information pertinent to the appropriate application of multiresidue methodology Method is applicable to residues with an N-methylcarbamate structure in either nonfatty or fatty foods when determinative step includes post-column hydrolysis and derivatization. See Table 401-a, following method description, for chemicals tested through the method Method is applicable to naturally fluorescent residues when post-column hydrolysis and derivatization are not performed. See Table 401-b, following Table 401-a. Certain commodities, e.g., oranges, contain naturally fluorescent co-extractives that interfere with analysis. Limit of quantitation is 0.01 ppm carbofuran in high moisture products(fresh fruits and vegetables)and about 0. 02 ppm in dry products REFERENCE STANDARDS Dissolve reference standards of N-methylcarbamates in methanol to produce con- entrations of l ug/ mL. Store solutions in actinic glassware, and keep in refrigera- tor when not in use. Most carbamate standards stored in this manner are stable fo several months. However, methiocarb sulfone and sulfoxide degrade within hours and days, respectively, even with stated storage precautions STEPS OF THE METHOD Extraction E) Recommended use El (p. 401-3) Extraction with methanol high moisture products E2(P 401-4) Extraction with methanol, reduced low moisture products le size Cleanup(C) Cl (P 401-5) Two stage liquid-liquid pa artitioning products and charcoal/Celite column cleanup
Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 401–1 Pesticide Analytical Manual Vol. I SECTION 401 401: METHOD FOR N-METHYLCARBAMATES BASIC REFERENCES Krause, R.T. (1979) J. Chromatogr. 185, 615-624 Krause, R.T. (1980) J. Assoc. Off. Anal. Chem. 63, 1114-1124 Krause, R.T. (1985) J. Assoc. Off. Anal. Chem. 68, 734-741 GENERAL PRINCIPLES N-methylcarbamate insecticide residues, including carbamate metabolites, are extracted with methanol. The extract is cleaned up by partitioning and column chromatography on a charcoal/Celite column. Residues are selectively determined with an HPLC system consisting of a reverse phase (RP) column, post-column hydrolysis and derivatization, and detection of the resultant derivative with a fluorescence detector. Variations in the determinative step may be used for additional residues not of the N-methylcarbamate structure. APPLICABILITY Consult Guide to PAM I for additional information pertinent to the appropriate application of multiresidue methodology. Method is applicable to residues with an N-methylcarbamate structure in either nonfatty or fatty foods when determinative step includes post-column hydrolysis and derivatization. See Table 401-a, following method description, for chemicals tested through the method. Method is applicable to naturally fluorescent residues when post-column hydrolysis and derivatization are not performed. See Table 401-b, following Table 401-a. Certain commodities, e.g., oranges, contain naturally fluorescent co-extractives that interfere with analysis. Limit of quantitation is 0.01 ppm carbofuran in high moisture products (fresh fruits and vegetables) and about 0.02 ppm in dry products. REFERENCE STANDARDS Dissolve reference standards of N-methylcarbamates in methanol to produce concentrations of 1 µg/mL. Store solutions in actinic glassware, and keep in refrigerator when not in use. Most carbamate standards stored in this manner are stable for several months. However, methiocarb sulfone and sulfoxide degrade within hours and days, respectively, even with stated storage precautions. STEPS OF THE METHOD Extraction (E) Recommended Use E1 (p. 401-3) Extraction with methanol high moisture products E2 (p. 401-4) Extraction with methanol, reduced low moisture products sample size Cleanup (C) C1 (p. 401-5) Two stage liquid-liquid partitioning all products and charcoal/Celite column cleanup
SECTION 401 Pesticide Analytical Manual Vol I Determination D) Recommended use DLl (p 4101-9) HPLC, post-column hydrolysis and N-methylcarbamate derivatization. fluorescence detection residues DL2 (P. 401-13)HPLC, fluorescence detection naturally fluorescent Figure 401-a >75% water <75% water C1 DL1 N-methylcarbamates fluorescent VALIDATION The following combination has undergone interlaboratory validation and is recom- mended for use El+cl+ DLl Validation report: Krause, RT(1985)/. Assoc. Of. Anal. Chem. 68, 726-733. Collaborative study leading to AOAC official final action status for aldicarb, aldicarb sulfone, bufencarb, carbaryl, carbofuran, 3-hydroxycarbofuran, methiocarb, methomyl, and oxamyl in grapes and potatoes AOAC official method reference: official Methods of Analysis of the AOAC(1990) 15th 401-2 Form FDA 2905a (6/92]
SECTION 401 401–2 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I Determination (D) Recommended Use DL1 (p. 401-9) HPLC, post-column hydrolysis and N-methylcarbamate derivatization, fluorescence detection residues DL2 (p. 401-13) HPLC, fluorescence detection naturally fluorescent residues Figure 401-a Method for N-Methylcarbamates VALIDATION The following combination has undergone interlaboratory validation and is recommended for use: E1 + C1 + DL1 Validation report: Krause, R.T. (1985) J. Assoc. Off. Anal. Chem. 68, 726-733. Collaborative study leading to AOAC official final action status for aldicarb, aldicarb sulfone, bufencarb, carbaryl, carbofuran, 3-hydroxycarbofuran, methiocarb, methomyl, and oxamyl in grapes and potatoes. AOAC official method reference: Official Methods of Analysis of the AOAC (1990) 15th ed., 985.23. >75% water E1 <75% water E2 C1 DL2 naturally fluorescent chemicals DL1 N-methylcarbamates
Pesticide Analytical Manual Vol. I SECTION 401 E1 EXTRACTION WITH METHANOL Reference Krause, RT(1980)/ Assoc. Off. Anal. Chem. 63, 1114-1124 Principles Residues are extracted from high moisture products (>75% water)with methanol found to be the most effective extractant for N-methylcarbamates in tests using radiolabeled materials. The filtered extract is concentrated with a system that permits evaporation of the relatively high boiling point methanol without destroying heat e residues pparatus Buchner funnel(Buchner), porcelain, 12 cm diameter waporator, vacuum rotary with circulating chilled liquid (see Figure 401-b intain 1+I water/ethylene glycol solution in condensing coils and around receiving flask at -15C with 1/2 horsepower cooling unit. Insulate con- denser with Styrofoam or other material Control evaporator vacuum with vacuum pump and gauge; manometer may be used but is not preferred filter paper, Sharkskin, or 597 S&s to fit Buchner Figure401七 flask, round-bottom (r-b), 2 L, 5 Vacuum Rotary Evaporator homogenizer, Polytron Model T 10-35, with PT 35K genera tor containing knives, head equipped with metal (not Teflon)bushing homogenizer jar, four-side glass, I qt magnetic stirrer, star, 10 mm di ameter×8 mm height vacuum filtration flask. 500 mL Reagents bath]circulator methanol, distilled from all-glass To remove higher boiling solvents from solutions apparatus containing heat-labile residues. Water/ethylene Directions glycol at -15C cools receiving flask of rotary evaporator and (insulated] evaporator con- Add 150 g chopped high mois- denser ture product and 300 mL methanol to homogenizerjar Homogenize mixture 30 sec at about half speed(setting of 7)and then 60 sec at full speed Vacuum filter homogenate through Buchner fitted with filter paper and collect filtrate in 500 mL vacuum filtration flask. Reduce vacuum during filtration if filtrate begins to boil Transfer portion of filtrate equivalent to 100 g sample to 2 L r-b flask
Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 401–3 Pesticide Analytical Manual Vol. I SECTION 401 E1 EXTRACTION WITH METHANOL Reference Krause, R.T. (1980) J. Assoc. Off. Anal. Chem. 63, 1114-1124 Principles Residues are extracted from high moisture products (>75% water) with methanol, found to be the most effective extractant for N-methylcarbamates in tests using radiolabeled materials. The filtered extract is concentrated with a system that permits evaporation of the relatively high boiling point methanol without destroying heatlabile residues. Apparatus Buchner funnel (Buchner), porcelain, 12 cm diameter evaporator, vacuum rotary with circulating chilled liquid (see Figure 401-b). Maintain 1+1 water/ethylene glycol solution in condensing coils and around receiving flask at –15° C with 1/2 horsepower cooling unit. Insulate condenser with Styrofoam or other material. Control evaporator vacuum with vacuum pump and gauge; manometer may be used but is not preferred. filter paper, Sharkskin, or 597 S&S, to fit Buchner flask, round-bottom (r-b), 2 L, Ts 24/40 homogenizer, Polytron Model PT 10-35, with PT 35K generator containing knives, head equipped with metal (not Teflon) bushing homogenizer jar, four-side, glass, 1 qt magnetic stirrer, star, 10 mm diameter × 8 mm height vacuum filtration flask, 500 mL Reagents methanol, distilled from all-glass apparatus Directions • Add 150 g chopped high moisture product and 300 mL methanol to homogenizer jar. • Homogenize mixture 30 sec at about half speed (setting of 7) and then 60 sec at full speed. • Vacuum filter homogenate through Buchner fitted with filter paper and collect filtrate in 500 mL vacuum filtration flask. Reduce vacuum during filtration if filtrate begins to boil. • Transfer portion of filtrate equivalent to 100 g sample to 2 L r-b flask. Figure 401-b Vacuum Rotary Evaporator To remove higher boiling solvents from solutions containing heat-labile residues. Water/ethylene glycol at –15° C cools receiving flask of rotary evaporator and (insulated) evaporator condenser. Condenser (insulated) Motor Cooling unit Water bath 35° C Constant temperature bath circulator -15° C Needle valve Pump Manometer To vacuum pump
SECTION 401 Pesticide Analytical Manual Vol I mL water volume 100 g sample +200 mL methanol-10 mL contraction factor 100 g sample Add enough water to r-b flask to total 100 mL water Add star magnetic stirrer to r-b flask. Place 250 mL trap on 2L r-b flask and attach to vacuum rotary evaporator Circulate refrigerated (-150C)(1+1)water/ethylene glycol through evapo- rator condensing coils; maintain receiving flask at -15 C by immersion in refrigerated bath Apply vacuum slowly to minimize frothing by regulating with needle valve After full vacuum is applied, slowly place flask in 35 C water bath. Con centrate extract to 75 mL ALTERNAT/VE E2 EXTRACTION WITH METHANOL. REDUCED SAMPLE SIZE Principle Reduced sample size permits same amount of solvent to extract residues effectively from low moisture products (<75% water) Proceed as in El, except extract 75 g ground low moisture product with 300 mL methanol Transfer portion of filtrate equivalent to 50 g sample to 2 L I-b flask. nL water volume 50 +200 mL methanol le Continue as in el "Add enough water to r-b flask to total 100 mL water Form FDA 2905a (6/92]
SECTION 401 401–4 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I mL water 100 g sample volume 100 g sample = + 200 mL methanol – 10 mL contraction factor • Add enough water to r-b flask to total 100 mL water. • Add star magnetic stirrer to r-b flask. Place 250 mL trap on 2 L r-b flask and attach to vacuum rotary evaporator. • Circulate refrigerated (–15° C) (1+1) water/ethylene glycol through evaporator condensing coils; maintain receiving flask at –15° C by immersion in refrigerated bath. • Apply vacuum slowly to minimize frothing by regulating with needle valve. After full vacuum is applied, slowly place flask in 35° C water bath. Concentrate extract to 75 mL. ALTERNATIVE: E2 EXTRACTION WITH METHANOL, REDUCED SAMPLE SIZE Principle Reduced sample size permits same amount of solvent to extract residues effectively from low moisture products (<75% water). Directions • Proceed as in E1, except extract 75 g ground low moisture product with 300 mL methanol. • Transfer portion of filtrate equivalent to 50 g sample to 2 L r-b flask. mL water 50 g sample volume 50 g sample = + 200 mL methanol • Continue as in E1, "Add enough water to r-b flask to total 100 mL water
Pesticide Analytical Manual Vol. I SECTION 401 C1 LIQUID-LIQUID PARTITIONING AND CHARCOAL/CELITE COLUMN CLEANUP Reference Krause, RT(1980)/. Assoc Of. Anal. Chem. 63, 1114-1124 Principles Residues in aqueous extract are transferred to acetonitrile by liquid-liquid partition- ing in the presence of sodium chloride. Co-extractives are removed from the acetonitrile solution by partitioning them into petroleum ether, which is discarded Residues are partitioned from acetonitrile into methylene chloride. Methylene chloride solution is cleaned up on a charcoal/Celite column, and residues are eluted with toluene/acetonitrile Apparatus chromatographic column, 22 mm id x 300 mm, Teflon stopcock,coarse porosity fritted dise chromatographic column, 25 mm id, plain evaporator, vacuum rotary, as described in E-1 flasks, round-bottom(r-b), 250 and 500 mL, 1 L, T24/40 magnetic stirrer, star, 10 mm diameter x 8 mm height separatory funnel (separator), 250 and 500 mL vacuum adapter, side arm, with 324/40 joints agent acetonitrile, distilled from all-glass apparatus; see Section 204 for distillation Celite 545 harcoal(Nuchar S-N), produced by Westvaco Corp. and available from Eastman Kodak. Cat. No. 118 0454 1+4(w/w) charcoal/Celite, combined after each is prepared as directed below; mix thoroughly and store in sealed container dichlorodimethylsilane hydrochloric acid, concentrated, reagent grade isopropanol, distilled from all-glass apparatus methanol, distilled from all-glass apparatus methyl red thylene chloride, distilled from all-glass apparatus petroleum ether, distilled from all-glass apparatus sodium chloride, reagent grade :%(w/v)sodium chloride/water 20%(w/v) sodium chloride/water
Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 401–5 Pesticide Analytical Manual Vol. I SECTION 401 C1 LIQUID-LIQUID PARTITIONING AND CHARCOAL/CELITE COLUMN CLEANUP Reference Krause, R.T. (1980) J. Assoc. Off. Anal. Chem. 63, 1114-1124 Principles Residues in aqueous extract are transferred to acetonitrile by liquid-liquid partitioning in the presence of sodium chloride. Co-extractives are removed from the acetonitrile solution by partitioning them into petroleum ether, which is discarded. Residues are partitioned from acetonitrile into methylene chloride. Methylene chloride solution is cleaned up on a charcoal/Celite column, and residues are eluted with toluene/acetonitrile. Apparatus chromatographic column, 22 mm id × 300 mm, Teflon stopcock, coarse porosity fritted disc chromatographic column, 25 mm id, plain evaporator, vacuum rotary, as described in E-1 flasks, round-bottom (r-b), 250 and 500 mL, 1 L, Ts 24/40 magnetic stirrer, star, 10 mm diameter × 8 mm height separatory funnel (separator), 250 and 500 mL vacuum adapter, side arm, with Ts 24/40 joints Reagents acetonitrile, distilled from all-glass apparatus; see Section 204 for distillation directions Celite 545 charcoal (Nuchar S-N), produced by Westvaco Corp. and available from Eastman Kodak, Cat. No. 118 0454 1+4 (w/w) charcoal/Celite, combined after each is prepared as directed below; mix thoroughly and store in sealed container dichlorodimethylsilane glass wool, Pyrex hydrochloric acid, concentrated, reagent grade isopropanol, distilled from all-glass apparatus methanol, distilled from all-glass apparatus methyl red methylene chloride, distilled from all-glass apparatus petroleum ether, distilled from all-glass apparatus sodium chloride, reagent grade 2% (w/v) sodium chloride/water 20% (w/v) sodium chloride/water
SECTION 401 Pesticide Analytical Manual Vol I Odium sulfate, anhydrous, granular, reagent grade; see Section 204 for han- dling directions toluene, distilled from all-glass apparatus eluant: 25%(v/v)toluene/acetonitrile Preparation of Silanized Celite 545 Slurry 150 g Celite 545 with IL (1+I)hydrochloric acid/ water in 2L beaker, over with watch glass, and stir magnetically while boiling 10 min Cool slurry, filter, and wash with distilled or HPLC grade water until filtrate is neutral Wash Celite with 500 mL methanol followed by 500 mL methylene chloride and then air dry celite in hood on watch glass to remove solvent Transfer Celite to I L glass-stoppered (g-s) Erlenmeyer flask. Heat unstop- pered flask in 120 C oven overnight and then cool flask in desiccator Place flask in hood and carefully pipet 3 mL dichlorodimethylsilane onto Celite. Stopper flask, mix well, and let flask remain at room temperature 4 Add 500 mL methanol to flask. mix. and let stand 15 min. d Celite and wash with isopropanol until neutral Air dry silanized Celite in hood to remove isopropanol Dry silanized Celite in 105C oven 2 hr and cool in desiccator. Store sila nized Celite in g-s container. Test Celite for total silanization with two tests Place about i g celite in 50 mL water; silanized Celite will float Place second 1 g Celite in 20 mL toluene saturated with methyl red; silanized Celite will appear yellow. If particles of Celite are dispersed in water and/or appear pink with methyl red/ toluene solution, active sites still exist on Celite; repeat silanization Purification of charcoal s y urry 100 g Nuchar S-N with 700 mL hydrochloric acid, cover with watch glass, and stir magnetically while boiling 1 h Add 700 mL water. stir. and boil additional 30 min Cool slurry, filter, and wash with water until neutral Wash Nuchar S-N with 500 mL methanol followed by 500 mL methylene chloride, and air dry Nuchar S-N in hood to remove solvent. Dry Nuchar S-N in 120C oven 4 hr. Cool in desiccator. Store NucharS-N in g-s container. Testing of Charcoal/ Celite Prepare cleanup column of (1+4)(w/w) charcoal/Celite as described be- Prepare methanol solution of 5 ug/mL each carbaryl, methiocarb, methio- carb sulfoxide, and methomyl Use freshly prepared mixed standard solu- 401-6 Form FDA 2905a (6/92]
SECTION 401 401–6 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for handling directions toluene, distilled from all-glass apparatus eluant: 25% (v/v) toluene/acetonitrile Preparation of Silanized Celite 545 • Slurry 150 g Celite 545 with 1 L (1+1) hydrochloric acid/water in 2 L beaker, cover with watch glass, and stir magnetically while boiling 10 min. • Cool slurry, filter, and wash with distilled or HPLC grade water until filtrate is neutral. • Wash Celite with 500 mL methanol followed by 500 mL methylene chloride, and then air dry Celite in hood on watch glass to remove solvent. • Transfer Celite to 1 L glass-stoppered (g-s) Erlenmeyer flask. Heat unstoppered flask in 120° C oven overnight and then cool flask in desiccator. • Place flask in hood and carefully pipet 3 mL dichlorodimethylsilane onto Celite. Stopper flask, mix well, and let flask remain at room temperature 4 hr. • Add 500 mL methanol to flask, mix, and let stand 15 min. • Filter silanized Celite and wash with isopropanol until neutral. • Air dry silanized Celite in hood to remove isopropanol. • Dry silanized Celite in 105° C oven 2 hr and cool in desiccator. Store silanized Celite in g-s container. • Test Celite for total silanization with two tests. Place about 1 g Celite in 50 mL water; silanized Celite will float. Place second 1 g Celite in 20 mL toluene saturated with methyl red; silanized Celite will appear yellow. If particles of Celite are dispersed in water and/or appear pink with methyl red/toluene solution, active sites still exist on Celite; repeat silanization. Purification of Charcoal • Slurry 100 g Nuchar S-N with 700 mL hydrochloric acid, cover with watch glass, and stir magnetically while boiling 1 hr. • Add 700 mL water, stir, and boil additional 30 min. • Cool slurry, filter, and wash with water until neutral. • Wash Nuchar S-N with 500 mL methanol followed by 500 mL methylene chloride, and air dry Nuchar S-N in hood to remove solvent. • Dry Nuchar S-N in 120° C oven 4 hr. Cool in desiccator. Store Nuchar S-N in g-s container. Testing of Charcoal/Celite • Prepare cleanup column of (1+4) (w/w) charcoal/Celite as described below. • Prepare methanol solution of 5 µg/mL each carbaryl, methiocarb, methiocarb sulfoxide, and methomyl. Use freshly prepared mixed standard solution; methiocarb sulfoxide degrades in solution
