《美国FDA农残分析手册》（第一卷英文版）Chapter 4 Selective MRMs

Pesticide Analytical Manual Vol. I SECTION 401 Pipet 5 mL solution into 250 mL I-b flask and 5 mL into 25 mL actinic volumetric flask Dilute solution in volumetric flask to 25 mL with methanol: use as hPlc reference standard Evaporate standard solution in r-b flask just to dryness with vacuum rotary evaporator as described below. After last trace of methanol has evaporated remove r-b flask from evaporator and dissolve carbamate residue in 10 m ethylene chlorido Transfer methylene chloride solution in r-b flask to prepared adsorbent column and elute as described bele After evaporation ofeluate in r-b flask, dissolve residue in 25 mL methanol filter aliquot through filtration device, and quantitate recovery of carba- mates as in DLl. Nuchar S-n is considered satisfactory if recovery is 295% Directions Partitioning Transfer concentrated extract from El or E2 to 500 mL separator con- taining 15 g sodium chloride. Shake separator until sodium chloride is Wash r-b flask with three 25 mL portions acetonitrile, transferring each to 500 mL separator; shake separator 30 sec, and let layers separate 5 min Drain aqueous phase into 250 mL separator containing 50 mLacetonitrile shake 20 sec, let layers separate, and discard aqueous layer Add 25 mL 20%o aqueous sodium chloride solution to acetonitrile in 500 mL separator, shake 20 sec, let layers separate, and transfer aqueous olution to 250 mL separato Shake 250 mL separator 20 sec, let layers separate, and discard aqueous Add 100 mL petroleum ether to 500 mL separator, shake 20 sec, let layers separate, and drain acetonitrile layer into second 500 mL separator Transfer acetonitrile in 250 mL separator to first 500 mL separator which contains petroleum ether, shake 20 sec, let layers separate, and transfer acetonitrile to second 500 mL separator Add 10 mL acetonitrile to first 500 mL separator, shake, let layers separate, and transfer acetonitrile to second 500 mL separator. Discard petroleum Add 50 mL% aqueous sodium chloride solution to acetonitrile in second 500 mL separator Extract mixture successively with 100, 25, and 25 mL methylene chloride, shaking each 20 sec(shake 25 mL portions gently) Drain lower methylene chloride/acetonitrile layers through 22 mm id column containing about 5 cm sodium sulfate. Collecteluate in I Lr-bflask Add star magnetic stirrer to r-b flask Place 250 mL trap on I L r-b flask and tach to vacuum rotary evaporator. Circulate refrigerated (-15C)(1+1)water/ethylene glycol through evapo rator condensing coils, maintain receiving flask at -15C by immersion in refrigerated bath

Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 401–7 Pesticide Analytical Manual Vol. I SECTION 401 • Pipet 5 mL solution into 250 mL r-b flask and 5 mL into 25 mL actinic volumetric flask. • Dilute solution in volumetric flask to 25 mL with methanol; use as HPLC reference standard. • Evaporate standard solution in r-b flask just to dryness with vacuum rotary evaporator as described below. After last trace of methanol has evaporated, remove r-b flask from evaporator and dissolve carbamate residue in 10 mL methylene chloride. • Transfer methylene chloride solution in r-b flask to prepared adsorbent column and elute as described below. • After evaporation of eluate in r-b flask, dissolve residue in 25 mL methanol, filter aliquot through filtration device, and quantitate recovery of carba￾mates as in DL1. Nuchar S-N is considered satisfactory if recovery is ≥95%. Directions Partitioning • Transfer concentrated extract from E1 or E2 to 500 mL separator con￾taining 15 g sodium chloride. Shake separator until sodium chloride is dissolved. • Wash r-b flask with three 25 mL portions acetonitrile, transferring each to 500 mL separator; shake separator 30 sec, and let layers separate 5 min. • Drain aqueous phase into 250 mL separator containing 50 mL acetonitrile, shake 20 sec, let layers separate, and discard aqueous layer. • Add 25 mL 20% aqueous sodium chloride solution to acetonitrile in 500 mL separator, shake 20 sec, let layers separate, and transfer aqueous solution to 250 mL separator. • Shake 250 mL separator 20 sec, let layers separate, and discard aqueous layer. • Add 100 mL petroleum ether to 500 mL separator, shake 20 sec, let layers separate, and drain acetonitrile layer into second 500 mL separator. • Transfer acetonitrile in 250 mL separator to first 500 mL separator which contains petroleum ether, shake 20 sec, let layers separate, and transfer acetonitrile to second 500 mL separator. • Add 10 mL acetonitrile to first 500 mL separator, shake, let layers separate, and transfer acetonitrile to second 500 mL separator. Discard petroleum ether layer. • Add 50 mL 2% aqueous sodium chloride solution to acetonitrile in second 500 mL separator. Extract mixture successively with 100, 25, and 25 mL methylene chloride, shaking each 20 sec (shake 25 mL portions gently). • Drain lower methylene chloride/acetonitrile layers through 22 mm id column containing about 5 cm sodium sulfate. Collect eluate in 1 L r-b flask. • Add star magnetic stirrer to r-b flask. Place 250 mL trap on 1 L r-b flask and attach to vacuum rotary evaporator. • Circulate refrigerated (–15° C) (1+1) water/ethylene glycol through evapo￾rator condensing coils; maintain receiving flask at –15° C by immersion in refrigerated bath

SECTION 401 Pesticide Analytical Manual Vol I Apply vacuum slowly to minimize frothing by regulating with needle valve After full vacuum is applied, slowly place flask in 35 C water bath Remove r-b flask from evaporator immediately after last traces of solution have evaporated and add 10 mL methylene chloride to r-b flask. Charcoal/Celite Cleanup Fit one-hole No 5 rubber stopper onto tip of chromatographic column with stopcock, add side-arm vacuum adapter and 500 mL r-b flask, open stopcock, and connect apparatus to vacuum line Place 0.5 g silanized Celite in chromatographic column, tamp, add 5 g charcoal/Celite mixture, and tamp again Add 1-2 cm glass wool plug on of adsorbent Prewash column with 50 mL 25% toluene/acetonitrile eluant. Close stol P sh solution is about o5 cm fr Disconnect vacuum discard solution in r-b flask. and reconnect flask to Transfer 10 mL methylene chloride extract from partitioning steps to column and elute column at 5 mL/min Wash I L r-b flask with 10 mL methylene chloride and then with 25 mL eluant. Transfer each separately to column and elute each to top of glass wool before adding next solution Add 100 mL eluant and elute column at 5 mL/min. Turn off stopcock when top of eluant reaches top of glass wool. Evaporate solution in 500 mL r-b flask just to dryness using vacuum evap- orator as ab Remove flask from evaporator immediately after all solu- Immediately pipet 5 mL methanol into 500 mL T-b flask to dissolve resi- dt Cleaned up extract contains 20 g sample equivalent/mL solution for high moisture products and 10 g sample equivalent/mL solution for low mois- 401-8 Form FDA 2905a (6/92]

SECTION 401 401–8 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I • Apply vacuum slowly to minimize frothing by regulating with needle valve. After full vacuum is applied, slowly place flask in 35° C water bath. • Remove r-b flask from evaporator immediately after last traces of solution have evaporated and add 10 mL methylene chloride to r-b flask. Charcoal/Celite Cleanup • Fit one-hole No. 5 rubber stopper onto tip of chromatographic column with stopcock, add side-arm vacuum adapter and 500 mL r-b flask, open stopcock, and connect apparatus to vacuum line. • Place 0.5 g silanized Celite in chromatographic column, tamp, add 5 g charcoal/Celite mixture, and tamp again. Add 1-2 cm glass wool plug on top of adsorbent. • Prewash column with 50 mL 25% toluene/acetonitrile eluant. Close stop￾cock when prewash solution is about 0.5 cm from top of glass wool. • Disconnect vacuum, discard solution in r-b flask, and reconnect flask to apparatus. • Transfer 10 mL methylene chloride extract from partitioning steps to column and elute column at 5 mL/min. • Wash 1 L r-b flask with 10 mL methylene chloride and then with 25 mL eluant. Transfer each separately to column and elute each to top of glass wool before adding next solution. • Add 100 mL eluant and elute column at 5 mL/min. Turn off stopcock when top of eluant reaches top of glass wool. • Evaporate solution in 500 mL r-b flask just to dryness using vacuum evap￾orator as above. Remove flask from evaporator immediately after all solu￾tion has evaporated. • Immediately pipet 5 mL methanol into 500 mL r-b flask to dissolve resi￾due. • Cleaned up extract contains 20 g sample equivalent/mL solution for high moisture products and 10 g sample equivalent/mL solution for low mois￾ture products

Pesticide Analytical Manual Vol. I SECTION 401 DL1 HPLC POST-COLUMN DER/VATIZATON FLUORESCENCE DETECTION Reference Krause, RT(1978)/. Chromatogr. Sci. 16, 281-288 Principles Residues in methanol solution are separated on a C-8 reverse phase HPLC column using acetonitrile/water gradient mobile phase Residues eluting from the column are hydrolyzed in-line to methylamine under alkaline conditions. Methylamine is reacted, also in-line, with o-phthalaldehyde and 2-mercaptoethanol to form a fluorophore that is measured by a fluorescence detector. This post-column derivatization- fluorescence detection determinative step is very selective for residues containing the N-methylcarbamate structure. Apparatus either(a)13 mm diameter Swinny stainl Se with Luer-Lok tip, fitted with filtration device for solutions, 10 mL syring ss steel filter holder and 13 mm diameter filters, 5.0 um LS-type, or(b)disposable membrane filters, 13 mm diameter,0.22 um nylon membrane, encased in polypropylene (Preassembled devices that do not require a syringe are also available. HPLCsystem( Figure 401-c)must meet system suitability test below. Complete system consists of 1)mobile phase delivery system, programmable HPLC gradient system 2)injector, automatic sampler with 10 uL injection loop 3)guard column, stainless steel, containing 25-37 um pellicular C-8 or C-18 4)column oven or heater Figure 401-C HPLC System for Determination of N-Methylcarbamates Water Acetonitrile ner→ OPA-MERC Guard 25 cm d chamber ecorder/data

Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 401–9 Pesticide Analytical Manual Vol. I SECTION 401 DL1 HPLC, POST-COLUMN DERIVATIZATION, FLUORESCENCE DETECTION Reference Krause, R.T. (1978) J. Chromatogr. Sci. 16, 281-288 Principles Residues in methanol solution are separated on a C-8 reverse phase HPLC column using acetonitrile/water gradient mobile phase. Residues eluting from the column are hydrolyzed in-line to methylamine under alkaline conditions. Methylamine is reacted, also in-line, with o-phthalaldehyde and 2-mercaptoethanol to form a fluorophore that is measured by a fluorescence detector. This post-column derivatization-fluorescence detection determinative step is very selective for residues containing the N-methylcarbamate structure. Apparatus filtration device for solutions, 10 mL syringe with Luer-Lok tip, fitted with either (a) 13 mm diameter Swinny stainless steel filter holder and 13 mm diameter filters, 5.0 µm LS-type, or (b) disposable membrane filters, 13 mm diameter, 0.22 µm nylon membrane, encased in polypropylene. (Preassembled devices that do not require a syringe are also available.) HPLC system (Figure 401-c) must meet system suitability test below. Complete system consists of: 1) mobile phase delivery system, programmable HPLC gradient system 2) injector, automatic sampler with 10 µL injection loop 3) guard column, stainless steel, containing 25-37 µm pellicular C-8 or C-18 packing 4) column oven or heater Figure 401-c HPLC System for Determination of N-Methylcarbamates Water Acetonitrile Fluorescence detector Recorder/data Post-column handling reaction Hydrolysis chamber NaOH OPA-MERC 100° C Valve injector Guard column C-8 analytical column 25 cm

SECTION 401 Pesticide Analytical Manual Vol I 5)analytical column, 25 cmx4.6 mm id, containing 6 um Zorbax C-8 spherical particles. Column packing should consist of 5 or 6 um spherical silica particles bonded with monofunctional octyl silane reagent to form mono- molecular bond 6) connecting tubing, No. 304 stainless steel (1.6 mm od x 0. 18 mm id)to connect injector, column, and first tee 7)post-column derivatization unit, as shown in telescoped portion of Figure 401-b. Units used during collaborative study of od were assembled from the following parts a)reservoirs for sodium hydroxide and OPA-MERC reaction solutions, 60 cmx25 mm id glass columns with Teflon fittings; pressurize reservoirs with nitrogen gas, adjusted to create appropriate reagent flow(Pumps can be substituted for nitrogen gas pressure. b) 6mx0.5 mm id Teflon restriction coil, to connect each reservoir to 15 cm x 0.18 mm id stainless steel tubing, which in turn is connected to 0.74 mm id stainless steel mixing tee(Valco Instruments Co, Cat. No ZVT-062)for connection to flow of mobile phase c)carbamate hydrolysis chamber, stainless steel tubing, 3 mx0. 48 mm id No. 321, coiled to fit in small oven capable of maintaining constant, uniform100°C d) reaction tube, 25 cm stainless steel tubing between tee that delivers OPA-MERC solution and 1.5 cm x0.8 mm id detector cell tubing Commercial post-column derivatization units that replace these compo- nents are now available from several manufacturers (abl, Pickering Instru- ments,Waters). Systems with dual piston pumps are preferred 8)fl nce detector dual monoch or, equipped with $20 uL cell 9)recorder, strip chart recorder or computing integrator compatible wit detecto acetonitrile, UV grade distilled from all-glass apparatus. Before use, degas acetonitrile in glass bottles by applying vacuum and slowly stirring with mag netic stirrer 5 min. Acetonitrile other than HPlC grade may cause broad 2-mercaptoethanol(MERC), 98+% methanol, distilled from all-glass apparatus o-phthalaldehyde (OpA), chromatographic grade sodium borate buffer solution, 0.05 M. Add 19. 1 g aCs grade sodium tetrabor- ate decahydrate(Na, BO.10 H,O)and about 500 mL degassed HPLC grade water to 1 L volumetric flask. Heat flask in steam bath to dissolve sodium tetraborate, cool to room temperature, and dilute to volume with degassed HPLC grade water. Mix well, but gently, to minimize re-incorporation of airinto solution odium hydroxide solution, 0.05 N Prepare clear supernate sodium hydroxide as follows: to one part sodium hydroxide(reagent quality containing <5% sodium carbonate), add one part water and swirl until solution is complete Stopper and set aside until sodium carbonate has settled, leaving clear liquid 401-10 Form FDA 2905a (6/92]

SECTION 401 401–10 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I 5) analytical column, 25 cm × 4.6 mm id, containing 6 µm Zorbax C-8 spherical particles. Column packing should consist of 5 or 6 µm spherical silica particles bonded with monofunctional octyl silane reagent to form mono￾molecular bond. 6) connecting tubing, No. 304 stainless steel (1.6 mm od × 0.18 mm id) to connect injector, column, and first tee 7) post-column derivatization unit, as shown in telescoped portion of Figure 401-b. Units used during collaborative study of method were assembled from the following parts: a) reservoirs for sodium hydroxide and OPA-MERC reaction solutions, 60 cm × 25 mm id glass columns with Teflon fittings; pressurize reservoirs with nitrogen gas, adjusted to create appropriate reagent flow. (Pumps can be substituted for nitrogen gas pressure.) b) 6 m × 0.5 mm id Teflon restriction coil, to connect each reservoir to 15 cm × 0.18 mm id stainless steel tubing, which in turn is connected to 0.74 mm id stainless steel mixing tee (Valco Instruments Co., Cat. No. ZVT-062) for connection to flow of mobile phase c) carbamate hydrolysis chamber, stainless steel tubing, 3 m × 0.48 mm id No. 321, coiled to fit in small oven capable of maintaining constant, uniform 100° C d) reaction tube, 25 cm stainless steel tubing between tee that delivers OPA-MERC solution and 1.5 cm × 0.3 mm id detector cell tubing Commercial post-column derivatization units that replace these compo￾nents are now available from several manufacturers (ABI, Pickering Instru￾ments, Waters). Systems with dual piston pumps are preferred. 8) fluorescence detector, dual monochromator, equipped with ≤20 µL cell 9) recorder, strip chart recorder or computing integrator compatible with detector Reagents acetonitrile, UV grade distilled from all-glass apparatus. Before use, degas acetonitrile in glass bottles by applying vacuum and slowly stirring with mag￾netic stirrer 5 min. Acetonitrile other than HPLC grade may cause broad, nonreproducible peaks in chromatograms. 2-mercaptoethanol (MERC), 98+% methanol, distilled from all-glass apparatus o-phthalaldehyde (OPA), chromatographic grade sodium borate buffer solution, 0.05 M. Add 19.1 g ACS grade sodium tetrabor￾ate decahydrate (Na2 B4 O7 •10 H2 O) and about 500 mL degassed HPLC grade water to 1 L volumetric flask. Heat flask in steam bath to dissolve sodium tetraborate, cool to room temperature, and dilute to volume with degassed HPLC grade water. Mix well, but gently, to minimize re-incorporation of air into solution. sodium hydroxide solution, 0.05 N. Prepare clear supernate sodium hydroxide as follows: to one part sodium hydroxide (reagent quality containing <5% sodium carbonate), add one part water and swirl until solution is complete. Stopper and set aside until sodium carbonate has settled, leaving clear liquid

Pesticide Analytical Manual Vol. I SECTION 401 (about 10 days). Pipet 27 mL clear supernate sodium hydroxide into 100 mL volumetric flask. Dilute to volume with water and mix(5 N sodium hydroxide) ipet 10 mL5 N sodium hydroxide into I l volumetric flask. Dilute to volume with degassed HPLC grade water and mix well, but gently, to minimize re- incorporation of air into solution water,HPLC grade, commercial product or prepared from water purification equipment that produces distilled, deionized water For HPLC, degas water s described for acetonitrile. Water must be adequately purified to prevent plugging HPLC column and extraneous peaks in chromatograms. All water used in HPLC procedure must be HPLC grade. ("Water"that does not specify HPLC grade means distilled water. flask. add 10 mL methanol and swirl to dissolve opa. add about 500 mL 0.05 M sodium borate buffer solution and l mL 2-mercaptoethanol. Dilute to vol- Ime with sodium borate buffer solution. Mix well, but gently, to minimize re- incorporation of air into solution.(Borate solution purchased in plastic bottles or low purity grades of OPa may cause excessively high background fluorescence. Solution is acceptable for about 2 days when stored at room mperature, about I week when stored in refrigerator or under helium System Operation Adjust mobile phase flow rate to 1.50+0.02 mL/ min with 50% acetonitrile/ hPLC grade wate Adjust flow rates of 0.05 N sodium hydroxide and oPA-merc reaction solution to 0.50+0.02 mL/min each. Operate column oven at 35C and hydrolysis chamber at 100C Set fluorescence detector excitation and emission wavelengths to 340 and 455 nm, respectively, and slit widths to 15 and 12 nm, respectively. Set detector photomultiplier tube gain to low and time constant to l sec Equilibrate system 10 min with 12% acetonitrile/HPLC grade water, inject sample, and begin $0 min linear gradient to 70% acetonitrile/HPLCgrade water Adjust sensitivity so that 10 ng carbofuran produces 50+5% full scale deflection on printer-plotter. Baseline noise should be <2%, and carbam- ates should elute as shown in Figure 401-c If system will not be used for several days, replace aqueous mobile phase with methanol and pump through system Drain sodium hydroxide and OPA-MERC reaction solutions from their reservoirs. and wash reservoir and associated tubing with water, then methanol. When starting system change mobile phase to HPLC grade water, and wash reservoirs and associated tubing with water before adding reaction solutions System Suitability Test See Chapter 6, HPLC, for further information about evaluating HPLC systems Prepare mixed standard solution containing l ug/mL each aldicarb sul- foxide, aldicarb sulfone, carbofuran, and carbaryl Chromatograph solution, using HPLC system operation described above Retention times will be about 6.5.8. 20. and 21 min 401-11

Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 401–11 Pesticide Analytical Manual Vol. I SECTION 401 (about 10 days). Pipet 27 mL clear supernate sodium hydroxide into 100 mL volumetric flask. Dilute to volume with water and mix (5 N sodium hydroxide). Pipet 10 mL 5 N sodium hydroxide into 1 L volumetric flask. Dilute to volume with degassed HPLC grade water and mix well, but gently, to minimize re￾incorporation of air into solution. water, HPLC grade, commercial product or prepared from water purification equipment that produces distilled, deionized water. For HPLC, degas water as described for acetonitrile. Water must be adequately purified to prevent plugging HPLC column and extraneous peaks in chromatograms. All water used in HPLC procedure must be HPLC grade. (“Water” that does not specify HPLC grade means distilled water.) OPA-MERC reaction solution. Weigh 500 mg OPA, transfer to 1 L volumetric flask, add 10 mL methanol, and swirl to dissolve OPA. Add about 500 mL 0.05 M sodium borate buffer solution and 1 mL 2-mercaptoethanol. Dilute to vol￾ume with sodium borate buffer solution. Mix well, but gently, to minimize re￾incorporation of air into solution. (Borate solution purchased in plastic bottles or low purity grades of OPA may cause excessively high background fluorescence.) Solution is acceptable for about 2 days when stored at room temperature, about 1 week when stored in refrigerator or under helium. System Operation • Adjust mobile phase flow rate to 1.50±0.02 mL/min with 50% acetonitrile/ HPLC grade water. • Adjust flow rates of 0.05 N sodium hydroxide and OPA-MERC reaction solution to 0.50±0.02 mL/min each. Operate column oven at 35° C and hydrolysis chamber at 100° C. • Set fluorescence detector excitation and emission wavelengths to 340 and 455 nm, respectively, and slit widths to 15 and 12 nm, respectively. Set detector photomultiplier tube gain to low and time constant to 1 sec. • Equilibrate system 10 min with 12% acetonitrile/HPLC grade water, inject sample, and begin 30 min linear gradient to 70% acetonitrile/HPLC grade water. • Adjust sensitivity so that 10 ng carbofuran produces 50±5% full scale deflection on printer-plotter. Baseline noise should be <2%, and carbam￾ates should elute as shown in Figure 401-c. • If system will not be used for several days, replace aqueous mobile phase with methanol and pump through system. Drain sodium hydroxide and OPA-MERC reaction solutions from their reservoirs, and wash reservoirs and associated tubing with water, then methanol. When starting system, change mobile phase to HPLC grade water, and wash reservoirs and associated tubing with water before adding reaction solutions. System Suitability Test See Chapter 6, HPLC, for further information about evaluating HPLC systems. • Prepare mixed standard solution containing 1 µg/mL each aldicarb sul￾foxide, aldicarb sulfone, carbofuran, and carbaryl. • Chromatograph solution, using HPLC system operation described above. Retention times will be about 6.5, 8, 20, and 21 min, respectively