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1、10.1.01 AOAC Official Method 970.52 Organochlorine and Organophosphorus Pesticide Residues General Multiresidue Method A. Principle Thoroughly mixed test portion is extracted with CH3CN (high-H2O foods) or aqueous CH3CN (low-H2O or high sugar foods). Fat is extracted from fatty foods and partitioned
2、 between petroleum ether and CH3CN. Aliquot (nonfatty foods) or entire solution (fatty foods) of CH3CN is diluted with H2O and residues are extracted into petroleum ether. Residues are purified by chro- matography on Florisil column, eluting with mixture of petro- leum and ethyl ethers. Residues in
3、concentrated eluates are measuredbyGasChromatography(GC)andidentifiedbycombi- nations of gas, thin layer, or paper chromatography. Analystcompetenceinapplyingmethodfortraceresiduesshould beassuredbeforeanalysis.Recoveriesofaddedcompoundsthrough method should be 80%. Absenceofinterferencesarisingfrom
4、laboratoryandreagentcon- taminationshouldalsobeassuredbyregularperformanceofreagent blanks. Solvents in particular, because of their concentration during methods, can contribute significant interferences if not sufficiently purified. Solvents of adequate purity are commercially available from severa
5、l manufacturers, but each batch must be tested under conditions of method in which it will be used. Other reagents and apparatus (rubber, plastics, glass wool, etc.) arealsopotentialsourceofinterferences.Seereferencesforrecover- ies obtained during collaborative and validation studies and Ta- ble 97
6、0.52A for commodities approved. See H, introductory para- graph,andH(c)forGCperformancerequirements:sensitivity,sepa- ration capability, and linearity. Behavior of 200 pesticides and industrial chemicals in method is given in JAOAC 61, 640(1978). B. General Reagents Solvents must be purified and fin
7、al distillation conducted in all-glass apparatus See A. Solvent purity test.Electron capture GC requires absence of substancescausingdetectorresponseasindicatedbyfollowingtest: Place 300 mL solvent in Kuderna-Danish concentrator fitted with 3-ball Snyder column and calibrated collection vessel, and
8、evapo- rate to 5 mL. Inject 5 L concentrate from 10 L syringe into gas chromatograph, using conditions described in H(c). Concentrate must not cause recorder deflection 1 mm from baseline for 260 min after injection. (a) Acetonitrile.See solvent purity test. Purify technical CH3CNasfollows:To4LCH3CN
9、add1mLH3PO4,30gP2O5,and boilingchips,anddistilinall-glassapparatusat8182C.Donotex- ceed 82C. Some lots of reagent grade CH3CN are impure and require distil- lation. Generally vapors from such lots will turn moistened red lit- mus paper blue when held over mouth of storage container. Pronounced amine
10、 odor is detectable. (b) Acetonitrile saturated with petroleum ether.Saturate CH3CN, (a), with redistilled petroleum ether, (m). (c) Alcohol.USP, reagent grade, or methanol, ACS. (d) Alcoholic alkali solution.2%. Dissolve 2 g KOH in alco- hol, and dilute to 100 mL. (e) Eluting solvent, 6%.Dilute 60
11、mL ethyl ether, (h), to 1 L with redistilled petroleum ether, (m). (f) Eluting solvent, 15%.Prepare as in (e), using 150 mL ethyl ether. (g) Eluting solvent, 50%.Prepare as in (e), using 500 mL ethyl ether. (h) Ethylether.Seesolventpuritytest.Redistilledat3435C, and stored under N2. Add 2% alcohol (
12、c). Must be peroxide-free by test in Definitions of Terms and Explanatory Notes. (i) Florisil.60/100 PR grade, activated at 675C (1250F), available from Floridin Co., 3 Pennsylvania Center, Pittsburgh, PA 15235 USA. When 675C activated Florisil is obtained in bulk, transfer immediately after opening
13、 to ca 500 mL (1 pt) glass jars, or bottles, with glass-stoppered or foil-lined, screw-top lids, and store in dark. Heat 5 h at 130C before use. Store at 130C in glass-stopperedbottlesorinair-tightdesiccatoratroomtemperature and reheat at 130C after 2 days. Preparemixedpesticidestandardsolutioninhex
14、anecontaining1, 4,1,2,1,2,and4g/mL,respectively,ofronnel,ethion,heptachlor epoxide, parathion, dieldrin, endrin, and malathion. TesteachbatchofactivatedFlorisilbyplacing1mLmixedpesti- cide standard on prepared column and eluting as in Cleanup, O. Concentrate eluates from Florisil column to 10 mL. In
15、ject aliquot (seeH)ofeacheluateintogaschromatographanddeterminequanti- tativerecoveryofeachcompoundasinR.Florisilthatquantitatively elutes heptachlor epoxide, ronnel, and ethion in 6% eluate; dieldrin, 2000 AOAC INTERNATIONAL Table 970.52ACompounds and commodities to which general method applies Com
16、poundOfficial Final Action Dieldrin (CAS-60-57-1) Heptachlor epoxide (CAS-1024-57-3) Group I nonfatty foods, dairy products, fish, vegetable oils, whole eggs BHC (CAS-608-73-1) DDE (CAS-72-55-9) DDT (CAS-8017-34-3) TDE (CAS-72-54-8) Group I nonfatty foods, dairy products, fish, whole eggs Lindane (C
17、AS-58-89-9) Methoxychlor (CAS-72-43-5) Ethylan (CAS-72-56-0) Group I nonfatty foods, dairy products, whole eggs Aldrin (CAS-309-00-2) Endrin (CAS-72-20-8) Heptachlor (CAS-76-44-8) Mirex (CAS-2385-85-5) Group I nonfatty foods, whole eggs o,p-DDT (CAS-8017-34-3)Group I nonfatty foods, dairy products D
18、iazinon (CAS-333-41-5) Ethion (CAS-563-12-2) Malathion (CAS-121-75-5) Methyl parathion (CAS-298-00-0) Parathion (CAS-56-38-2) Ronnel (CAS-299-84-2) Group II nonfatty foods PCBs (CAS-12767-79-2)Poultry fat, fish, dairy products Group I nonfatty foods: apples*, apricots, barley*, beets, bell peppers,
19、broc- coli*, cabbage*, cantaloupes, cauliflower*, celery, collard greens, corn meal and silage, cucumbers*, eggplant, endive, grapes*, green beans, hay, kale*, mustard greens*, oats*, peaches, pears, peas, plums, popcorn, potatoes*, radishes, radish tops, spinach, squash*, strawberries, sugar beets,
20、 sweet po- tatoes, tomatoes*, turnips*, turnip greens*, wheat*. endrin,andparathionin15%eluate;andmalathionin50%eluate,is satisfactory. Adsorptivity of lots of Florisil may be tested with lauric acid and size of column adjusted to compensate for variation in adsorptivity JAOAC 51, 29(1968). Test adj
21、usted column before use by per- forming elution test above. (j) Hexane.Seesolvent purity test. Redistilled in all-glass ap- paratus. (k) Magnesium oxide.Adsorptive magnesia (Fisher Scientific Co. No. S-120). Treat as follows: Slurry ca 500 g with H2O, heat on steam bath ca 30 min, and filter with su
22、ction. Dry overnight at 105130C and pulverize to pass No. 60 sieve. Store in closed jar. (l) MagnesiaCelite mixture.Mix treated MgO (k) with Celite 545, 1 + 1 by mass. Petroleum ether extract of Celite should be free of electron-capturing substances. (m) Petroleum ether.See solvent purity test. Redi
23、stilled in all-glass apparatus at 3060C. (n) Sodium sulfate.Anhydrous, granular. C. Reagents for Thin-Layer Chromatography Surplus 1994 (a) Aluminum oxide.Neutral Al2O3“G” (Type E, EM Science No. 1090), or equivalent, for thin-layer chromatography (TLC). (b) Developing solvents for organochlorine pe
24、sticides.(1) n-Heptane, commercial grade. (2) n-Heptane containing 20 mL/L reagent grade acetone. (c) Chromogenic agent for organochlorine pesticides.Dis- solve 0.100 g AgNO3in 1 mL H2O, add 20 mL 2-phenoxyethanol (Practical, Eastman Kodak Co.), dilute to 200 mL with acetone,add verysmalldrop30%H2O2
25、,andmix.Storeindarkovernightandde- cant into spray bottle. Discard after 4 days. (d) Developing solvents for organophosphorus pesticides.(1) Immobile.150 or 200mL/L N,N-dimethylformamide (DMF) in ether. Dilute 75 or 100 mL DMF respectively to 500 mL with ether B(m) and mix. (2) Mobile.Methylcyclohex
26、ane. (e) Chromogenic agents for organophosphorus pesticides.(1) Stock dye solution.Dissolve 1 g tetrabromophenolphthalein ethyl ester(EastmanNo.6810)in100mLacetone.(2)Dyesolution.Di- lute 10 mL stock dye solution (1) to 50 mL with acetone. (3) Silver nitratesolution.Dissolve0.5gAgNO3in25mLH2Oanddilu
27、teto 100mLwithacetone.(4)Citricacidsolution.Dissolve5ggranu- lar citric acid in 50 mL H2O and dilute to 100 mL with acetone. D. Reagents for Paper Chromatography Surplus 1980 See 29.004, 13th Ed. E. General Apparatus (a) High-speed blender.Waring, or equivalent. (b) Chromatographictubes.WithTeflonst
28、opcocksandcoarse fritted plate or glass wool plug; 22 mm id 300 mm. (c) Chromatographictubeswithoutstopcocks.22mmid300 or 400 mm. (d) Filter tubes.Ca 22 mm id 200 mm with short delivery tube and coarse fritted plate or glass wool plug. (e) KudernaDanish concentrators.500 and 1000 mL with Snyderdisti
29、llingcolumnand5or10mLplain,volumetric,andgrad- uated receiving flasks (Kontes Glass Co. No. 570000, 621400, and 570050, or equivalent). (f) Separators.1000 and 125 mL with Teflon stopcocks. (g) Micro-Snyder column.2-ball (Kontes Glass Co. No. 569001, or equivalent). (h) Micro-Vigreaux column.Kontes
30、Glass Co. No. 569251, or equivalent. F. Apparatus for Thin Layer Chromatography Surplus 1994 (a) Standard model applicator.Desaga/Brinkmann or equiva- lent. (b) Standard mounting board.Desaga/Brinkmann or equiva- lent. (c) Dryingrack.Desaga/Brinkmannorequivalent.Accommo- dates ten 8 8 in (ca 20 20 c
31、m) plates. (d) Stainless steel desiccating cabinet.Desaga/Brinkmann Model 51, or equivalent. (e) Windowglass.88in(ca2020cm),doublestrengthwin- dowglassplatesofuniformwidthandthickness;smoothoffcorners and edges with file or other tool. (f) Chromatographictankandaccessories.Withmetalinstead of glass
32、troughs. (g) Dipping tank and accessories.Stainless steel, 81281214 316in(ca22220.60.5cm)insidewidthwithmetalsupportsand close-fitting U-shaped cover ca 9 12in (ca 23 1 cm). Capacity ca 300 mL. (h) Spotting pipets.1 L. (i) Spray bottle.8 oz(237 mL) (Thomas Scientific No. 2753-J10 or Lurex Scientific
33、, Inc., No. 131-0514, 250 mL). (j) Chromatographysprayflask.250mL(MicrochemicalSpe- cialties Co., 1825 Eastshore Hwy, Berkeley, CA 94710 USA, No. S-4530-D). (k) Tank liner.Cut 2 pieces, 1214834in (31 22 cm), from desk blotter, white or colored, and bend into L-shape to fit tank. (l) Strong ultraviol
34、et light source.Such as germicidal lamps (General Electric Co., Nela Park, Cleveland, OH 44112 USA), ei- ther (1) two 30 watt, 36 in (91 cm) tubes, No. G30T8, mounted in standard 30 watt reflector fixture ca 20 cm above papers; or (2) two 15watt,18 (46cm)tubes,No.G15T8,mountedinstandard15watt 2000 A
35、OAC INTERNATIONAL Figure 970.52AKCl thermionic detector coil for in-series dual detection system. desk lamp fixture placed ca 10 cm above papers. Shield to protect eyes and skin at all times. G. Apparatus for Paper Chromatography Surplus 1980 See 29.007, 13th Ed. H. Apparatus for Gas Chromatography
36、See also JAOAC 47, 326342(1964); 49, 821(1966). GCsystemwhenoperatedwithcolumn,(b),andapproximatecon- ditions described in Gas Chromatography, R, should be capable of producing ca 12scale deflection for 1 ng heptachlor epoxide by elec- tron capture detection and for 2 ng parathion by KClthermionic d
37、e- tection, and should resolve mixture of heptachlor, aldrin, heptachlor epoxide, ethion, and carbophenothion into separate peaks. Retention time for aldrin should be ca 4.5 min. Compounds of interest must not bedegradedbyanypartofGCsystem,e.g.,endrinshouldexhibitsin- gle peak. (a) Gas chromatograph
38、.Instrument consisting of on-column injection system, all-glass column in oven controlled to 0.1C, electron capture and thermionic detectors, each with independent power supply, electrometer, and appropriate mV recorder. (b) Column.Glass, 1.85 m (6 ft) 4 mm id packed with 10% DC-200 (w/w) on solid s
39、upport: (1) 80100 mesh Chromosorb W HP (Manville Filtration and Minerals, manufacturer, but available through many GC distributors); (2) 80100 mesh Gas-Chrom Q (Alltech-Applied Science Laboratories, Inc.); (3) 8090 mesh AnakromABS(Analabs,Inc.).DC-200maybereplacedbyOV-101 (available from many GC dis
40、tributors). Weigh2gDowCorning200siliconefluid(12 500centistokes) orOV-101intobeaker.DissolveinCHCl3andtransferto300mL Morton-type flask, using total of ca 100 mL CHCl3. Add 18 g solid support, (1), (2) or (3), to flask. Swirl, and let stand ca 10 min. Place flask on rotary evaporator and remove solv
41、ent slowly with intermittent rotation, using 50C H2O bath and slight vacuum. (Foaming may occur initially.) When solids appear damp, increase vacuum. Remove last traces of CHCl3without ro- tation or by air drying. Use only free-flowing material to fill col- umn. Use care at all stages of column prep
42、aration to prevent fracturingsolidsupport.Conditioncolumn at 250260Cwith2 flow of ca 100 mL/min 48 h or until endrin exhibits single peak. (c) Electron capture detector (ECD; Surplus 1995).Concen- tric design, for use with dc voltage supply and 3H source (ca 150 mCi (5.55106Bq) 3H,U.S.NuclearRegulat
43、oryCommissionlicenseisre- quired.) Surplus 1995.Determine detector operating characteristics as follows: Apply dc voltage to detector. After system becomes sta- ble (overnight), determine current-voltage relationship at various voltagesbetween200and0V.(Currentmeasurementsatvoltagesof 200, 150, 100,
44、75, 50, 40, 30, 25, 20, 15, 10, 8, 6, 4, 2, and 1 provide points for smooth curve.) Slightly lower, stable, standing current may be obtained after detector has been at operating temperature several hours. This is probably due to loss of some easily removed radioactive material. Determine and plot re
45、sponse-voltage relation- ship at 1 109A full scale sensitivity for 1 ng injections of heptachlor epoxide at same voltages used in obtaining cur- rent-voltagecurve.Selectasoperatingvoltagethatvoltageatwhich heptachlorepoxidecausesca4050%fullscalerecorderdeflection. Check linearity of system from 0.22
46、.0 ng heptachlor epoxide. Otherelectroncapturedetectorsmaybesubstitutedfordcvoltage concentric design 3H detector, which is no longer marketed. Con- stant current, variable frequency 63Ni electron capture detectors are acceptable substitutes when operated at conditions to produce sta- ble, reproduci
47、ble, linear responses. Optimum conditions may pro- duce more sensitive response than from 3H detector. To maintain same method limit of quantitation of 3H detector, inject proportion- ately smaller equivalent test portion weight into 63Ni detector sys- tem. The 63Ni electron capture detector may pro
48、vide different 2000 AOAC INTERNATIONAL Figure 970.52BKCl thermionic detector coil for par- allel and in-series split dual detection systems. Surplus 1995. Figure 970.52CIn-series split dual detection sys- tem. Surplus 1995. Figure 970.52DParallel dual detection system. Sur- plus 1995. Figure 970.52E
49、In-series dual detection system. Surplus 1995. relative responses for pesticides than those obtained with 3H elec- tron capture detector. Use of ArCH4carrier gas, as recommended for most 63Ni detectors, precludes use of KClTD dual detection sys- tem, (d), (h)(j). (d) Potassium chloride thermionic detector (KClTD; Surplus 1995).Flameionizationdetectormodifiedtoincorporatecoilwith KCl coating prepared as in (1) or (2). Detector voltage is 300 V d