AOAC Official Method 2004
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D7C69FF7B0D54FEBA1A00B48FAE2E4D1 |
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0.49 |
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3 |
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日期： |
2024-7-31 |
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AOAC OFFICIAL METHODS OF ANALYSIS (2007) NATURAL TOXINS,Chapter 49, p. 63,test solution, mL (1 mL); V4 = volume of aliquot of test solution,injected on column, naL(0.1 mL); Ms = mass of test portion extracted,g(15 g); 1000 = fectorto convert g to kg;ス顿=fector to convert ng to,咯,Reference: J. AOACInt. 84, 444(2001).,49.6.02B,AOAC Official Method 2004.10,Ochratoxin A in Green Coffee,Immunoaffinity Column Cleanup and Liquid Chromatography,First Action 2004,(Applicable to the determination of ochratoxin A [OTA] in green,coffee at S2.60 ng/g.),Caution'. OTA is a potent nephrotoxin with immunotoxic,teratogenic, and potential genotoxic properties. The,International Agency for Research on Cancer (IARC),has classified OTA as a possible human carcinogen,(group 2B). Protective cloihing, gloves, and safety,glasses should be worn at all times, and all standard and,sample preparation steps shotild be carried out in a finne,hood. Swab accidental spills of OTA with 1 % NaOCl,bleach solution, and wait 10 min before adding 5%,solution of acetone in water. Rinse all glassware,exposed to OTA with methanol, add 1% NaOCl,solution, and after 2 h add acetone to bring the acetone,concentration to 5% of the total volume. Let reaction,proceed for 30 min, and then wash glassware,thoroughly. Toluene is toxic. Operations involving fliis,solvent must be performed in a fiime hood. Methanol is,hazardous. The blending step must be carried out by,using an explosion-proof blender in a fiime hood. All,analyses should be carried out in a fume hood. Fallow,applicable environmental rules and regulations for,disposal of waste solvents.,See Table 2004.10A for the results of the interlaboratory study,supporting acceptance of the method.,A. Principe,A test portion is blended with methanol-3% aquedu&iggdium,bicarbonate solution (50 + 50). The extract is filtered, dihiteR，M的,phosphate-bufifered saline (PBS), and applied to an immunoa^nity Zz,column containing antibodies specific for OTA. After washing, the,toxin is eluted from Ihe column with methanol and quantified^hjt,liquid chromatography (LC) with fluorescence detection. : ノ,B. Apparatus,(a) Immunoqffinity columns.—Containing OTA. antibodies. The,columns should have a capacity of >100 ng OTA. and should recover,>85% OTA when 100 mL standard solution containing 5 ng toxin in,methanol-3% sodium bicarbonate solution (50+50)/PBS solution (4,+ 96) is passed through the column. Immunoaffinity columns should,be brought to room temperature before use.,(b) Homogenizer/blender.—Motor speed: 0-16 000 rpm;,equipped with glass vials with capacity of946 mL (mason type) and,stainless steel helix; Omnimixer Sorvall Model 17105, or equivalent,(c) Ultrasonic bath.,(d) Fast qualitative filter paper.一24 cm diameter, folded;,Whatman No. 4, or equivalent,(e) Fiber glass membrane.—5.5 cm diameter, 1 gm retention,Whatman GF/B, or equivalent,(f) Membrane.-0.45 pm Cellulose or cellulose nitrate for,aqueous organic solvent.,(g) Calibrated volumetric flasks.—50,100,1000, and 2000 mL.,(h) Analytical balance.—Readability 0.1 mg.,(i) Laboratory balance.—Readability 0.01 g.,(j) Disposable syringe barrels.—For use as reservoirs (60 and,10 mL capacity); Luer locks; with attachments and adapters to fit to,immunoaffinity columns.,(k) Water bath or block heater,一Maintaining temperature at,400-45℃; equipped with gas flow adapter.,(1) l^hcuum system.―With firnnel top, fennel membrane support,aluminium clamp, stopper, and side-arm flask; Millipore type, or,equivalent,(m) Calibrated volumetric pipets.—4 and 25 mL.,(n) Vortex mixer.,Table 2004.10A. Interlaboratory study results for determination of OTA in green coffee,Test,material,Mean value,found for OTA,ng/g Recovery, %,No. of sets of,acceptable,results r, ng/g % ng/g RSDr, % R ng/g sR. ng/g rsdRj %,No. of,outlier labs HorRat,1a NCb — 18 NC NC NC NC NC NC NC,2C 4.48 92.8 15d 0.93 0.33 7.42 2.05 0.73 16.34 0.45,3a 2.60 一15 1.22 0.44 16.78 1.50 0.53 20.51 0.52,4a 6.32 一17 3.70 1.32 20.94 5.16 1.84 29.17 0.85,5fl 12.89 — 15 3.33 1.19 9.24 7.30 2.60 21.15 0.65,Naturally contaminated green coffee samples.,"NC = Not calculated (because the OTA level was below the limit of detection).,c Blank green coffee (OTA at <0.12 ng/g) spiked with OTA at 4.82 ng/g.,d Data from Laboratory A were not included in the statistical analysis (noncompliance, results reported as <0.05 ng/g).,e Outliers = Laboratories C and K.,f Outliers = Laboratories A, K, and N,9 Outlier = Laboratory K.,"Outliers = Laboratories A, D, and K.,Gccrv? IMTFRMATIONAL,NATURAL TOXINS AOAC OFFICIAL METHODS OF ANALYSIS (2007),Chapter 49, p. 64,(o) Spectrophotometer,一Measuring from 200 to 400 nm, with,qua……
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