AOAC Official Method 2003
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D9D3AD47AD2745429C0073A4D153D295 |
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0.07 |
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3 |
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日期： |
2016-2-28 |
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文本摘录（文本识别可能有误，但文件阅览显示及打印正常，pdf文件可进行文字搜索定位）：
49.2.31,AOAC Of fi cial Method 2003.02,Af la toxin B1 in Cat tle Feed,Immunoaffinity Column Liquid Chromatogra phy Method,First Ac tion 2003,Final Action 2006,(Applicable for the determination of aflatoxin B1 >1 ng/g in cat tle,feed.),Cau tion: This method re quires the use of so lu tions of af la toxin,B1. Aflatoxins are car ci no genic to hu mans. Aflatoxins,are sub ject to light deg ra da tion. Pro tect an a lyt i cal work,ad e quately from the day light, and keep af la toxin,stan dard so lu tions pro tected from light by us ing am ber,vi als or alu minium foil. Use acid-washed glass ware,(e.g., vi als, tubes, flasks) to pre vent loss of af la toxin.,Take spe cial care with new glass ware. Soak new,glass ware in di lute acid (e.g., 2M sul fu ric acid) for,sev eral hours and rinse ex ten sively with dis tilled wa ter,to re move all traces of acid (check with pH pa per).,See Ta ble 2003.02A for the re sults of the interlaboratory study,sup port ing ac cep tance of the method.,A. Prin ci ple,An ex tract of the test por tion is ap plied to an af fin ity col umn,con tain ing an ti bod ies spe cific to af la toxin B1. Af ter elu tion,af la toxin B1 is quan ti fied by LC with post-col umn derivatization,and flu o res cence de tec tion.,B. Per for mance Stan dard and Gen eral Pro ce dures for,Immunoaffinity Col umns,The immunoaffinity col umns must con tain an ti bod ies raised,against af la toxin B1. The col umns must have a ca pac ity of not less,than 40 ng af la toxin Bl and must give a re cov ery of not less than 80%,when ap plied as a stan dard so lu tion in CH3OH/H2O con tain ing,0.25 ng toxin.,Ad just col umns to room tem per a ture be fore con di tion ing. For,con di tion ing ap ply 10 mL phos phate-buf fered sa line (PBS) on top,of col umn and let it pass at a speed of 2–3 mL/min through the,col umn (grav ity). Make sure that a small por tion (0.5 mL) of the,PBS re mains on the col umn un til ex tract is ap plied. Note: Meth ods,for load ing onto af fin ity col umns, wash ing the col umn, and eluting,vary slightly be tween col umn man u fac tur ers; fol low pre cisely the,spe cific in struc tions sup plied with the col umns.,C. Apparatus,(a) Ver ti cal or hor i zon tal shaker.,(b) Fil ter pa per.—24 cm, prefolded.,(c) Erlenmeyer flask.—500 mL screw-top or glass stop per.,(d) Glass microfiber fil ter pa per.—Re ten tion 1.6 mm.,(e) Res er voir.—75 mL with Luer tip con nec tor for af fin ity,col umn.,(f) Hand pump.—20 mL sy ringe with Luer lock or rub ber,stop per.,(g) Vol u met ric flask.—5, 10, and 20 mL (ac cu racy of at least,0.5%).,(h) LC pump.—With flow rates be tween 0.20 and 1.00 mL/min.,(i) In jec tion sys tem.—To tal loop in jec tion valve with loop,be tween 100 and 1000 mL. De ter mine RSD (n = 10) for rep li cate,in jec tions of 0.1 ng/mL af la toxin B1. RSD must be <5% for op tion,A; if this can not be met, use op tion B, D(q).,(j) Re versed-phase LC col umn.—4.6 mm ′ 25 cm, 5 mm, e.g.,LC-18 or ODS-2.,(k) Post-col umn derivatization sys tem.—Ei ther with PBPB,(sec ond LC pulseless pump, zero-dead vol ume T-piece, re ac tion,tub ing min i mum 45 cm ′ 0.5 mm id PTFE) or with,elec tro chem i cally gen er ated bro mine (e.g., Kobraa cell–Rh鬾e,Di ag nos tics Ltd., Lyon, France; www.r-biopharmrhone.com).,(l) Flu o res cence de tec tor.—Wave length of 8 = 360 nm,ex ci ta tion fil ter and a wave length of 8 > 420 nm cut-off emis sion,fil ter, or equiv a lent.,(m) Dis pos able fil ter unit.—Cel lu lose or cel lu lose ni trate,0.45 mm.,(n) Pipet.—Marked with 1–10 mL ca pac ity.,(o) An a lyt i cal bal ance.—Read abil ity 0.1 mg.,(p) Lab o ra tory bal ance.—Read abil ity 0.01 g.,(q) Cal i brated microliter sy ringe(s) or microliter,pipet(s).—20–500 mL ca pac ity.,(r) Immunoaffinity col umns spe cific for af la toxin B1,cleanup.—See B for per for mance stan dard for col umn. For ex am ple,c o l u m n s f r o m Vi c a m ( Wa t e r t o w n , M A , U S A ;,www.vicam.com/products/mycotoxins.html) and Rh鬾e Diagnostics,(Glas gow, UK; now R-Biopharm Rh鬾e Ltd) have been found to,meet these cri te ria.,D. Re agents,All re agents shall be of rec og nized an a lyt i cal grade.,Un less oth er wise stated, use wa ter com ply ing with grade 3 of ISO,3696.,?2006 AOAC IN TER NA TIONAL,Ta ble 2003.02A. Interlaboratory study re sults for af la toxin B1 in cat tle feed,Added, ng/g No. of labsa Av er age, ng/g Mean rec., % sr, ng/g sR, ng/g RSDr, % RSDR, % r, ng/gb R, ng/gb HorRat,1.2 20 (1) 1.33 111 0.08 0.26 5.9 19.4 0.22 0.72 1.3,3.6 20 (1) 3.89 106 0.25 0.67 6.4 17.5 0.69 1.87 1.3,NCc 21 (0) <0.02 — — — — — — — —,NC 18 (3) 0.54 — 0.04 0.10 7.2 17.9 0.11 0.27 1.0,NC 19 (2) 0.87 — 0.08 0.1……
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