AOAC Official Method 997
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5AC99F192B84455EA96D9F97D500F93B |
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0.2 |
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4 |
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日期： |
2016-2-28 |
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5.2.05,AOAC Of fi cial Method 997.04,Monensin in Pre mix and An i mal Feeds,Liquid Chromatographic Method,First Ac tion 1997,Final Action 2004,(Applicable for de ter mi na tion of monensin activity in range of,5–200 000 mg/kg in all types of pre mixes and an i mal feeds.),See Ta ble 997.04 for the re sults of the interlaboratory study,sup port ing ac cep tance of the method.,A. Prin ci ple,Monensin is ex tracted from pre mixes and feeds with,methanol–water (90 + 10, v/v) and isolated by liq uid,chro ma tog ra phy with post-column derivatization. Acid-reaction,prod uct of monensin with vanillin is mea sured by vari able,wave length de tec tor at 520 nm. There is no in ter fer ence from,narasin, salinomycin, and lasalocid.,B. Apparatus,(a) Liq uid chromatograph.—With re corder or in te gra tor,(man ual or com puter), pulse-dampened LC pump [for LC mo bile,phase, C(g)] and re agent pump [for vanillin re agent, C(h)] both,op er at ing at 0.7 mL/min; vari able wave length absorbance de tec tor,op er at ing at 520 nm with rise time of 1.0 and range of 0.2 (or as,needed); 90° Tee po si tioned in such way so in let flows di rectly,op pose one an other; and stain less steel coil (6.1 m ′ 0.51 mm id),en closed in 98.0°C heater. (Com pa ra ble com po nents may be,sub sti tuted. Pa ram e ters may be mod i fied to com pen sate for daily,vari a tions in in stru ment per for mance.),Op er ating con di tions: in jec tion vol ume, 200 mL; col umn,temper ature, ambient; reaction coil temper ature, 98°C; flow rate,(both pumps), 0.7 mL/min. Approximate retention times: monensin,A, 640 s; monensin B, 560 s; (see Fig ure 997.04A for chromatogram,of typical system suitability test, Figure 997.04B for chromatogram of,rumensin medicated article, and Figure 997.04C for chromatogram of,monensin medicated feed.),(Note: When not us ing sys tem for 2–3 days, or at reg u lar in ter vals,dur ing use pe ri ods, flush sys tem with meth a nol to pro long life of,com po nents.),(b) LC col umn.—250 ′ 4.6 mm id (Whatman Partisil 5 ODS-3,or equiv a lent). C18 guard col umn may be used to ex tend life of,an a lyt i cal col umn.,(c) LC vi als.,(d) Me chan i cal shaker.—Wrist ac tion shaker, or equiv a lent.,(e) Fil ter.—0.2–0.5 mm po ros ity; Tef lon or chem i cal re sis tant.,(f) Con tainers.—500–1000 mL, glass or poly propy lene.,(g) Vol u met ric flasks.—100 and 200 mL.,(h) Mag netic stir rer.,(i) Tef lon-coated stir bars.,(j) Bal ances.—(1) Top load ing and (2) an a lyt i cal.,C. Re agents,(a) Sol vent.—Meth a nol, LC grade.,(b) Pu rified water.—e.g., LC grade.,(c) Sul fu ric acid.—An a lyt i cal grade.,(d) Gla cial ace tic acid.—ACS grade.,(e) Vanillin.—99% pu rity.,(f) Neu tral ized meth a nol.—Add 1 g NaHCO3 to 4 L meth a nol.,Mix well and, if needed, fil ter through 11 mm pore size fil ter pa per.,(g) LC mo bile phase.—Methanol–H2O–glacial ace tic acid,so lu tion (94 + 6 + 0.1, v/v/v). De gas with He prior to use.,(h) Vanillin re agent.—Methanol–sulfuric acid–vanillin solution,(95 + 2 + 3, v/v/w). While stir ring gently, add slowly and care fully,20 mL con cen trated H2SO4 to 950 mL methanol, C(a). (Caution:,Avoid splat ter ing when add ing H2SO4 to meth a nol, us ing pipet or,other suit able equip ment. Do not add H2SO4 to meth a nol by pour ing,from beaker or grad u ated cyl in der.) Let meth a nol–H2SO4 solution,cool to room tem per a ture. Add 30.0 g vanillin while stir ring gently.,Vanillin re agent is sta ble up to 4 weeks at room tem per a ture in dark,am ber or foil-covered con tainer.,(i) Ex trac tion so lu tion.—Methanol–H2O so lu tion (90 + 10, v/v).,(j) Monensin standard solutions.—(1) Monensin stan dard stock,solution.—1 mg/mL. Ac cu rately weigh 100 mg monensin reference,stan dard (avail able from Eli Lilly and Co., Lilly Cor po rate Cen ter,Indianapolis, IN 46285, USA) into 100 mL volumetric flask and,di lute to vol ume with meth a nol, C(a). (2) Monensin work ing stan dard,solutions.—(i) For rations with theoreti cal monensin concentra tions,>40 ppm. Quantitatively dilute monensin stan dard stock so lu tion with,extraction solution to obtain monensin con cen tra tions of 5.0, 10.0,20.0, and 40.0 mg/mL. (ii) For rations with theoreti cal monensin,concentra tions <40 ppm. Quantitatively dilute monensin stan dard,stock so lu tion with ex trac tion so lu tion to ob tain monensin,con cen tra tions of 0.25, 1.25, 2.5, 3.75, and 5.0 mg/mL.,Monensin stan dard so lu tions are sta ble up to 4 weeks in,re frig er a tor or at room tem per a ture pro tected from di rect sun light.,(k) Res o lu tion so lu tion.—Con taining 5–40 mg monensin/mL,and 5–40 mg narasin/mL. Prepare resolution solution as follows……
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