AOAC Official Method 2005
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AFBB938C355C4295BDC6B14761B69D87 |
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0.12 |
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6 |
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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）：
51.3.01,AOAC Of fi cial Method 2005.07,b-Car o tene in Sup ple ments and Raw Ma te ri als,Re versed-Phase High Pres sure,Liquid Chromatogra phic Method,First Ac tion 2005,(Ap pli ca ble to the de ter mi na tion of all-trans- and to tal b-car o tene,in raw ma te ri als and sup ple ments from 0 to 200 mg/g ex cept for,beadlet ma te ri als and tab let ma te ri als made from beadlets.),See Tables 2005.07A–D for the results of the interlaboratory,study supporting acceptance of the method.,A. Prin ci ple,Wa ter dispersible for mu la tions such as pow ders, emul sions,tab lets, and cap sules are di gested with pro te ase and ex tracted with,di chloro methane and al co hol. Oily sus pen sions are dis solved,di rectly in di chloro methane and al co hol. The ex tract is,chromatographed on a C18 isocratic high-per for mance liq uid,chro ma tog ra phy (LC) sys tem that sep a rates the pre dom i nant,geo met ri cal iso mers of b-car o tene from each other and from other,ca rot en oids such as all-trans-a-car o tene, lycopene, cryptoxanthin,lutein, and zeaxanthin. In the case of prod ucts with rel a tively high,a-car o tene con tent, where the cis-isomers of a-car o tene are known,to in ter fere with the quantitation of b-car o tene, the ex tract is fur ther,chromatographed with a more se lec tive C30 re versed-phase LC,system in the gradient mode that minimizes this interference.,B. Re agents,(a) b-Car o tene, type II.––Sigma-Aldrich (St. Louis, MO; USA;,CAS 7235-40-7). Pu rity >95%, grade: type II.,(b) Al ka line pro te ase R.—Bio-Cat, Inc. (Troy, VA, USA;,www.bio-cat.com).,(c) n-Hex ane.––CAS 110-54-3, pu rity 399%, per ox ide free,(GC).,(d) Cyc lo hex ane.––CAS 110-82-7, pu rity 99.5% (GC).,(e) Tetrahydrofuran.––CAS 109-99-9, pu rity 399.5%, per ox ide,free (GC).,(f) Eth a nol.––CAS 64-17-5, pu rity 399.5% (GC).,(g) Di chloro methane.––CAS 75-09-2, pu rity 399.5% (GC).,(h) Pro te ase.––CAS 9014-01-1, bac te rial al ka line pro te ase,en zyme prep a ra tion in wa ter, Syn onym: subtilisin, IUB 3.4.21.62.,(i) Meth a nol.––CAS 67-56-1, pu rity 99.8% (GC).,(j) Butylated hydroxytoluene (BHT).––CAS 128-37-0, pu rity,399%.,(k) N-ethyldiisopropylamine.––CAS 7087-68-5, pu rity 398%,(GC).,(l) 2-Propanol.––CAS 67-63-0, pu rity 398% (GC).,(m) Am mo nium ac e tate.––CAS 631-61-8, pu rity 398% (NT).,(n) Acetonitrile.––CAS 75-05-08, pu rity 399.9% (GC).,(o) Methyl-tert-butyl ether (MTBE).––CAS 1634-04-4, pu rity,399.5% (GC).,(p) Wa ter.––Dis tilled or de min er al ized.,(q) LC con trol so lu tion.—(1) Preparation.—Dis solve about 3,mg b-car o tene ref er ence sub stance and 1 g BHT in 50 mL,tetrahydrofuran. Add 200 mL eth a nol and re flux for 2 h in a wa ter,bath at 80°C. Cool, di lute to 500 mL with eth a nol, and trans fer the,so lu tion to a dis penser bot tle. Mix well, leave over night at room,tem per a ture, and dis pense so lu tion into a large num ber of LC vi als.,Care fully seal the vi als im me di ately af ter fill ing with,Tef lon/sil i cone septa, and store at ca 5°C in the dark. (2) Use of the,LC con trol so lu tion.––Mea sure the ini tial b-car o tene con tent of the,con trol so lu tion when the LC sys tem is cal i brated. In ject in par al lel,with the stan dard so lu tion, at least six 20 mL aliquots into the LC,sys tem. Cal cu late the mean b-car o tene con tent of the con trol,so lu tion from the re sult ing chromatograms us ing the newly,de ter mined re sponse fac tor. Sub se quently, in ject the con trol,so lu tion to gether with each se ries of test ex tracts. The re sponse,fac tor is re garded as con stant as long as the mea sured b-car o tene,con tent of the con trol so lu tion cor re sponds to the ini tial value within,±2%. As long as the re sponse fac tor re mains within these lim its, the,orig i nal re sponse fac tor can be used for cal cu la tions. But the LC,sys tem must be recalibrated if the mea sured b-car o tene con tent,ex ceeds the tol er ance.,(r) Mo bile phase for LC Sys tem A.—In a 1 L vol u met ric flask,dis solve 50 mg BHT in 20 mL 2-propanol and add 0.2 mL,N-ethyldiisopropylamine, 25 mL 0.2% am mo nium ac e tate,so lu tion, (m), 455 mL acetonitrile, and ca 450 mL meth a nol.,Mix ture cools and con tracts. Warm to room tem per a ture and,di lute to vol ume with meth a nol.,(s) Mo bile phase for LC Sys tem B.––(1) Sol vent 1.—In a 1 L,vol u met ric flask, dis solve 200 mg vi ta min C in 500 mL methanol,(sonicate if necessary), add 100 mL MTBE, and dilute to volume,with methanol. Discard after 2 days. (2) Solvent 2.—MTBE.,C. Apparatus,(a) An a lyt i cal bal ance.––Readability ±0.0005 g.,(b) Spectrophotometer.––Dual beam, wave length range of,190–900 nm, 1.5 nm……
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