AOAC Official Method 985
|
ID： |
C971A29362E14E81B725246923EC2BB5 |
|
文件大小(MB)： |
0.07 |
|
页数： |
1 |
|
文件格式： |
|
|
日期： |
2016-2-28 |
|
购买： |
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）：
45.4.05,AOAC Of fi cial Method 985.28,Sul fur Amino Acids,in Food, Feed In gre di ents,and Processed Foods,Ion Exchange Chromatographic Method,First Ac tion 1985,Fi nal Ac tion 1986,Re vised First Ac tion 1988,A. Prin ci ple,Pro tein is ox i dized with performic acid to form cysteic acid and,methionine sulfone, then hy dro lyzed with 6M HCl. Cysteic acid and,methionine sulfone are mea sured by ion ex change chro ma tog ra phy.,B. Reagents and Apparatus,(a) Performic acid.—Add 1 vol ume of 30% H2O2 to 9 vol umes,of 88% for mic acid in glass-stoppered Erlenmeyer, and let mix stand,at room tem per a ture 1 h, swirl ing fre quently. Af ter 1 h, place flask in,ice bath 30 min. Pre pare just be fore use.,(b) Buffer.—As recommended by amino acid analyzer supplier.,(c) So dium ci trate elu tion buffer 2.—pH 4.25, 0.2M (Pierce,Chem i cal Co.). Fil ter twice through 0.45 mm fil ter.,( d ) C y s t e i c a c i d – m e t h i o n i n e s u l f o n e s t a n d a rd,so lu tion.—2.5 mmole/mL. Dis solve 93.6 mg L-cysteic acid (Sigma,Chem i cal Co.), 90.6 mg L-methionine sulfone (ICN K&K,Lab o ra tories Inc., Division of ICN Biomedicals, PO Box 28050,Cleve land, OH 44128-0250, USA, or equivalent), and 1 drop of,“pHix” buffer pre ser va tive; di lute to 200 mL with H2O.,(e) Norleucine in ter nal stan dard so lu tions.—(1) Stock,so lu tion.—10 mmole/mL. Dis solve 0.328 mg norleucine in H2O and,di lute to 250 mL with H2O. (2) Working so lu tion.—1.5 mmole/mL.,Di lute 75 mL stock so lu tion to 500 mL with H2O.,(f) Amino acid as sorted stan dard so lu tion.—Dis solve 1.77 g,L-as par tic acid, 0.40 g L-threonine, 0.53 g L-serine, 0.58 g L-valine,0.66 g L-isoleucine, 1.32 g L-leucine, 0.38 g glycine, and 0.45 g,L-alanine in H2O and di lute to 1 L with H2O.,(g) Amino acid an a lyzer.—Op er ated in ac cor dance with,man u fac turer’s in struc tions.,C. Prepa ration of Test Portion,Grind laboratory sample in Brinkmann mill fit ted with 1 mm,screen, and mix thor oughly. Weigh test por tion con tain ing,7.5–25 mg pro tein, but not >75 mg test por tion, into 125 mL boil ing,flask. Pipet 2.00 mL cysteic acid–methionine sulfone stan dard,so lu tion into each of two 125 mL boil ing flasks and evap o rate to,dry ness at 37°C in vac uum ro tary evap o ra tor.,D. Prep a ra tion of Ox i dized Hydrolysate,Place flasks con tain ing test por tions and stan dards in ice bath,30 min. Add 10 mL cold performic acid to each, do not swirl, stop per,flasks, and place in ice bath 16 h.,Add to each flask 3 mL 48% HBr and 3 drops of 1-octanol, swirl,and let stand in ice bath 30 min. Evap o rate to dry ness at 37°C in,vac uum ro tary evap o ra tor. Place flasks on vac uum man i fold and,evac u ate to <25 mm Hg for 315 min , or un til com pletely dry.,Re move flasks from man i fold, add 15 mL 6M HCl, purge with dry,N2 1 min, stop per, and place in oven at 110° ± 0.5°C for 18 h.,Let flasks cool, add 4.0 mL in ter nal stan dard work ing so lu tion,8 mL H2O, and 3 drops of 1-octanol, and evap o rate to dry ness in,vac uum ro tary evap o ra tor at <50°C. Re peat ad di tion of 8 mL H2O,and evap o ra tion 2 ad di tional times. Add 15 mL so dium ci trate,load ing buffer and shake on or bital shaker 30 min. Ad just pH to 2.2 ±,0.05 with 2M NaOH. Add 1.5 mL amino acid as sorted stan dard,so lu tion to flasks con tain ing cysteic acid–methionine sulfone,stan dards. Quan ti ta tively trans fer test so lu tion and stan dards to,25 mL vol u met ric flasks, rins ing with so dium ci trate loa ding buffer,and di lute to vol ume. Fil ter test so lu tion and stan dard so lu tions,through glass fi ber pa per (Whatman GF/A), trans fer fil trate to,cen tri fuge tube, and cen tri fuge 5 min at 15 000 rpm.,E. De ter mi na tion,Load 40 mL test so lu tion or stan dard so lu tion into amino acid,an a lyzer sam ple hold ing unit. Op er ate amino acid an a lyzer,ac cord ing to man u fac turer’s in struc tions and in ject cysteic,acid-methionine sulfone stan dard so lu tion. Ad just chro mato graphic,con di tions as re quired to ob tain >90% res o lu tion for methionine,sulfone peak. In ject test so lu tions and stan dards, in ject ing stan dards,af ter ev ery 5 test so lu tions and at be gin ning and end of test set.,F. Cal cu la tions,Mea sure peak ar eas (not height) for cysteic acid and methionine,sulfone for test por tions and stan dards. Cal cu late re sponse fac tor for,cysteic acid (KC) and methionine sulfone (Km).,KC = (area of in ter nal stan dard/area of cysteic acid),′ (0.20 mmole/mL ′ 25 mL ′ MW ′ 100),Cal cu late re sponse fac tor for methionine (Km) in sim i lar man ner.,For cysteine, use 121.2 mg/mmole for MW. For methionine, use,149.2 mg/……
……