AOAC45.2.02维生素B12的检测

45.2.02

AOAC Official Method952.20

Cobalamin(Vitamin B12Activity)

in Vitamin Preparations

Microbiological Methods

First Action1952

Final Action1960

(Applicable to materials containing ca≥0.1μg of vitamin B12activ-ity/g or mL.)

A.Basal Medium Stock Solution

Using ingredients in amounts prescribed for cobalamin,Ta-ble960.46A(a)(see45.2.01),proceed as directed below. Dissolve L-cystine and D,L-tryptophan in10mL1M https://www.360docs.net/doc/6113433594.html,ing solutions prepared as in A,add,with mixing,and in following order: adenine–guanine–uracil solution,(b);xanthine solution,(r);vita-min solution I,(l);vitamin solution II,(m);salt solution A,(i);salt solution B,(j);asparagine solution,(c);and acid-hydrolyzed casein solution,(a).Add ca100mL H2O and add,with mixing,anhydrous glucose,NaCH3COO?3H2O,and ascorbic acid.When solution is complete,adjust to pH6.0with NaOH solution,add,with mixing, polysorbate80solution,(h),and dilute with H2O to250mL.

Titrimetric Method

B.Cyanocobalamin Standard Solutions

(a)Stock solution.—100ng/mL.Accurately weigh,in closed system,USP Cyanocobalamin Reference Standard equivalent to 50–60μg cyanocobalamin,that has been dried to constant weight and stored in dark over P2O5in desiccator.Dissolve in25%alcohol, and dilute with additional25%alcohol to make cyanocobalamin concentration exactly100ng/mL.Store in dark at ca10°C.

(b)Intermediate solution.—1ng/mL.Dilute10mL stock solu-tion,(a),with25%alcohol to1L.Store in dark at ca10°C.

(c)Working solution.—Dilute suitable volume intermediate so-lution,(b),with H2O to measured volume such that after incubation as in960.46D and960.46E(see45.2.01),response at5.0mL level of this solution is equivalent to titration[as described in960.46E(see 45.2.01)]of ca8–12mL.Designate this as standard solution.(This concentration is usually0.01–0.04ng cyanocobalamin/mL standard solution.)Prepare fresh standard solution for each assay.

C.Inoculum

Make transfer of cells from stock culture of Lactobacillus leichmannii,960.46C(a)(see45.2.01),to sterile tube containing 10mL liquid culture medium,960.46B(a)(see45.2.01).Incubate 6–24h at any selected temperature between30and40°C held con-stant to within±0.5°C.Under aseptic conditions,centrifuge culture and decant supernate.Wash cells with3ca10mL portions sterile 0.9%NaCl solution or sterile suspension medium,960.46B(c)(see 45.2.01).Resuspend cells in10mL sterile0.9%NaCl solution or sterile suspension medium.Dilute aliquot with sterile0.9%NaCl so-lution or sterile suspension medium to give T equivalent to that for dried cell weight[as described in960.46F(see45.2.01)]of 0.50–0.75mg/tube when read against suspension medium set at 100%T.Cell suspension so obtained is inoculum.

D.Test Solution

Prepare aqueous extracting solution just before use containing, in each100mL,1.3g anhydrous Na2HPO4,1.2g citric acid?H2O,and 1.0g anhydrous sodium metabisulfite,Na2S2O5.Place measured amount of vitamin preparation in flask containing≥25mL extracting solution for each g or mLvitamin preparation taken.If vitamin prepa-ration is not readily soluble,comminute to disperse it evenly in liquid; then agitate vigorously and wash down sides of flask with H2O. Autoclave mixture10min at121–123°C and cool.If lumping oc-curs,agitate mixture until particles are evenly dispersed.Dilute mix-ture to measured volume with H2O,and let any undissolved particles settle,or filter or centrifuge if necessary.Take aliquot of clear solu-tion,add H2O,adjust to pH6.0,and dilute with additional H2O to measured volume containing,per mL,cobalamin activity ca equiva-lent to that of standard solution,B(c).Designate this as test solution. Excess of bisulfite may affect test organism.Therefore,test solution must contain≤0.03mg Na2S2O5/mL.

E.Test

Using standard solution,B(c),test solution,D,basal medium stock solution,A,and inoculum,C,proceed as in960.46D,E,and H (see45.2.01).

Turbidimetric Method

F.Cyanocobalamin Standard Solution

Dilute suitable volume cyanocobalamin intermediate standard so-lution,B(b),with H2O to measured volume such that after incuba-tion as in960.46D and960.46G(see45.2.01),with inoculated blank set at100%T,%T at5.0mL level of this solution is equivalent to that for dried cell weight[as described in960.46F(see45.2.01)]of ≥1.25mg.Designate this as standard solution.(This concentration is usually0.01–0.04ng cyanocobalamin/mL standard solution.)Pre-pare fresh standard solution for each assay.

G.Test Solution

Proceed as in D,except that where reference is made to concentra-tion of cyanocobalamin activity ca equivalent to that of standard so-lution,B(c),replace by concentration of cyanocobalamin activity ca equivalent to that of standard solution,F.Designate solution so ob-tained as test solution.

H.Assay

Using standard solution,F,test solution,G,basal medium stock solution,A,and inoculum,C,proceed as in960.46D,G,and H(see 45.2.01).

References:JAOAC35,161,169,726(1952);36,846(1953);

37,781(1954);38,711(1955);39,167,172(1956);

40,856(1957);41,61,587(1958);42,529(1959). CAS-68-19-9(cobalamin;cyanocobalamin;vitamin B12)

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