AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

can also be used for the determination of 13- cis vitaminA palmitate and 13- cis vitamin A acetate using the following correction factors.

have an RSD of ≤2.0% and that the cis vitamin A palmitate and α -tocopherol peaks are not cut off. ( 3 ) Inject the remaining working standards, a control sample, sample preparations, and a second set of standards. The column switch time should be checked before and after each set of standard and sample injections, every 24 h, or when the mobile phase is changed. H. Determination ( a )  Quantitative determination .— ( 1 )  Resolution .—Visually inspect each standard and sample chromatogram and verify that the vitamin A and vitamin E peaks are adequately resolved. Compare the vitamins A and E standard responses to the vitamins A and E sample responses and verify that the sample responses are within the range of the standard responses. Calculate the R of the 13- cis vitamin A and all-trans vitamin A peaks. Resolution of these isomers must be >1.85 ( see Appendix II for example calculations). ( 2 )  Linearity .—The linear correlation coefficient, r, for all standard curves must be ≥0.9996, and the difference in peak responses of the same standards injected before and after a set of samples must be ≤4.0%. I. Calculations and Expression of Results ( a )  Working standard concentration calculations .—The concentrations of all- trans vitamin A palmitate, vitamin A acetate, vitamin E acetate, and α-tocopherol in each of the working standards are calculated from the amounts of standard weighed and the declared or determined potencies or purities of the standards using the following formula: C= (Wt × P × B × T)/D where B= biological activity in IU/mg (B= 1817 IU/mg for vitaminA palmiate and 2904 IU/mg for vitamin A acetate), T = trans fraction, C = vitamin A or E concentration in IU/L or mg/L, Wt = weight of reference standard weighed in g, P = purity in mg/mg, and D = dilution factor. Dilution factors for all of the standards are listed in Table  2012.09 . ( b )  Standard curves .—Prepared for all- trans vitamin A palmitate, all- trans vitamin A acetate, vitamin E acetate, and α-tocopherol by performing a linear least-squares routine on standard peak areas versus corresponding standard concentrations. The all- trans vitaminAstandard curves are used for the quantitation of both 13- cis vitamin A and all- trans vitamin A in samples. The r values for all standard curves must be ≥0.9996, and the difference in peak responses of the same standards injected before and after a set of samples must be ≤4.0%. ( c )  Vitamin A calculations.— Since 13- cis vitamin A palmitate and 13- cis vitaminAacetate standards are not available, the all- trans vitamin A palmitate and all- trans vitamin A acetate standard curves

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where E 13 are the molar absorptivity of all- trans and 13- cis vitamin A, respectively, at the wavelength employed for the determination. This ratio has been estimated to be 1.075. B is the biological potency of 13- cis vitamin A relative to that of all- trans vitamin A (0.75). ( d )  Sample concentration calculations.— The vitamin A and vitamin E concentrations for products can be calculated from the following equation: Vitamin concentration, IU/kg, IU/L, mg/kg, or mg/L = (C × 10 × df)/SS where C = the vitamin concentration of the sample preparation obtained from the vitaminAand E standard curves in IU/L or mg/L. For total vitamin A, the sum of the trans vitamin A concentration and the 13- cis vitamin A concentration are added together. SS = Sample size in g, 10 = sample dilution volume in mL, if applicable, and df = dilution factor of the reconstituted powder (if applicable): ( e )  Units conversions.— ( 1 ) Vitamin A results can be converted from IU to weights or retinol equivalents (RE) using the following conversion factors: ( a )  All-trans retinyl acetate .—1 μg all- trans retinyl acetate is equivalent to 2.904 IU or 0.871 RE. ( b )  All-trans retinyl palmitate. —1 μg all- trans retinyl palmitate is equivalent to 1.817 IU or 0.545 RE. ( 2 ) Vitamin E results can be converted from weights to IU or tocopherol equivalents (TE) using the following conversion factors: ( a )  All-rac α -tocopheryl acetate.— 1 mg is equivalent to 1 IU or 0.671 TE. ( b )  Natural (RRR) α -tocopheryl acetate.— 1 mg is equivalent to 1.36 IU or 0.913 TE. ( c )  All-rac α -tocopherol. —1 mg is equivalent to 1.1 IU or 0.738 TE. ( d )  RRR α -tocopherol.— 1 mg is equivalent to 1.49 IU or 1.0 TE. References: J. AOAC Int . 96 , 1407(2013) DOI: 10.5740/jaoacint.13-203 ݂݀ ൌ ݓ ݄݁݅݃ ݐ ݌ ݎ ݋݀ ݑ ܿ ݐ ൅ ݓ ݄݁݅݃ ݓ ݐ ܽ ݐ ݁ ݎ ݓ ݄݁݅݃ ݐ ݋݂ ݌ ݎ ݋݀ ݑ ܿ ݐ all and E

AOAC SMPR 2011.003 J. AOAC Int. 95, 291(2012). AOAC SMPR 2011.010 J. AOAC Int . 96 , 485(2013)

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