AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

Figure 2016.13D. Chromatogram of a toddler formula sample. Lut = Lutein, Zea = zeaxanthin, Apo = apocarotenal, AC = α-carotene, and BC = β-carotene.

β-carotene, and 471 nm for lycopene). Calculate the SP of each reference standard as the observed absorbance over the expected absorbance: SP = (Ab sMS × 50 000)/(E 1%,1cm × W) where Abs MS = absorbance of the standard measuring solution; 50000 = dilution factor; E 1%,1cm = extinction coefficient (lutein in MTBE, 2589 at 444 nm; β-carotene in MTBE, 2588 at 450 nm; lycopene in hexane, 3450 at 471 nm); and W = weight (mg) of reference standard. SP is typically greater than 0.90 (i.e., 90%). ( 2 )  Chromatographic purity (CP). —Inject standard working solutions, E(c) , at least three times. CP is calculated as

( 3 )  Reference standard purity .—Calculate the purity (P) of each reference standard: P = SP × CP × 100 where 100 = factor for converting decimal to percent. ( b ) Calculate the concentration (μg/100 mL) of each carotenoid analyte (e.g., C Lut for lutein) in the all- trans form in each calibration solution, E(f) : = reference standard purity of all- trans -lutein calculated in H(a) ( 3 ); 100 = conversion from percent to decimal; 1000 = conversion of milligrams to micrograms; (2/100) = dilution of stock solution tomixed carotenoid intermediate solution; V MC = volume of mixed carotenoid intermediate solution, E(e) , used; and V Total = dilution volume. ( c ) Calculate the concentration (μg/100 mL) of the apocarotenal internal standard (C A ) in each calibration solution, E(f) : C Lut = W Lut × 2 × (P Lut /100) × 1000 × (2/100) × (V MC /V Total ) where W Lut = weight (mg) of lutein used to make the stock solution; 2 = conversion of 50 mL to 100 mL; P Lut

CP = (area of the all- trans -carotenoid peak)/ (sum of areas of all relevant peaks)

Relevant peaks include all peaks in the HPLC chromatogram, with the exception of solvent peaks. CP is typically greater than 0.95 (i.e., 95%).

Figure 2016.13E. Chromatogram of an RTF adult nutritional sample. Lut = Lutein, Apo = apocarotenal, AC = α-carotene, and BC = β-carotene.

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