AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

2016.13 (SEPTEMBER 2017) CAROT-02 METHOD

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(a) Determine the purity of lutein, β-carotene, and lycopene standards by first determining the spectrophotometric purity and then the chromatographic purity of each. The overall purity is calculated as the product of the two measured purities (1) Spectrophotometric purity (SP) .—Measure each standard measuring solution, E(b) , against an MTBE blank at its absorbance maximum (444 nm for lutein, 450 nm for β- carotene, and 471 for lycopene). Calculate the spectrophotometric purity of each reference standard as the observed absorbance over the expected absorbance: SP = (Abs MS × 50 000)/(E 1%,1cm × W) where Abs MS = absorbance of the standard measuring solution; 50 000 = dilution factor; E 1%,1cm = extinction coefficient [lutein in MTBE: 2589 at 444 nm (18); β- carotene in MTBE: 2588 at 450 nm (18); lycopene in hexane: 3450 at 471 nm (17)]; W = weight of reference standard, in mg; spectrophotometric purity is typically greater than 0.90 (i.e., 90%). (2) Chromatographic purity (CP). —Inject standard working solutions, E(c) , at least 3 times. The CP is calculated as: 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. Chromatographic purity is typically greater than 0.95 (i.e., 95%). (3) Reference standard purity (P) .—Calculate the purity of each reference standard: P = SP × CP × 100 where 100 = factor for converting decimal to percent. (b) Calculate the concentration of each carotenoid analyte (e.g., lutein, C Lut ) in the all- trans form, in μg/100 mL, in each calibration solution, E(f) :