AOAC Final Action Methods in 2019

Figure 2016.13B. Chromatogram of β-carotene system suitability solution, E(g). AC = α-Carotene and BC = β-carotene.

reference standard as the observed absorbance over the expected absorbance: SP = (Abs MS × V Total × 1000)/(E 1%,1cm × V Stock × C W ) where Abs MS = absorbance of the standard measuring solution; V Total = total volume of standard measuring solution made, in mL; 1000 = factor for g to mg; E 1%,1cm = extinction coefficient (lutein in ethanol: 2550 at 446 nm; β-carotene in cyclohexane: 2505 at 456 nm; lycopene in cyclohexane: 3310 at 476 nm); V Stock = volume of standard stock solution used, in mL; C W = stock concentration by weight, in mg/100 mL; SP is typically >0.90 (i.e., 90%). ( 2 )  CP. —Inject standard working solutions, E(c) , at least three times. CP is calculated as:

( 4 ) Inject the calibration solutions before and after each set of sample injections (up to 12 samples in each set). Calculate the slope relative to the ISTD as shown in H(h) . The coefficient of determination (R 2 ) for each curve should be >0.995. The slopes from the two curves should not differ by more than 2% for lutein or β-carotene and not by more than 10% for lycopene. Use the average of the points from the two curves bracketing the samples for calculations. ( 5 ) Representative sample chromatograms are shown in Figures  2016.13C – F . H. Calculations ( a ) Determine the purity of lutein, β-carotene, and lycopene standards by first determining the spectrophotometric purity (SP) and then the chromatographic purity (CP) of each. The overall purity is calculated as the product of the two measured purities. ( 1 ) SP .—Measure each standard measuring solution, E(b) , against the appropriate solvent blank at its absorbance maximum (446 nm for lutein in ethanol, 456 nm for β-carotene in cyclohexane, and 476 nm for lycopene in cyclohexane). Calculate the SP of each

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 >0.95 (i.e., 95%).

Figure 2016.13C. Chromatogram of a milk-based infant formula sample. Lut = Lutein, Zea = zeaxanthin, Apo = apocarotenal, and BC = β-carotene.

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