6. AOACSPIFANMethods-2018Awards

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

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

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

coefficient (18; lutein in MTBE, 2589 at 444 nm; β-carotene in MTBE, 2588 at 450 nm); and W = the weight (mg) of reference standard. Spectrophotometric purity is typically greater than 0.90 (i.e., 90%). ( 2 )  Chromatographic purity. —Inject standard working solutions, E(c) , at least three times. The chromatographic purity (CP) is calculated as ( ) ( ) = CP area of theall - - carotenoid peak sumof areas of allrelevant peaks trans 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 .—Calculate the purity (P) of each reference standard: = × × P SP CP 100 where SP = the spectrophotometric purity; CP = the chromatographic purity; and 100 = the factor for converting decimal to percent.