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274 S CHIMPF ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 1, 2018

Figure 7. Chromatographic separation of carotenoids in a child milk powder (4052755861).

(b) Peak resolution. — Inject a sample containing all carotenoids of interest and confirm acceptable resolution between isomers using the guidelines in Table 2017.04C and the following equation: R = 2 ð t 2 − t 1 Þ = ð W 2 + W 1 Þ where R = resolution; t 1 = retention time of peak 1 in minutes; t 2 = retention time of peak 2 in minutes; W 1 = peak width of peak 1 in minutes; and W 2 = peak width of peak 2 in minutes ( see Table 2017.04C ). (c) Standard and sample analysis. — Once the system has equilibrated, inject each working standard, inject samples, and inject another set of working standards. (d) System shutdown. — After all samples and standards have been analyzed, store the column in mobile phase A. (a) Standard curve preparation. — ( 1 ) Visually inspect each standard and sample chromatogram and verify peak resolution from other peaks and correct identification of peaks. ( 2 ) Peak areas are measured with a data system. Before calculating the concentration of all - trans - b -carotene, cis isomers of b -carotene, all - trans -lutein, cis isomers of lutein, and all - trans - lycopene and lycopene isomers, verify that sample peak responses are within the range of standard peak responses. ( 3 ) Prepare standard curves by averaging the peak areas of standards injected at the beginning of a set of samples with the peak areas of standards of the same concentration injected at the end of the G. Calculations

set of samples and performing linear least-squares (regression) on concentration versus averaged peak areas. A standard curve must have a correlation of at least 0.999 to be considered acceptable for sample calculations. At each working standard concentration, the peak areas of standards injected at the beginning and end of a set of samples should not increase or decrease by more than 10%. (b) Sample concentration. — Note : All cis -lutein isomer concentrations are calculated from the trans -lutein curve. All cis- b -carotene isomer concentrations are calculated from the trans- b -carotene curve, and all cis- lycopene isomer concentrations are calculated from the trans- lycopene curve. — ( 1 ) trans-Lutein sample concentration. — C t - Lut = ð C DS × D 1 × D 2 × R TW Þ = ð A 1 × SW × R PW × 10 Þ where C t -Lut = trans -lutein concentration in µg/100 g; C DS = lutein concentration extrapolated from the calibration curve in µg/L; D 1 = first dilution volume in milliliters (10 mL extraction solution); D 2 = second dilution volume in milliliters (0.25 mL dilution solution); R TW = total reconstitution weight in grams, if applicable; A 1 = aliquot of the first dilution pipetted, in milliliters; SW = sample weight in grams; R PW = weight of powder reconstituted in grams, if applicable; and 10 = conversion to 100 g. ( 2 ) cis-Lutein sample concentration. — C cis - Lut = ð C DS × 1 : 43 × D 1 × D 2 × R TW Þ = ð A 1 × SW × R PW × 10 Þ where C cis -Lut = cis -lutein concentration in µg/100g; C DS = lutein concentration extrapolated from the calibration curve in µg/L; 1.43 = correction factor when calculating cis -lutein from the trans -

Figure 8. Chromatographic separation of carotenoids in an infant partially hydrolyzed milk powder (410057652Z).