6. AOACSPIFANMethods-2018Awards

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≥3.7 between all- trans -lutein and zeaxanthin and ≥2.5 between zeaxanthin and apocarotenal. See Figure 2016.13A . ( 3 )  Resolution between β-carotene cis and trans isomers and α-carotene .—Inject the β-carotene system suitability solution, E(g) , and determine the resolution between the two major cis isomers of β-carotene, all- trans -β-carotene, and α-carotene. Resolution should be ≥1.7 between 13- cis -β-carotene and cis/ trans -α-carotene and ≥2.6 between all- trans -β-carotene and 9- cis -β-carotene. See Figure 2016.13B . ( 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 internal standard as shown in H(d) . 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 2.0%. ( 5 ) Representative sample chromatograms are shown in Figures 2016.13C - E .

H. Calculations

(a)  Determine the purity of lutein and β-carotene 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 .—Measure each standard measuring solution, E(b) , against an MTBE blank at its absorbance maximum (444 nm for lutein and 450 nm for β-carotene). Calculate the spectrophotometric purity (SP) of each reference standard as the observed absorbance over the expected absorbance: SP Abs 50000 E W MS 1%,1cm ( ) ( ) = × ×

where Abs MS

= the absorbance of the standard measuring

solution; 50000 = the dilution factor; E 1%,1cm

= the extinction

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

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