AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

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

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% 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 – E . 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 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 an MTBE blank at its absorbance maximum (444 nm for lutein, 450 nm for

G. Chromatography ( a )  Chromatographic conditions.— Set up the UHPLC system according to the specifications in Table 2016.13C . Follow the manufacturer’s instructions for column installation, cleaning, and storage. ( b )  System suitability checks .—( 1 ) Resolution between lutein cis and trans isomers.— Inject the lutein system suitability solution, E(h) , and determine the resolution between the two major cis isomers and all- trans -lutein. Resolution should be ≥1.4 between 13- cis - and 13′- cis -lutein and ≥2.2 between 13′- cis - and all- trans - lutein using the half-width method. See Figure  2016.13A . ( 2 )  Resolution between all-trans-lutein, zeaxanthin, and apocarotenal .—From the chromatogram of the lutein system suitability solution, E(h) , determine the resolution between all- trans -lutein, zeaxanthin, and apocarotenal. Resolution should be ≥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

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

© 2017 AOAC INTERNATIONAL