AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

Carot-02 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

G. Chromatography (a) Conditions

Analytical column

YMC C30 3 μm, 250 x 2.0 mm YMC C30 3 μm, 10 x 2.0 mm

Guard column

Column temperature 30 °C Mobile phases

A: 20 mM ammonium acetate in MeOH B: MTBE

Gradient

Time (min)

%B

0 1

5

10 10

10 25

100

25.1

5 5

30

Flow rate

0.25 mL/min ca. 185 bar

Backpressure

Injection volume 5 μL UV/Visible detection 450 nm

(b) Resolution between isomers (1) Resolution between lutein cis and trans isomers. – Inject the qualitative cis / trans lutein solution and determine the resolution between the two major cis isomers and all- trans - lutein. (2) Resolution between β-carotene cis and trans isomers and α-carotene. – Inject the β- carotene system suitability solution and determine the resolution between the two major cis isomers, all- trans -β-carotene, and α-carotene. (3) Resolution between all- trans -lutein, zeaxanthin, and apocarotenal. – Inject the mixed carotenoid working solution and determine the resolution between all- trans -lutein, zeaxanthin, and apocarotenal. (c) Calibration. – Inject the mixed carotenoid WS three times before and after each set of sample injections. Calculate the response factor based on average peak areas of analytes and the internal standard. 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 (SP)

Measure each standard measuring solution against an MTBE blank at its absorbance maximum (444 nm for lutein and 450 nm for β-carotene). Calculate the spectrophotometric purity of each reference standard as the observed absorbance over the expected absorbance:

SP = (Abs MS

x 100 x 1000)/(E 1%,1cm

x W)