Analysis of Carotenoids in Vegetables by HPLC

Eight vegetables were pre-treated with and without sa­ ponification, and chromatographed by reversed-phase HPLC. Carote11Oid peaks were identified using seven reference standards. Samples extracts were also chromatographed llsing a mixed magnesia and diatomaceous earth column. Fractions were eluted using stepwise increases in the proportion of acetone in hexane, studied by UV-VIS ab­ sorption spectrophotometry, and re-chromatographed by HPLC. The major carotenoids, aand {3-carotenes, lutein and lycopene were thus identified. Saponification removed various nOH-carotenoid pigments in green vegetables, re­ sulting in HPLC profiles which were easier to quantitate, while prolonging the life of the HPLC column. The process did not result in a drastic loss of aand {3-carotelle, although there appeared to be some loss of lutein, which possesses 1lO pro-vitamin A activity. "Total carote11Oid content" of the saponified vegetables could be obtained from the ab­ sorbance reading of the extracts at 450 111n.