High performance liquid chromatographic determination of vitamin A in margarine, milk, partially skimmed milk, and skimmed milk.
Saponification and subsequent evaporation of extracts can cause losses of vitamin A during analysis and thus cause erratic results. These steps were therefore avoided by measuring retinyl palmitate and beta-carotene directly in hexane extracts of milk and margarine. Extracts were purified before high performance liquid chromatography (HPLC) by washing with aqueous alcohol. Retinyl palmitate was measured at 325 nm after chromatography on LiChrosorb Si60, 5 micrometer, using ethyl ether-hexane (2+98) as the solvent system. Milk (2 mL) was shaken with absolute ethanol (5 mL) and hexane (5 mL). Water (3 mL) was added and, after mixing and centrifugation, 100 microL hexane hexane layer was injected. Margarine (5 g) was dissolved in hexane (100 mL). After 5 mL of the hexane solution was washed with 60% ethanol and centrifuged, a 50 microL aliquot was injected. The results of the retinyl palmitate estimation in milk agreed with those obtained by a fluorometric method; the results in margarine agreed with those obtained by saponification, extraction, and HPLC. The coefficients of variation on analysis of 10 replicate samples of milk and margarine were 3 and 4.5%, respectively. The analysis of margarine for vitamin A was completed by measuring beta-carotene with a detector set at 453 nm; the coefficient of variation of this measurement was 3% (10 replicates).