Reverse Phase High Pressure Liquid Chromatography of Vitamin A in Margarine, Infant Formula, and Fortified Milk

Abstract

Retinal was determined in margarine (50 mg), infant formula (1 ml), and fortified milk (1 ml) by saponification in centrifuge tubes, extraction of the unsaponifiable lipid with hexane, and high pressure liquid chromatography (HPLC) in 90% methanol on a 25 cm x 3.2 mm column containing 10 micrometer LiChrosorb reverse phase. beta-Carotene was determined using the same column and 99% methanol as eluant. The vitamins in the eluate were identified and measured from their absorption at 325 (retinol) and 453 nm (beta-carotene), using a computing integrator. Retinol and carotene were prominent peaks in the chromatograms from properly fortified samples and were satisfactorily separated from other materials. In a survey of 12 different margarines for retinol, the results from liquid chromatography agreed with those of a variety of spectrophotometric and fluorometric procedures but they were obtained with greater ease and they could be interpreted more confidently. The recovery of retinol from oil was better than 99%. The coefficients of variation was 6.8% for 12 replicate analyses of a margarine for retinol and 5.4% for 10 replicate analyses of a margarine for beta-carotene.