A rapid saponification procedure for the simultaneous HPLC determination of the natural levels of vitamins A, E and beta-carotene in milk

Abstract

A rapid saponification procedure was developed for the simultaneous extraction of vitamins A, E and beta-carotene in milk, prior to HPLC analysis. A high accuracy, sensitivity and ruggedness were achieved by the use of an internal standard, beta-tocopherol, which enabled the determination of the low levels of vitamins occurring naturally. Vitamin losses during extraction and the subsequent HPLC analysis were minimized by the use of antioxidants and by preconditioning the silica column with alpha-tocopherol in order to prevent irreversible adsorption of vitamin E on the column. The results compared well with those obtained from a non-saponification approach using a modified Rose-Gottlieb extraction. Relative standard deviations for repeatability of 4.9, 3.5 and 3.3% were achieved for vitamins A, E and beta-carotene respectively. The rapid, single extraction and simultaneous HPLC analysis used for the 3 vitamins made the method well suited for routine analytical work