Retinol determination in milk by HPLC and fluorescence detection

A simple and rapid method for determination of retinol in whole milk is described. Milk (400 mg) was diluted with a water-methanol-ethanol mixture (55:9:36 by vol.) and saponified with potassium hydroxide in a culture tube. Retinol was extracted into 10 ml heptane-di-isopropyl ether (3:1) of which 100 microliters was injected into the HPLC. Fluorescence detection ensured the specificity and sensitivity. The critical points in the analysis were the extraction of retinol from fatty samples and the instability of retinol towards photo-oxidation and air oxidation in alkaline solvents. There was an average recovery of 98.5 +/- 2.4% and a CV of 3.6%.