Simultaneous HPLC analysis of fat-soluble vitamins in selected animal products after small-scale extraction

A method for simultaneous determination of fat-soluble vitamins in animal products is presented. Milk, and two fish products containing many ingredients, were used as test materials. The vitamins determined were tocopherols, β-carotene, all-trans-retinol and in the case of fish, cholecalciferol. The sample preparation procedure, consisting of saponification and extraction by n-hexane–ethyl acetate was carried out in small-scale. To cope with the highly different composition of the test materials, some modifications were needed. Normal-phase HPLC separation using diisopropyl ether-n-hexane gradient elution with UV and fluorescence detections for tocopherols, β-carotene and all-trans-retinol was used. Cholecalciferol was determined from the same extract by reverse-phase HPLC (with UV detection) after semi-preparative HPLC purification. Recoveries of spiked samples varied from 80 to 111% for all determined fat-soluble vitamins. The day-to-day repeatability was in most cases under 7%, and the within-day variation of this method was small, under 5.5% for all vitamins. The described analytical method is effective and fast, enabling the processing of a large number of samples.