Determination of vitamins E (α-, γ- and δ-tocopherol) and D (cholecalciferol and ergocalciferol) by liquid chromatography in milk, fruit juice and vegetable beverage

Concentrations of fat-soluble vitamins in food products vary in relation to origin, preparation and/or storage of the products and this makes it hard to predict the exact concentration of these micronutrients in the processed product. In order to determine their content in new food matrixes is necessary to adapt and validate a routine method. A simple method for simultaneous extraction, identification and determination of alpha-, delta- and gamma-tocopherol, ergocalciferol and cholecalciferol in different beverages was established. Saponification was performed with 15 mL of KOH in ethanol (50%, w/v) and BHT was added as antioxidant. The subsequent extraction was carried out with hexane. This was followed by a liquid chromatographic separation on a reversed-phase C-18 (5 mu m, 150 x 4.6 mm) column with acetonitrile-methanol (90:10, v/v) as the mobile phase. UV detection was sufficiently sensitive for the detection of all compounds at the same lambda (265 nm). It was shown that the HPLC method provides baseline separation of all these compounds with good resolution values in 25 min. Recovery of analytes and reproducibility of HPLC proved to be good. Furthermore, the usefulness of this method was demonstrated by its successful application to the analysis of various beverages from different natural origins (milk, infant formula, orange juice, fruit juice with milk and vegetable beverage).