Development and Validation of a High Performance Liquid Chromatographic Method for Simultaneous Determination of Vitamins A and D3 in Fluid Milk Products

An HPLC method for simultaneous determination of vitamins A and D-3 in fluid milk was developed and validated. Saponification and extraction conditions were studied for optimum recovery and simplicity. An RP HPLC system equipped with a C-18 column and diode array detector was used for quantitation. The method was subjected to a single-laboratory validation using skim, 2% fat, and whole milk samples at concentrations of 50, 100, and 200% of the recommended fortification levels for vitamins A and D-3 for Grade "A" fluid milk. The method quantitation limits for vitamins A and D-3 were 0.0072 and 0.0026 mu g/mL, respectively, Average recoveries between 94 and 110% and SD values ranging from 2.7 to 6.9% were obtained for both vitamins A and D-3. The accuracy of the method was evaluated using a National Institute of Standards and Technology standard reference material (1849a) and proficiency test samples.