Development and validation of a high performance liquid chromatographic method for simultaneous determination of vitamins A and D3 in fluid milk products.

Abstract

An HPLC method for simultaneous determination of vitamins A and D3 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 C18 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 D3 for Grade "A" fluid milk. The method quantitation limits for vitamins A and D3 were 0.0072 and 0.0026 μg/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 D3. The accuracy of the method was evaluated using a National Institute of Standards and Technology standard reference material (1849a) and proficiency test samples.