Optimization of Sample Preparation Procedure for Determination of Fat-Soluble Vitamins in Milk and Infant Food by HPLC Technique

Background: The analysis of vitamins in baby food is a challenging task given the complexity of the food matrix, vitamin stability, and strict regulations of the European Union regarding permissible deviations from declared values. Vitamins in food exist in different concentrations and forms and have different stabilities; thus, the preparation of samples for a reliable analysis using the same procedure is not straightforward. Therefore, significant attention has been devoted to optimizing sample preparation in the analysis of vitamins. Methods: This study aims to determine which of the sample preparation and extraction methods is the most efficient for the simultaneous determination of vitamins A, D, E, and K in milk and baby food using high-performance liquid chromatography (HPLC). Different samples of baby food were prepared in seven different ways based on four methods (saponification, enzymatic hydrolysis, solvent extraction, and solid-phase extraction). Results and Conclusions: According to the validation parameters, the optimal preparation method proved to be solid-phase extraction with a C18 stationary phase, with recoveries of 97.4%, 96.1%, 98.3%, and 96.2% for vitamins A, D, E, and K, respectively, and HPLC with a UV–Vis detector was identified as a sufficiently sensitive technique for the identification and quantification of fat-soluble vitamins in milk and baby food.