Method for the extraction of riboflavin for high-performance liquid chromatography and application to casein

Three different approaches, including the commonly used acid digestion, were compared for the efficient extraction of very low concentrations of riboflavin (vitamin B2) from casein. An extraction method that used pepsin to release riboflavin was the most efficient. The pepsin extraction method was then further optimized using a factorial design to yield the maximum amount of riboflavin. The enzyme takadiastase was used for the conversion of all forms of riboflavin to the free form, and this conversion was found to be time dependent. A reversed-phase HPLC system with fluorescence detection and a methanol-water mobile phase containing acetate buffer were used for the separation and quantification of riboflavin in the extracts. A limit of detection of less than 0.1 mg kg-1 casein and a repeatability RSD of 3% were achieved. The recovery of the method was satisfactory.