Research Report: Determination of Riboflavin Content in Goat and Cow Milk by High Performance Liquid Chromatography (HPLC)
AbstractA high performance liquid chromatography method has been developed to determine riboflavin content in goat and cow whole milk. The present method involves acidification of the milk to precipitate bulk of the proteins and centrifugation affording a supernatant, which was diluted with 2% acetic acid to a constant volume. This solution was subjected to HPLC analysis using an ODS column. The method is simple, rapid and sensitive for riboflavin determination. The results obtained by the method compared well with the results of analysis by the \"Official Methods of Analysis\" of the Association of Official Analytical Chemists (AOAC).
Several analytical methods for the determination of riboflavin have been reported (Kamman, 1980). Most of the procedures involve a pretreatment to liberate the protein bound vitamin from flavoproteins. Fluorometric methods of detection are considered the most desirable because of their sensitivity, rapidity, and specificity. However, they require extra treatments to separate riboflavin from interfering fluorescent compounds. In the official methods of analysis of the Association of Official Analytical Chemists (AOAC, 1996, method 970.65), samples are treated with potassium permanganate (KMnO4) and hydrogen peroxide (H2O2) to remove background fluorescence. Rashid and Potts (1980) treated milk samples with acidified lead acetate solution to separate extraneous protein and minimize background fluorescence. Fellman et al. (1982) have reported a C18 reversed phase separation for the simultaneous determination of thiamin and riboflavin in selected foods including milk. This method required the removal of interfering substances using a 0.01M phosphate buffer.
Journal of Agriculture, Science and Technology Vol.3(2) 2001: 96-101