HPLC Determination of Riboflavin in Fortified Foods

Various foods of plant and animal origin contain riboflavin or vitamin B2. Major sources are protein-rich foods, such as milk and dairy products, and also meat products. Riboflavin occurs in foods in free form or bound in the flavins FMN (fla- vin mononucleotide) and FAD (flavin adenine dinucleotide). A complete analysis of riboflavin needs combined extraction with dilute mineral acids and enzymatic hydrolysis. In milk and dairy products, free riboflavin is the main component. For the simultaneous determination of flavins plus free riboflavin, extraction with methanol and dichloromethane followed by partitioning with citrate buffer, pH 5.5, or by using dilute formic acid containing 2 mol l−1 urea is practicable. The high- performance liquid chromatographic separation of riboflavin and flavins is mainly carried out by using reversed-phase (RP) C18 columns and in most cases by isocrat- ic elution with mobile phases based on acidified methanol–water or acetonitrile followed by fluorimetric detection (λex = 444 nm; λem = 530–565 nm). Quantifica- tion of the extracted riboflavin after irradiation to lumiflavin and fluorescence de- tection is rarely practiced. riboflavin, vitamin B2, flavins, high-performance liquid chromatography, fluorimetric detection