Indirect Competitive Immunoassay for Detection of Vitamin B2 in Foods and Pharmaceuticals

An indirect immunoassay for the determination of vitamin B-2 in food samples and vitamin tablets was developed. A carbodiimide-modified active ester method was used to synthesize the immunogen for vitamin B-2. The coupling ratio of vitamin B-2 to carrier protein in immunogen was 19.98:1. The titer of the polyclonal antibody was 1:64000, and the antibody showed high specificity in the presence of vitamin B-2 photolytic products and other B group vitamins. The immunoassay showed detection limits (LODs) of 1.07 ng/mL in PBS, 24.6 ng/g in vitamin drink, and 0.50 mg/kg in milk powder. Recovery was 99.58-110.91% in milk powder and 70.20-100.5% in vitamin drink Vitamin B-2 samples were analyzed by high-pressure liquid chromatography (HPLC) and the immunoassay, and results showed good agreement. Finally, this method was applied to detect vitamin B-2 in commercial milk powder and vitamin tablets, and the detected amount correlated well with the labeled amount.