Immunoassays for riboflavin and flavin mononucleotide using antibodies specific to d-ribitol and...

Riboflavin (vitamin B₂), a water-soluble vitamin, plays a key role in maintaining human health. Though, numerous methods have been reported for the determination of total riboflavin (TRF) content in foods and biological samples, very few methods are reported for quantifying riboflavin and its coenzymes [flavin mononucleotide (FMN); flavin adenine dinucleotide (FAD)] individually. Recently, we have demonstrated that antibodies specific to d-ribitol and d-ribitol-5-phosphate also recognize riboflavin and FMN, respectively, and not vice-versa. In this study, we have evaluated these two antibodies for the analysis of riboflavin and FMN by indirect competitive ELISA (icELISA) in selected foods and pharmaceuticals. Under the optimal assay conditions, 50% inhibition concentration (IC₅₀) and limit of detection (LOD, IC₁₀) were 3.41ng/mL and 0.02ng/mL for riboflavin, and 7.84ng/mL and 0.24ng/mL for FMN, respectively, with detectable concentration range between 0.1 and 100ng of analytes and <0.1% cross-reactivity with other water-soluble vitamins. The amounts of TRF in food samples, as analyzed by icELISA using ribitol antibody, were 90-95% of the reported values in the literature or label values. Quantification of individual flavins (riboflavin and FMN) from the same food samples showed variation in their values compared to TRF, and were in good agreement with values obtained from HPLC and AOAC methods. Further, spiking and recovery analysis of food samples and pharmaceuticals showed no significant matrix effects. The immunoassays were validated in terms of accuracy and precision using inter- and intra-assays. The immunoassays developed in this study are sensitive and appears feasible for screening a large number of samples in the quantification of riboflavin and FMN in various biological samples, pharmaceuticals and natural/processed foods.