Rapid determination of vitamin B2 (riboflavin) in plasma by HPLC

Background: Riboflavin (vitamin B-2), as the exclusive source for the coenzymes Flavin mononucleotide (FMN) and Flavin adenine dinucleotide (FAD) in humans, is a water-soluble vitamin critical for metabolism and energy production. In its coenzyme forms, riboflavin is involved in essential oxidation-reduction reactions. Deficiency leads to skin and mucosa! disorders. Measurement of plasma riboflavin can be used to assess vitamin B2 status in at-risk individuals. Methods: Proteins are removed from plasma by acid precipitation. An aliquot of the resulting supernatant is analyzed by reversed-phase HPLC. Impurities are separated from riboflavin isocratically and the target material is detected fluorometrically (excitation 450 nm; emission 520 nm). Results: The method was validated for linearity, limit of quantification, accuracy, precision, and interference. The method was accurate and correlated well (R-2=0.993) to expected concentrations of spiked pooled plasma samples. Imprecision was <10%. Riboflavin concentrations were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements. The reference interval established by nonparametric analysis was 6.7-50.1 nmol/l. Conclusions: This HPLC method allows separation and measurement of riboflavin in plasma in 7 min. Results from the assay may be used for clinical diagnosis of deficiency and to monitor therapeutic vitamin supplementation regimes. (C) 2010 Elsevier B.V. All rights reserved.