Quantification of Riboflavin, Flavin Mononucleotide, and Flavin Adenine Dinucleotide in Human Plasma by Capillary Electrophoresis and Laser-induced Fluorescence Detection

BACKGROUND: Riboflavin is the precursor of flavin mononucleotide (FMN) and FAD, which serve as cofactors for several redox enzymes. We have developed a capillary electrophoresis method for the determination of riboflavin and its two coenzyme forms in human plasma. METHODS: Trichloroacetic acid-treated plasma was subjected to solid-phase extraction on reversed-phase columns. The analytes were separated by micellar electrokinetic capillary chromatography in uncoated fused- silica capillaries filled with borate buffer containing 50 mmol/L sodium dodecyl sulfate, methanol, and N-methylformamide. Native fluorescence was monitored at 530 nm, using an argon laser operating at 488 nm as excitation source. RESULTS: The assay was linear over a concentration range of two orders of magnitude, and the limit of detection was far below physiological concentrations for all vitamers. The within-day and between-day coefficients of variation were 4-9% and 6-12%, respectively. The reference values (median, 5-95 percentiles) obtained by analyzing plasma from 63 healthy subjects were 8.6 nmol/L (2.7-42.5 nmol/L) for riboflavin, 7.0 nmol/L (3.5-13.3 nmol/L) for FMN, and 57.9 nmol/L (44.5-78.1 nmol/L) for FAD. CONCLUSIONS: Capillary electrophoresis with laser-induced fluorescence detection allows determination of all riboflavin vitamers far below physiological concentrations. The method may become a useful tool for the assessment of riboflavin status in humans.