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 e xtraction on reversed-phase columns. The analytes were separated by micella r electrokinetic capillary chromatography in uncoated fused-silica capillar ies filled with berate buffer containing 50 nmol/L sodium dodecyl sulfate, methanol, and N-methylformamide. Native fluorescence was monitored at 530 n m, using an argon laser operating at 488 nm as excitation source. Results: The assay was linear over a concentration range of two orders of m agnitude, and the limit of detection was far below physiological concentrat ions for all vitamers. The within-day and between-day coefficients of varia tion 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/I. (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 dete ction allows determination of all riboflavin vitamers far below physiologic al concentrations. The method may become a useful tool for the assessment o f riboflavin status in humans. (C) 1999 American Association for Clinical C hemistry.