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Clinical Chemistry 45: 862-868, 1999;
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(Clinical Chemistry. 1999;45:862-868.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

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

Steinar Hustada, Per Magne Ueland and Jørn Schneede

Department of Pharmacology, University of Bergen, Armauer Hansens Hus, 5021 Bergen, Norway.
a Author for correspondence. Fax 47-55-974605; e-mail steinar.hustad{at}farm.uib.no

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.© 1999 American Association for Clinical Chemistry




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Multianalyte Quantification of Vitamin B6 and B2 Species in the Nanomolar Range in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry
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Phenotypic expression of the methylenetetrahydrofolate reductase 677C->T polymorphism and flavin cofactor availability in thyroid dysfunction
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S. Hustad, M. C. McKinley, H. McNulty, J. Schneede, J.J. Strain, J. M. Scott, and P. M. Ueland
Riboflavin, Flavin Mononucleotide, and Flavin Adenine Dinucleotide in Human Plasma and Erythrocytes at Baseline and after Low-Dose Riboflavin Supplementation
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S. Hustad, P. M. Ueland, S. E. Vollset, Y. Zhang, A. L. Bjorke-Monsen, and J. Schneede
Riboflavin as a Determinant of Plasma Total Homocysteine: Effect Modification by the Methylenetetrahydrofolate Reductase C677T Polymorphism
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