Liquid Chromatographic Determination of B2 Vitamers in Human Plasma and Whole Blood

Riboflavin (RF; vitamin B2) participates in redox reactions in 2 coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (1). Quantification of these 3 flavins in plasma and erythrocytes is essential for studying vitamin B2 metabolism (1)(2). We describe here an HPLC method for quantifying these flavins in both matrices. Riboflavin and FMN sodium salt were from Sigma, FAD disodium salt and acetonitrile were from Merck, and the internal standard galactoflavin was from Merck & Dohme. All chemicals were analytical grade. For quality control, we used 3 ClinChek serum and whole-blood controls (Recipe), reconstituted and stored at −80 °C. Aliquots of aqueous (9 g/L NaCl) flavin stock solutions (50 μmol/L) were stored at −20 °C in the dark. Stock solution concentrations were verified by measurement of absorbances at 450 nm (3). The HPLC system consisted of a Thermoquest SP 8700 X pump, Rheodyne Model 7125 100-μL loop injector, Jasco FP-920 fluorescence detector (excitation, 456 nm; emission, 512 nm), and Shimadzu Model C-R1B Chromatopac Integrator. A Phenomenex Onyx Monolithic C18 column [50 × 4.6 mm (i.d.)] …