Analysis of riboflavin and riboflavin cofactor levels in plasma by high-performance liquid chromatography

We describe an assay which determines simultaneously riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plasma, using galactoflavin (GF) as an internal standard. The flavins were extracted on a C18 Sep-Pack cartridge after protein precipitation with 10% trichloroacetic acid, and were analyzed on a C18 RP-HPLC with 85% phosphate–magnesium acetate buffer (pH 3.4) and 15% acetonitrile. FAD, FMN, GF and RF extraction recoveries were 101.0±5.6, 97.0±6.5, 97.0±2.0 and 95.0±4.1%, and reproducibilities were 5.9, 6.8, 2.1 and 4.3%, respectively. FAD, FMN and RF values in infant and adolescent plasma were in the range 53.5–108.2, 9.0–25.1 and 12.7–53.4 nM, and 36.5–157.20, 7.1–24.6 and 8.2–57.8 nM, respectively. Using GF as an internal standard improved the quantification of these B2 vitamers.