RESUMO
A high-performance liquid chromatographic method with fluorimetric detection for the quantification of riboflavin (RB), riboflavin 5'-phosphate (FMN), and flavin adenine dinucleotide (FAD) in plasma, whole blood, and urine is described. Under isocratic conditions with a reversed-phase column, the compounds are completely resolved and eluted within 9 min. Plasma proteins are precipitated with acetonitrile followed by shaking the aqueous phase with chloroform. Urine samples are diluted and injected directly. The reproducibility of this method for the quantification of RB in plasma has a between-day coefficient of variation of 6%. The application of this method is illustrated by analyzing plasma and urine samples from a human subject who received an intravenous dose of FMN equivalent to 25 mg of RB.
Assuntos
Mononucleotídeo de Flavina/análise , Flavina-Adenina Dinucleotídeo/análise , Riboflavina/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida de Alta Pressão , Mononucleotídeo de Flavina/sangue , Mononucleotídeo de Flavina/urina , Flavina-Adenina Dinucleotídeo/sangue , Flavina-Adenina Dinucleotídeo/urina , Humanos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Masculino , Pessoa de Meia-Idade , Riboflavina/sangue , Riboflavina/urina , Espectrometria de FluorescênciaRESUMO
A simple, specific, and sensitive high-performance liquid chromatographic (HPLC) method for the determination of riboflavin directly in urine samples using a fixed-wave-length spectrofluorometer is described. Centrifuged raw urine samples (50 microliter) are injected onto a reversed-phase microparticulate C18 column. The eluent is 0.01 M KH2PO4 (pH 5.0)-methanol (65:35). This method is capable of differentiating riboflavin from riboflavin-5-phosphate, non-riboflavin fluorescing components in urine, and photo-degraded riboflavin. The method shows good reproducibility and is linear to at least 12 micrograms/ml. The sensitivity of this procedure, at the 95% confidence limit, determined by linear regression analysis, is estimated to be 0.05 micrograms/ml using peak height and 0.07 micrograms/ml using peak area. This HPLC method is compared to an automated fluorometric method for riboflavin. The coefficient of linear regression of this comparison is Y = 0.858 + 0.893X, where X is the HPLC method and Y is the fluorometric method.
