Rapid method for determination of riboflavin in urine by high-performance liquid chromatography

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.