Critique of methods for the determination of riboflavin in urine.

Both fluorometric and microbiological measurements of urinary riboflavin excretion have served as a basis for evaluation of nutritional status, detection of deficiency states, measurement of physiological availability, and determination of daily requirements. Fluorometric assay methods have commonly followed three basic procedures; namely, (a) that of Najjar (l), based on the extraction of riboflavin into a butanol-pyridine medium; (b) the double hydrosulfite reduction method of Hodson and Norris (2); and (c) the Florisil method of Ferrebee (3). Most microbiological assays have been based on the Lactobacillus casei assay of Snell and Strong (4). However, the literature shows that considerable disagreement exists in regard to the reliability and relative advantages of the commonly used methods. For example, in a series of availability tests, Melnick et al. (5) report Strong and Carpenter’s (6) modification of the Snell and Strong method to be more specific for urinary riboflavin than the method of Najjar with the blank correction omitted, but these authors prefer the fluorometric method because of the better reproducibility. Keys et al. (7) compared the microbiological method of Snell and Strong with a modification of the Conner and Straub (8) Florisil procedure and found the latter considerably more satisfactory for urinary riboflavin assay. Recently, Slater and Morel1 (9) have developed a modification of the Najjar procedure and found it to show agreement for urine assay with an unpublished microbiological method with Lactobacillus casei. In a comparative study of fluorometric methods, Morel1 and Slater (10) found many methods to be non-specific for urinary riboflavin. These included a Florisil method utilizing the adsorptionelution technique of Barton-Wright and Booth (1 l), the direct and indirect methods of Najjar (l), and Hodson and Norris (2) procedure, the direct method of Ferrebee (3) with an added internal standard, and a method involving oxidation with cold KMn04 and HzOz, followed by a hydrosulfite blank.