Multicomponent spectrofluorimetric method for the assay of formylmethylflavin and its hydrolytic products: Kinetic applications.

A multicomponent spectrofluorimetric method has been developed for the simultaneous assay of formylmethylflavin (FMF), an intermediate product in the photolysis of riboflavin (vitamin B2 ), and its side-chain hydrolytic products, lumichrome (LC) in acidic solution and LC and lumiflavin (LF) in the alkaline solution as well as its ring cleavage products, 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxo-quinoxaline (DQ) in alkaline solution. The assay method also takes into account an oxidation product of FMF, i.e. carboxymethylflavin (CMF), in both acid and alkaline solutions. The method involves adjustment of the pH of hydrolysed solution to 2.0 to convert FMF to its protonated form, extraction of LC (acid solution) or LC and LF (alkaline solution) with chloroform and their simultaneous assay by fluorescence measurement at 478 and 530 nm, respectively. The aqueous phase is readjusted to pH 6.5, extracted with chloroform to remove undegraded FMF and used for the assay of CMF, KA and DQ at 530, 443 and 420 nm, respectively. The chloroform extract is used for the assay of FMF at 530 nm. The proposed method has been validated and applied to the study of the kinetics of a hydrolysis reaction of FMF at pH 11.0. The calibration curves for FMF and degradation products are linear in the range of 0.1-1.0 × 10-6  M. The limit of detection (LOD) and limit of quantification (LOQ) range from 2.54-5.75 × 10-8  M and 0.78-1.74 × 10-7  M, respectively, for these compounds. The mean recovery ranges from 99.3-102.1% with a RSD of 0.14-0.35%. Judging from the molar balance of FMF and the hydrolytic products, uniformity of analytical data during the reactions and linearity of kinetic plot, the method gives accurate results for the assay of FMF and all of its degradation products. It can be conveniently used for the assay of these compounds and for the kinetics and stability studies of FMF.