Multicomponent spectrofluorimetric method for the assay of carboxymethylflavin and its hydrolytic products: kinetic applications.

The simultaneous assay of carboxymethylflavin (CMF), an intermediate in the photolysis of riboflavin, and its hydrolytic side-chain cleavage products, lumichrome (LC) (acid solution) and LC and lumiflavin (LF) as well as isoalloxazine 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) (alkaline solution) has been carried out by a multicomponent spectrofluorimetric method. The method is based on the adjustment of pH of the degraded solutions to 2.0 and extraction of LC and LF with chloroform. The chloroform extract is evaporated to dryness under reduced pressure, the residue dissolved in pH 6.5 citro-phosphate buffer and LC and LF determined at their fluorescence maxima at 478 and 530 nm, respectively. The pH of the aqueous phase is re-adjusted to 6.5 and the solution used for the determination of CMF, KA and DQ at the wavelengths of 530, 443 and 420 nm, respectively. The proposed method has been validated according to ICH guidelines. The calibration curves for CMF and its hydrolytic products are linear in the concentration range of 0.5-5.0 × 10-6  M. The mean recovery ranges from 99.0-102.0% with relative standard deviation (RSD) of 0.19-0.99%. The limit of detection (LOD) and the limit of quantification (LOQ) are in the range of 1.17-1.78 × 10-7  M and 3.55-5.40 × 10-7  M, respectively. The uniformity of molar balance of CMF and degradation products during hydrolytic reactions indicates the accuracy of the proposed method for the spectrofluorimetric assay of the compounds. It has been applied to study the kinetics of hydrolytic reactions of CMF.

Photolysis of carboxymethylflavin in aqueous and organic solvent: a kinetic study.

This is the first study on the photolysis of carboxymethylflavin (CMF), an intermediate in the photolysis of riboflavin (RF). CMF is photodegraded by removal of side-chain to lumichrome (LC) in acid solution and to LC and lumiflavin (LF) in alkaline solution. It also undergoes alkaline hydrolysis to 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxoquinoxaline (DQ) by cleavage of isoalloxazine ring. CMF degrades to LC in organic solvents. The formation of LC in acid solution and organic solvents takes place by second-order reaction and those of LC, LF, KA and DQ in alkaline solution by first-order reactions. The values of second-order rate constants for the photolysis of CMF at pH 2.0 to 7.0 are in the range of 1.13 to 2.45 M-1 s-1 and those of first-order rate constants (k obs) at pH 8.0-12.0 from 1.53 to 4.18 × 10-4 s-1 and for the formation of photoproducts from 0.37 to 16.6 × 10-5 s-1. The photolysis of CMF is enhanced, with pH, in the alkaline region since the excited state is sensitive to alkaline hydrolysis. The photolysis and fluorescence quantum yields of CMF in aqueous and organic solvents have been reported. CMF and photoproducts have been assayed spectrofluorimetrically. The mode of CMF photolysis is discussed.

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.