Stability-indicating spectrofluorimetric method for the assay of riboflavin and photoproducts: Kinetic applications.

A stability-indicating spectrofluorimetric method has been developed for the simultaneous assay of riboflavin (RF) and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF) in aqueous solution. The method is based on the extraction of LC formed in acid solution and LC and LF formed in alkaline solution with chloroform at pH 2.0 and their assay by fluorescence measurements at 478 and 530 nm, respectively. The aqueous phase, on readjustment of the pH to 6.5, is used to extract FMF with chloroform and its assay is carried out at 530 nm. The aqueous phase is then used for the assay of RF at 530 nm. The proposed method gives more accurate results for the assay of RF compared to those of the United States Pharmacopeia (USP) spectrofluorimetric method which does not take into account the presence of RF photoproducts having similar fluorescence characteristics. The proposed method along with the USP method has been applied to the study of the kinetics of photolysis of RF, assay of stored commercial vitamin preparations and their radiated samples. The results show that the USP method does not distinguish between the fluorescence of RF and its photoproducts, and, therefore, gives erroneous results with about 11% excess in the quantity of the vitamin compared to that of the proposed method. This is due to the interference of the fluorescence of photoproducts in the assay of RF. The method has been validated for various analytical parameters according to the guideline of the International Council for Harmonization (ICH).

Photodegradation and stabilization of betamethasone-17 valerate in aqueous/organic solvents and topical formulations.

The effects of solvent [acetonitrile, methanol, and acetonitrile/water mixture (20:80, v/v)], buffer concentration (phosphate buffer, pH 7.5), ionic strength and commonly employed adjuvants on the photodegradation of betamethasone-17 valerate in cream and gel formulations have been studied on exposure to UV light (300-400 nm). A validated high-performance liquid chromatography method has been used to determine the parent compound and its photodegraded products. The photodegradation data in the studied solvents showed greater decomposition of the drug in solvents with a lower dielectric constant. A comparatively higher rate of photodegradation was observed in the cream formulation compared to that for the gel formulation. The kinetic treatment of the photodegradation data revealed that the degradation of the drug follows first-order kinetics and the apparent first-order rate constants for the photodegradation reactions, in the media studied, range from 1.62 to 11.30×10(-3) min(-1). The values of the rate constants decrease with increasing phosphate concentration and ionic strength which could be due to the deactivation of the excited state and radical quenching. The second-order rate constant (k') for the phosphate ion-inhibited reactions at pH 7.5 has been found to be 5.22×10(-2) M(-1) s(-1). An effective photostabilization of the drug has been achieved in cream and gel formulations with titanium dioxide (33.5-42.5%), vanillin (21.6-28.7%), and butyl hydroxytoluene (18.2-21.6%).

In vitro evaluation of betamethasone esters for phototoxic potential.

The phototoxicity of Betamethasone valerate and betamethasone dipropionate has been investigated on irradiation in the wavelength range of 300-400 nm, using some basic in vitro phototoxicity tests. Both esters cause photohemolysis of mouse red blood cells and photoperoxidation of linoleic acid. The photoproducts of both esters have also been found to be phototoxic. The toxicity of these photoproducts increases with further irradiation; however, they exhibit toxicity, even in the dark. The efficient inhibition of photohemolysis by the well-known free radical scavengers, such as butylhydroxyanisole and reduced glutathione, and minor inhibition by singlet-oxygen quenchers, such as sodium azide, suggests that cellular damage is mainly mediated by free radicals whereas singlet oxygen plays a minor role.

Photolysis of thiochrome in aqueous solution: A kinetic study.

The photolysis of thiochrome (THC), an oxidation product of thiamine (vitamin B1) (THE), used for its fluorimetric assay, has been studied in the pH range 7.0-12.0. THC undergoes photooxidation to oxodihydrothiochrome (ODTHC) which is oxidized to a non-fluorescent compound (OP1) on UV irradiation. The kinetics of the consecutive first-order reactions: THC→k1ODTHC→k2OP1, has been evaluated and the values of first-order rate constants, k1 (0.58-4.20 × 10-5, s-1) and k2 (0.05-2.03 × 10-5, s-1), at pH 7.0-12.0 have been determined. The rates of degradation of THC and ODTHC are enhanced with pH and the second-order rate constants k1' and k2' for the OH- ion-catalyzed reaction are in the range of 0.002-58.3 M-1 s-1. The quantum yields of the photolysis of THC and ODTHC in the pH range 7.0-12.0 have been determined. THC, ODTHC and OP1 have been identified by chromatographic, spectrometric and fluorimetric methods. THC and ODTHC have similar fluorescence characteristics and emit at 450 and 445 nm, respectively. THC, ODTHC and OP1 with distinct absorption maxima (370, 344 and 290 nm, respectively) have been determined by a newly developed and validated multicomponent spectrometric method during the photolysis reactions. The on-line formation of THC by the photooxidation of THE may lead to the degradation of THC and give erroneous results in the fluorimetric assay of THE. A scheme for the photolysis reactions of THC in aqueous solution is presented.

Metal ion mediated photolysis reactions of riboflavin: A kinetic study.

The effect of metal ion complexation on the photolysis of riboflavin (RF) using various metal ions (Ag+, Ni2+, Co2+, Fe2+, Ca2+, Cd2+, Cu2+, Mn2+, Pb2+, Mg2+, Zn2+, Fe3+) has been studied. Ultraviolet and visible spectral and fluorimetric evidence has been obtained to confirm the formation of metal-RF complexes. The kinetics of photolysis of RF in metal-RF complexes at pH7.0 has been evaluated. The apparent first-order rate constant (kobs) for the photolysis of RF and the formation of lumichrome (LC) and lumiflavin (LF) (0.001M phosphate buffer) and LC, LF and cyclodehydroriboflavin (CDRF) (0.2-0.4M phosphate buffer) have been determined. The values of kobs indicate that the rate of photolysis of RF is promoted by divalent and trivalent metal ions. The second-order rate constants (k' ) for the interaction of metal ions with RF are in the order: Zn2+>Mg2+>Pb2+>Mn2+>Cu2+>Cd2+>Fe2+>Ca2+>Fe3+>Co2+>Ni2+>Ag+. In phosphate buffer (0.2-0.4M), an increase in the metal ion concentration leads to a decrease in the formation of LC compared to that of CDRF by different pathways. The photoproducts of RF have been identified and RF and the photoproducts have simultaneously been assayed by a multicomponent spectrometric method. The mode of photolysis of RF in metal-RF complexes has been discussed.