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.

Effect of acetate and carbonate buffers on the photolysis of riboflavin in aqueous solution: a kinetic study.

The photolysis of riboflavin (RF) in the presence of acetate buffer (pH 3.8-5.6) and carbonate buffer (pH 9.2-10.8) has been studied using a multicomponent spectrophotometric method for the simultaneous assay of RF and its photoproducts. Acetate and carbonate buffers have been found to catalyze the photolysis reaction of RF. The apparent first-order rate constants for the acetate-catalyzed reaction range from 0.20 to 2.86 × 10(-4) s(-1) and for the carbonate-catalyzed reaction from 3.33 to 15.89 × 10(-4) s(-1). The second-order rate constants for the interaction of RF with the acetate and the carbonate ions range from 2.04 to 4.33 × 10(-4) M(-1) s(-1) and from 3.71 to 11.80 × 10(-4) M(-1) s(-1), respectively. The k-pH profile for the acetate-catalyzed reaction is bell shaped and for the carbonate-catalyzed reaction a steep curve. Both HCO3(-) and CO3(2-) ions are involved in the catalysis of the photolysis reaction in alkaline solution. The rate constants for the HCO3(-) and CO3(2-) ions catalyzed reactions are 0.72 and 1.38 × 10(-3) M(-1) s(-1), respectively, indicating a major role of CO3(2-) ions in the catalysis reaction. The loss of RF fluorescence in acetate buffer suggests an interaction between RF and acetate ions to promote the photolysis reaction. The optimum stability of RF solutions is observed in the pH range 5-6, which is suitable for pharmaceutical preparations.

Photolysis of tolfenamic acid in aqueous and organic solvents: a kinetic study.

Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug that was studied for its photodegradation in aqueous (pH 2.0-12.0) and organic solvents (acetonitrile, methanol, ethanol, 1-propanol, 1-butanol). TA follows first-order kinetics for its photodegradation, and the apparent first-order rate constants (k obs) are in the range of 0.65 (pH 12.0) to 6.94 × 10-2 (pH 3.0) min-1 in aqueous solution and 3.28 (1-butanol) to 7.69 × 10-4 (acetonitrile) min-1 in organic solvents. The rate-pH profile for TA photodegradation is an inverted V (∧) or V-top shape, indicating that the cationic form is more susceptible to acid hydrolysis than the anionic form of TA, which is less susceptible to alkaline hydrolysis. The fluorescence behavior of TA also exhibits a V-top-shaped curve, indicating maximum fluorescence intensity at pH 3.0. TA is highly stable at a pH range of 5.0-7.0, making it suitable for formulation development. In organic solvents, the photodegradation rate of TA increases with the solvent's dielectric constant and solvent acceptor number, indicating solute-solvent interactions. The values of k obs decreased with increased viscosity of the solvents due to diffusion-controlled processes. The correlation between k obs versus ionization potential and solvent density has also been established. A total of 17 photoproducts have been identified through LC-MS, of which nine have been reported for the first time. It has been confirmed through electron spin resonance (ESR) spectrometry that the excited singlet state of TA is converted into an excited triplet state through intersystem crossing, which results in an increased rate of photodegradation in acetonitrile.

Riboflavin (vitamin B2) sensitized photooxidation of ascorbic acid (vitamin C): A kinetic study.

Ascorbic acid (AH2) photoxidation sensitized by riboflavin (RF) has been studied between pH 2.0 and 12.0 in ambient air and anaerobic environment using UV and visible irradiation sources. The kinetics of AH2 degradation in aqueous medium along with RF is found to be first-order for its photodegradation. AH2 photolysis rate constants in aerobic and anaerobic conditions with RF (1.0-5.0 × 10-5 M) are 0.14-3.89 × 10-2 and 0.026-0.740 × 10-2 min-1, respectively. The rate constants (k2) of second-order kinetics for AH2 and RF photochemical interaction in aerobic and anaerobic conditions are in the range of 0.24-3.70 to 0.05-0.70 × 10-3 M-1 min-1, respectively, which manifests that increasing the RF concentration also increases the rate of photodegradation (photooxidation) of AH2. The k2 versus pH graph is bell-shaped which indicates that increasing the pH increases photolytic degradation rate of AH2 with RF. Increasing the pH results in the increased ionization of AH2 (ascorbyl anion, AH-) and redox potential which leads to the higher rates of photodegradation of AH2. Two-component spectrophotometric (243 and 266 nm, AH2 and RF, respectively) and high-performance liquid chromatography (HPLC) methods have been used to determine the concentration of AH2 and RF in pure and degraded solutions. The results obtained from these two methods are compared using a student t-test which showed no noteworthy difference between them.

Correction for irrelevant absorption in multicomponent spectrophotometric assay of riboflavin, formylmethylflavin, and degradation products: kinetic applications.

In the spectrophotometric assay of multicomponent systems involved in drug degradation studies, some minor or unknown degradation products may be present. These products may interfere in the assay and thus invalidate the results due to their absorption in the range of analytical wavelengths. This interference may be eliminated by the application of an appropriate correction procedure to obtain reliable data for kinetic treatment. The present study is based on the application of linear and non-linear irrelevant absorption corrections in the multicomponent spectrophotometric assay of riboflavin and formylmethylflavin during the photolysis and hydrolysis studies. The correction procedures take into account the interference caused by minor or unknown products and have shown considerable improvement in the assay data in terms of the molar balance. The treatment of the corrected data has led to more accurate kinetic results in degradation studies.

Vitamin B6: deficiency diseases and methods of analysis.

Vitamin B6 (pyridoxine) is closely associated with the functions of the nervous, immune and endocrine systems. It also participates in the metabolic processes of proteins, lipids and carbohydrates. Pyridoxine deficiency may result in neurological disorders including convulsions and epileptic encephalopathy and may lead to infant abnormalities. The Intravenous administration of pyridoxine to patients results in a dramatic cessation of seizures. A number of analytical methods were developed for the determination of pyridoxine in different dosage forms, food materials and biological fluids. These include UV spectrometric, spectrofluorimetric, mass spectrometric, thin-layer and high-performance liquid chromatographic, electrophoretic, electrochemical and enzymatic methods. Most of these methods are capable of determining pyridoxine in the presence of other vitamins and complex systems in µg quantities. The development and applications of these methods in pharmaceutical and clinical analysis mostly during the last decade have been reviewed.

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.

Photodegradation of levofloxacin in aqueous and organic solvents: a kinetic study.

The kinetics of photodegradation of levofloxacin in solution on UV irradiation in the pH range 2.0-12.0 has been studied using a HPLC method. Levofloxacin undergoes first-order kinetics in the initial stages of the reaction and the apparent first-order rate constants are of the order of 0.167 to 1.807×10-3 min-1. The rate-pH profile is represented by a curve indicating the presence of cationic, dipolar and anionic species during the reaction. The singly ionized form of the molecule is non-fluorescent and is less susceptible to photodegradation. The increase in the degradation rate in the pH range 5.0-9.0 may be due to greater reactivity of the ionized species existing in that range. The rate appears to vary with a change in the degree of ionization of the species present in a particular pH range and their susceptibility to photodegradation. Above pH 9, the decrease in the rate of photodegradation may be a result of deprotonation of the piperazinyl group. The levofloxacin molecule is more stable in the pH range around 7, which is then suitable for formulation purposes. The photodegradation of levofloxacin was found to be affected by the dielectric constant and viscosity of the medium.

Photoinitiated polymerization of 2-hydroxyethyl methacrylate by riboflavin/triethanolamine in aqueous solution: a kinetic study.

The polymerization of 1-3 M 2-hydroxyethyl methacrylate (HEMA) initiated by riboflavin/triethanolamine system has been studied in the pH range 6.0-9.0. An approximate measure of the kinetics of the reaction during the initial stages (~5% HEMA conversion) has been made to avoid the effect of any variations in the volume of the medium. The concentration of HEMA in polymerized solutions has been determined by a UV spectrophotometric method at 208 nm with a precision of ±3%. The initial rate of polymerization of HEMA follows apparent first-order kinetics and the rates increase with pH. This may be due to the presence of a labile proton on the hydroxyl group of HEMA. The second-order rate constants for the interaction of triethanolamine and HEMA lie in the range of 2.36 to 8.67 × 10(-2) M(-1) s(-1) at pH 6.0-9.0 suggesting an increased activity with pH. An increase in the viscosity of HEMA solutions from 1 M to 3 M leads to a decrease in the rate of polymerization probably as a result of the decrease in the reactivity of the flavin triplet state. The effect of pH and viscosity of the medium on the rate of reaction has been evaluated.

Stabilizing effect of citrate buffer on the photolysis of riboflavin in aqueous solution.

In the present investigation the photolysis of riboflavin (RF) in the presence of citrate species at pH 4.0-7.0 has been studied. A specific multicomponent spectrophotometric method has been used to assay RF in the presence of photoproducts during the reactions. The overall first-order rate constants (k obs ) for the photolysis of RF range from 0.42 to 1.08×10(-2) min(-1) in the region. The values of k obs have been found to decrease with an increase in citrate concentration indicating an inhibitory effect of these species on the rate of reaction. The second-order rate constants for the interaction of RF with total citrate species causing inhibition range from 1.79 to 5.65×10(-3) M(-1) min(-1) at pH 4.0-7.0. The log k-pH profiles for the reactions at 0.2-1.0 M citrate concentration show a gradual decrease in k obs and the value at 1.0 M is more than half compared to that of k 0, i.e., in the absence of buffer, at pH 5.0. Divalent citrate ions cause a decrease in RF fluorescence due to the quenching of the excited singlet state resulting in a decrease in the rate of reaction and consequently leading to the stabilization of RF solutions. The greater quenching of fluorescence at pH 4.0 compared to that of 7.0 is in accordance with the greater concentration of divalent citrate ions (99.6%) at that pH. The trivalent citrate ions exert a greater inhibitory effect on the rate of RF photolysis compared to that of the divalent citrate ions probably as a result of excited triplet state quenching. The values of second-order rate constants for the interaction of divalent and trivalent citrate ions are 0.44×10(-2) and 1.06×10(-3) M(-1) min(-1), respectively, indicating that the trivalent ions exert a greater stabilizing effect, compared to the divalent ions, on RF solutions.