Comparison of derivative spectrophotometric and liquid chromatographic methods for the determination of rofecoxib.

Two different UV spectrophotometric methods were developed for the determination of rofecoxib in bulk form and in pharmaceutical formulations. The first method, an UV spectrophotometric procedure, was based on the linear relationship between the rofecoxib concentration and the lambdamax amplitude at 279 nm. The second one, the first derivative spectrophotometry, was based on the linear relationship between the rofecoxib concentration and the first derivative amplitude at 228, 256 and 308 nm. Calibration curves were linear in the concentration range using peak to zero 1.5-35.0 microg x ml(-1). HPLC was carried out at 225 nm with a partisil 5 ODS (3) column and a mobile phase constituted of acetonitrile and water (50 :50 v/v). A linear range was found to be 0.05-35.0 microg x ml(-1). The developed methods were successfully applied for the assay of pharmaceutical dosage form. The statistics of the analytical data is also presented. The results obtained by first derivative spectrophotometry were compared with HPLC and no significant difference was found.

Glutathione S-conjugation of the sevoflurane degradation product, fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A) in human liver, kidney, and blood in vitro.

Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE) is a fluorinated alkene formed by degradation of the volatile anesthetic sevoflurane in anesthesia machines. FDVE is nephrotoxic in rats and undergoes glutathione-dependent conjugation to form two alkane (G1, G2) and two alkene glutathione S-conjugates (G3, G4), cleavage to cysteine S-conjugates, and beta-lyase-catalyzed metabolism to reactive thionoacyl fluorides, which may react with cellular macromolecules to cause nephrotoxicity. Although similar metabolites have been identified in human urine in vivo, little is known about sites and mechanisms of GSH conjugation in humans. This investigation quantified FDVE-GSH conjugates formed by human hepatic and renal microsomal and cytosolic fractions and blood in vitro. LC-MS/MS analysis identified all four GSH conjugates (G1-G4) formed in all human subcellular fractions. Quantitative analysis indicated that the relative order of formation was G2 > G1 > G4 > G3 with human liver and kidney subfractions. In blood, the order was G1 > G4 > G2 > G3. These results demostrate that FDVE undergoes GSH-dependent conjugation in human liver and kidney microsomes and cytosol as well as blood, which may account for the detection of corresponding mercapturic acids in the urine of patients exposed to FDVE.

Determination of tetrahydrozoline hydrochloride and fluorometholone in pharmaceutical formulations by HPLC and derivative UV spectrophotometry.

Two methods for the quantitative determination of tetrahydrozoline hydrochloride (1) and fluorometholone (2) in pharmaceutical eye drops (Efemoline) are described. The procedures are based on derivative UV spectrophotometry and HPLC. In the former method, d2A/d lambda 2 values were measured in methanol at 226 and 282 nm for 1 and 2, respectively. The relative standard deviations for the method were found to be 1.06% for 1 and 0.98% for 2. The latter method based on a reversed phase HPLC system using a Partisil 5 ODS analytical column. The mobile phase used for the separation of 1, 2 and internal standard (lidocaine) was methanol/acetonitrile/water (50:50:10 v/v) and the compounds in the eye drops were detected at 220 nm using an UV detector. The relative standard deviations for the HPLC method were determined to be 0.61% and 0.50% for 1 and 2, respectively. The proposed methods, which give thoroughly comparable data, are simple, rapid, and allow precise and accurate results and could be used for commercial formulations containing tetrahydrozoline hydrochloride and fluorometholone in combination.

Quantitative determination of acrivastine and pseudoephedrine hydrochloride in pharmaceutical formulation by high performance liquid chromatography and derivative spectrophotometry.

In this study, fourth derivative spectrophotometry and high performance liquid chromatography (HPLC) have been used and described for the quantitative determination of acrivastine (I) and pseudoephedrine hydrochloride (II) in their pharmaceutical capsules form (Duact). In the former method, d4A/d gamma 4 values were measured in methanol at 315 and 269 nm for (I) and (II) respectively. The relative standard deviations (RSD) for the method were found to be 1.16% for (I) and 0.94% for (II). The latter method based on reversed phase HPLC system using LiChrosorb C18 analytical column. The mobile phase used for separation of (I), (II) and internal standard (p-hydroxymethylbenzoate) were the water/acetonitrile/methanol/perchloric acid/n-octylamine (500:130:25:13:0.3 v/v) and the detection of the compounds in the capsules were at 260 nm using UV detector. The RSD for the HPLC method were determined to be 0.79 and 0.88% for (I) and (II) respectively. The proposed methods, which give thoroughly comparable data, are simple, rapid, and allow precise and accurate results and could be used for commercial formulations containing acrivastine and pseudoephedrine hydrochloride in combination.

Comutagenesis-III. In vitro metabolism of 2-amino-3-methylpyridine: the effect of various potential inhibitors, activators and inducers.

1. The effects of various potential inhibitors, activators and inducers on the metabolism of the comutagen 2-amino-3-methylpyridine (2A3MP) by rabbit hepatic microsomes and S9 supernatants have been studied. 2. The 1-N-oxidation of 2A3MP to 2-amino-3-methylpyridine-1-N-oxide (2A3M-PNO) was inhibited by 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525-A) and n-octylamine. 3. The C-oxidation products of 2A3MP, i.e. 2-amino-3-hydroxymethylpyridine (2A3HMP) and 2-amino-3-methyl-5-hydroxypyridine (2A3M5HP), were also inhibited by these compounds. 4. Pretreatment of animals with phenobarbitone (PB) resulted in an increase in the production of 2A3MPNO and 2A3HMP, whereas beta-naphthoflavone (BNF) pretreatment had a greater effect on the formation of 2A3M5HP. 5. Pretreatment with pyridine or pyrazine also had an appreciable effect on the formation of 2A3HMP. 6. It is suggested that different cytochrome P450 isozymes are responsible for the metabolic profile of 2A3MP. CYP2B was involved in the N-oxidation; 2E and/or 2B in the formation of 2A3HMP, and 3A and/or 1A in the formation of 2A3M5HP.

Comutagenesis-I: the in vitro metabolism of 2-amino-3-methylpyridine.

The metabolism of the comutagen 2-amino-3-methylpyridine has been studied in vitro using rat and rabbit hepatic preparations. 2-Amino-3-methylpyridine-N-oxide, 2-amino-3-hydroxymethylpyridine and 2-amino-5-hydroxy-3-methylpyridine were formed by both rat and rabbit hepatic preparations. No evidence was obtained for the formation of the corresponding 2-hydroxylamine, 2-nitroso, 2-nitro-3-methyl-pyridine or their condensation products i.e. azo, azoxy or hydrazo. The results are discussed in relation to the possible mechanism of action of the substrate.

Comutagenesis-II. Some factors influencing the in vitro metabolism of 2-amino-3-methylpyridine.

Factors affecting the metabolism of 2-amino-3-methylpyridine (2A3MP) in vitro have been studied and the conditions which allow maximal metabolism established. Ring nuclear and methyl hydroxylation, and 1-N-oxidation of 2A3MP were linear with respect to arochlor 1254 induced rat S9 supernatant (10,000 g fraction) up to 4.86 mg per ml. The results showed that 20 min incubation time was adequate to observe metabolites formed from 2A3MP. The rate of metabolite production increased with increase in substrate concentration up to 2 µmol per incubate. Using the data obtained the apparent K(m) and V(max) values were calculated using Hanes-Wolf and Lineweaver-Burk plot. No N-hydroxylation of the exo-amino group was observed.

Comutagenesis--V: rapid conversion of 2-hydroxyl-amino-3-methylpyridineto 2-amino-3-methylpyridine by a hepatic S9 preparation.

The in vitro metabolism of 2-hydroxylamino-3-methylpyridine has been investigated using arochlor 1254 pretreated rat S9 mixtures. 2-Hydroxylamino-3-methylpyridine is rapidly converted to the parent amine 2-amino-3-methylpyridine. No further oxidation products of 2-hydroxylamino-3-methylpyridine (i.e. nitroso or nitro) were detected under the HPLC conditions used. This observation may explain our previous findings on the in vitro metabolism of 2-amino-3-methylpyridine where no hydroxylamino metabolite was detected.

Comutagenesis-IV in vitro metabolism of the comutagen 2-amino-3-methylpyridine: species differences and metabolic interaction with norharman.

The metabolism of 2-amino-3-methylpyridine (2A3MP) in vitro has been investigated using the rat, rabbit, dog, marmoset, guinea pig and hamster hepatic microsomes and S9 supernatants (10,000 g fraction). Species differences were observed in the in vitro formation of 2-amino-3-methylpyridine-N-oxide (2A3MPNO), 2-amino-3-hydroxymethylpyridine (2A3HMP) and 2-amino-3-methyl-5-hydroxypyridine (2A3M5HP). The order of activity for 2A3MPNO using hepatic microsomes was dog > rat > rabbit > guinea pig > marmoset > hamster, for 2A3HMP dog > hamster > guinea pig > rat > rabbit > marmoset, for 2A3M5HP rabbit > hamster > dog > rat > guinea pig > marmoset. When hepatic S9 supernatants from various uninduced species were used, rabbit was more active for the production of 2A3MPNO and 2A3M5HP whilst hamster was more effective for the formation of 2A3HMP. Pretreatment of rats with arochlor 1254 enhanced the formation of all metabolites. No species produced a metabolite having the properties of 2-hydroxylamino-3-methylpyridine or further oxidation or condensation products. The present study did not show the presence of any new metabolite after co-incubation of 2A3MP with norharman.

Effect of various potential inhibitors, activators and inducers on the N-oxidation of isomeric aromatic diazines in vitro using rabbit liver microsomal preparations.

1. The effects of various potential inhibitors, activators and inducers on the N-oxidation of isomeric aromatic diazines (pyrazine, pyrimidine and pyridazine) by rabbit liver microsomes have been studied. 2. 2,4-Dichloro-6-phenylphenoxyethylamine (DPEA), SKF 525-A and N-octylamine decreased N-oxide formation at 10(-4) M concentrations. 3. Methimazole and carbon monoxide inhibited the N-oxidation of all three substrates studied. 4. The inhibitory effects were generally exaggerated when hepatic microsomal preparations from phenobarbitone-pretreated animals were used as enzyme source. 5. When phenobarbitone or pyridine were used as inducing agents, the N-oxidation of isomeric aromatic diazines showed considerable induction, whereas beta-naphthoflavone and Arochlor 1254 pretreatment had much weaker effects. 6. It is suggested that P4502E1 and/or 2B are the major subfamilies of P450 involved in the N-oxidation of isomeric diazines.

Factors involved in the N-oxidation of isomeric aromatic diazines by microsomal preparations.

Factors affecting the metabolism of isomeric aromatic diazines in vitro were studied and the conditions which allow maximal metabolism established. The N-oxidation of isomeric diazines was linear with respect to microsomal concentration up to 0.5 g original liver weight per ml using a rabbit microsomal suspension. N-Oxidation was also linear up to between 20-30 minutes depending on substrate studied. The rate of N-oxide production increased with increase in substrate concentration up to about 10 mumol/incubate, after which the rate declined. By using the data obtained the appropriate kinetic factors, Km and Vmax, for the N-oxidation of pyrazine, pyrimidine and pyridazine by rabbit hepatic microsomal preparations were calculated. Spectral binding constants, Ks, of substrates to cytochrome P450 were also calculated and appeared to be related to the Km values.

In vitro tuberculostatic activities of some 2-benzylbenzimidazole and 2-phenoxymethylbenzimidazole derivatives.

Some 2-benzylbenzimidazole and 2-phenoxymethylbenzimidazole derivatives were synthesized and tested for in vitro tuberculostatic activity against Mycobacterium tuberculosis H 37 Rv. and human type wild strain (protocol n degrees.4186). The synthesized compounds have one of the CH3, Cl, NO2 or OCH3 groups at position 5 and 4' and are prepared by heating appropriate o-phenylenediamines with the carboxylic acids in the presence of 4.5N HCl. The experiments indicate that 2-phenoxymethylbenzimidazoles were more active than the corresponding 2-benzylbenzimidazoles. The most active compound was 5-chloro-2-phenoxymethylbenzimidazole (IIc) (MIC: 125 micrograms/ml).