Spectrofluorometric determination of alkoxy-substituted benzylimidazolidinones in biological fluids.

Sensitive and specific spectrofluorometric assays were developed for the determination of d, 1-4-(3,4-dimethoxybenzyl)-2-imidazolidinone and d,1-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone in blood and urine. Each compound is selectively extracted from buffered blood or urine and oxidized with alkaline permanganate to its respective fluorescent benzoic acid derivative, which is quantitated in acetic acid in ethanol (1:99). The method was applied to the determination of blood levels and urinary excretion in the dog following the administration of single intravenous and oral doses of each compound.

Spectrofluorometric determination of substituted tetrahydrocarbazoles by a methylene blue sensitized photolytic reaction.

A spectrofluorometric method was developed for the determination of 6-chloro-9-[2-(2-methyl-5-pyridyl)ethyl]-1,2,3,4-tetrahydrocarbazole-2-methanol hydrochloride and its carboxylic acid analog in blood and urine. It involves extraction of both compounds at neutral pH, either from blood into ethyl acetate (the residue of which is dissolved in either) or from urine directly into ether. Both the alcohol and the acid are separated from each other by selective extraction into acid or base, respectively, and then reextracted into either from the respective aqueous medium by appropriate pH adjustment. The residues of the ether extracts containing the compounds are dissolved separately in 0.25 N NH4OH. Methylene blue is added to all samples, which are then exposed to UV energy for 15 min to produce the fluorophores. The fluorescence of the solutions is read at 370 nm, with excitation at 340 nm. The linear range of quantitation of both compounds is 0.02-10 mug/each/ml of final solution. The method was applied to the determination of blood levels and urinary excretion of the alcohol and its acid metabolite in a dog.

Luminescence analysis of anti-inflammatory agents in blood or plasma following thin-layer chromatographic separation.

The luminescence properties of some anti-inflammatory agents and of 6-chloro-1,2,3,4-tetrahydrocarbazole-2-carbox-ylic acid, 6-chloro-9-[2-(2-methyl-5-pyridyl)ethyl]-1,2,3,4-tet-rahydrocarbazole-2-methanol.HCl, and (d,l)-6-chloro-alpha-methyl-carbazole-2-acetic acid have been examined at both ambient and cryogenic temperatures. Assays were developed for indomethacin, the tetrahydrocarbazoles, and the carbazole based upon solvent extraction from blood or plasma, thin-layer chromatographic separation of the drugs from interfering materials and phosphorimetry of the eluted materials. The solvent elution may be accomplished either by manual scraping and elution or by the use of a semiautomated elution apparatus. These two techniques were compared with respect to overall recovery and precision using both fluorometry and phosphorimetry.

Determination of the anti-allergenic agent, 2-methoxy-11-oxo-11H-pyrido[2,1-b]quinazoline-8-carboxylic acid, in biological fluids by high-performance liquid chromatography.

A rapid, sensitive, and specific high performance liquid chromatographic (HPLC) assay was developed for the determination of 2-methoxy-11-oxo-11H-pyrido-[2,1-b]quinazoline-8-carboxylic acid (I) from biological fluids. The overall recovery from blood and plasma is 69 +/- 10% (S.D.) and 84 +/- 6% (S.D.), respectively, and the sensitivity limit of quantitation is 100 ng/ml by UV absorption and 5 ng/ml by fluorescence detection using a 1 ml specimen. The assay was used in the determination of blood levels of compound in the Rhesus monkey following intravenous administration of a 10 mg/kg dose, and of blood and urine levels of compound I in a dog following intravenous and oral administration of a 1 mg/kg dose.

Determination of diclofensine, an antidepressant agent, and its major metabolites in human plasma by high-performance liquid chromatography with fluorometric detection.

A sensitive and selective high-performance liquid chromatographic assay was developed for the determination of diclofensine (I) and its key metabolites in human plasma. The assay involves deproteinization of plasma, overnight Glusulase incubation to hydrolyze the major metabolite (I-B-glucuronide), extraction of the parent compound and its deconjugated metabolites (I-A, I-B and I-C) from the alkalinized aqueous phase into diethyl ether-ethanol (95:5), the residue of which (containing compounds I, I-A, I-B and I-C) is alkylated with 2-iodopropane dissolved in acetone, using solid potassium hydroxide as a catalyst. The compounds are extracted from the reaction mixture into diethyl ether, after adding ethanol-water-acetic acid (55:40:5), the residue of which is dissolved in 0.05 M sulfuric acid, and reacted with mercuric acetate at 100 degrees C, which oxidizes tertiary tetrahydroisoquinolines to their 3,4-dihydroisoquinoline derivatives, followed by a photochemical reaction in the same solution to form intensely fluorescent isoquinolinium derivatives. An aliquot of this reaction mixture is injected onto a reversed-phase high-performance liquid chromatography column (5-microns Nova-Pac C13 phase in a radial compression cartridge, 10 cm X 8 mm), using the mobile phase 0.25 M triethylammonium phosphate (pH 2.5)-0.25 M acetic acid-methanol-acetonitrile-tetrahydrofuran (150:350:125:375:25). The void volume (Vo) is approximately 1.4 min and the retention times (tR) of the respective isoquinolium derivatives of diclofensine (I) are ca. 3.5 min, internal standard (II) ca. 4.2 min, nordiclofensine (I-A) ca. 5 min, while the phenolic metabolites I-B and I-C give peaks at 6.4 min and 10.4 min, respectively. The derivatives are detected by fluorescence. The method was used to determine plasma concentrations of the parent drug (I) and its major phenolic metabolite I-B (aglycone) in plasma in two normal volunteers following a single oral 45-mg dose and following seven consecutive days of oral dosing of 45 mg three times a day as part of a multiple ascending dose tolerance study.

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H- pyrido [2,1-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection.

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[ 2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with acetonitrile--methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido [2,1-b]-quinazoline-8-carboxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 +/- 8.6% and 107.0 +/- 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I . 2HCl, a 10 mg/kg oral dose of I . 2HCl and of metabolite I-A.

Determination of the anti-tumor agent, 10-chloro-5-(2-dimethylaminoethyl)-7h-indolo[2,3-c] quinoline-6(5h)-one. HCl in blood or plasma by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic assay was developed for the determination of 10-chloro-5-(2-dimethylaminoethyl)-7H-indolo [2,3-C] quinolin-6(5H) one [I] in blood or plasma with an overall recovery of 100.3 +/- 9.1% and a limit of quantitation of 1.0 ng per ml of blood or plasma. the assay was used to determine blood concentrations of the drug in the rat following oral administration by intubation of a 1.17-mg dose of [I] . HCl.

Determination of coumermycin A1 in plasma by reversed-phase high-performance liquid chromatographic analysis.

Coumermycin A1 is an antibiotic isolated from Streptomyces hazeliensis var. hazeliensis nov. sp. as a sodium salt which exhibits antistaphylococcal activity. A sensitive and selective high-performance liquid chromatographic method was developed for the determination of the compound and three known homologues which are extracted from plasma buffered to pH 6.5 into methyl-tert.-butyl ether-2-propanol (97.5:2.5), the residue of which is dissolved in the mobile phase and analyzed by automated reversed-phase high-performance liquid chromatography using UV detection at 330 nm for quantitation. Novobiocin is used as the internal standard. The method was used to determine the plasma concentration--time profile of coumermycin A1 in the dog following a single intravenous administration of a 12 mg/kg dose of a solubilized dosage form of the bulk drug substance.

Determination of bromo-lasalocid in plasma by high-performance liquid chromatography with fluorimetric detection.

A rapid, sensitive, and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of bromo-lasalocid in plasma. The compound was extracted into isooctane-ethyl acetate (90:10) from plasma saturated with potassium chloride and adjusted to strongly alkaline pH. The residue of this extract was dissolved in methanol-2-methoxyethanol (95:5) and analyzed by HPLC on a 10-micrometer C18 column [mobile phase of methanol-water-2-methoxyethanol-1 M potassium phosphate buffer, pH 3.0 (90:10:2.5:0.2)] using fluorescence detection with excitation at 215 nm and emission at wavelengths greater than 370 nm. The overall recovery of the assay was 65%, with a limit of sensitivity of 0.1 microgram/ml. The method was used to obtain plasma concentration-time profiles in the dog following oral administration of bromo-lasalocid-ethanolate.