Norman Strojny

Determination of Chlordiazepoxide and Its Metabolites in Plasma by High Pressure Liquid Chromatography

Abstract A rapid, sensitive and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of chlordiazepoxide and its metabolites from plasma. The assay involves extraction of chlordiazepoxide and its metabolites into diethyl ether from plasma buffered to pH 9. The overall recovery of chlordiazepoxide is 80 ± 5.0% (S.D.) and the sensitivity limit of detection is 50 to 100 ng/ml of plasma, using a 1 ml specimen. The assay was used in the determination of plasma levels of chlordiazepoxide and its metabolites in man following oral administration of chlordiazepoxide. HCl. The chromatographic behavior of other clinically important benzodiazepines and their major metabolites is also reported.

Determination of chlordiazepoxide, diazepam, and their major metabolites in blood or plasma by spectrophotodensitometry.

An analytical procedure was developed for the determination of chlordiazepoxide, diazepam and their major metabolites in blood or plasma. Demoxepam, a metabolite of chlordiazepoxide, is determined by spectrofluorometry after extraction. The remaining compounds are determined by spectrophotodensitometry after thin-layer chromatographic separation. The sensitivity limit of the spectrofluorometric determination of demoxepam is 0.1 to 0.2 microgram while that of the spectrophotodensitometric determination of chlordiazepoxide, diazepam and their N-desmethyl metabolites is 0.05 to 0.2 microgram. The sensitivity and specificity of the assay renders it suitable for monitoring plasma levels of chlordiazepoxide and its major metabolites following single or chronic oral administration of chlordiazepoxide hydrochloride. The sensitivity limit for diazepam and nordiazepam, its major metabolite, renders the assay useful only for the determination of plasma concentrations resulting from high dosage of diazepam. The assay was used to determine chlordiazepoxide and its metabolites following oral administration of Librium. The data showed a significant correlation to those obtained on the same specimens by differential pulse polarography and by radioimmunoassay.

Determination of (d,l)-6-chloro-α-methylcarbazole-2-acetic acid in blood and urine by spectrofluorimetry and electron-capture gas chromatography

Abstract A sensitive and specific fluorimetric assay was developed for the determination of (d,l)-6-chloro-α-methyl-carbazole-2-acetic acid [I] in blood and urine, based on the intrinsic fluorescence of the compound in 1%, acetic acid in ethanol. The compound can also be determined by gas chromatography based on the response of its methyl ester to electron-capture detection. A t.l.c. step is used in the assay, not only to ensure specificity, but also as a necessary clean-up step. The fluorimetric method was applied to the determination of blood levels and the urinary excretion of the compound in a dog after intravenous administration of a single 10 mg kg-1 dose.

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.

The determination of 2-hydroxynicotinic acid and its major metabolite in blood and urine by spectro-photofluorimetry and differential pulse polarography

Abstract A sensitive and specific spectrofluorimetric assay was developed for the determination of 2-hydroxynicotinic acid (1) and its major metabolite, the N-1-riboside (11) in blood and urine. Both compounds exhibit strong fluorescence in acidic media. Thin-layer chromatography is employed to separate the drug from its metabolite; the compounds are eluted from the silica gel into methanol—0.1 M hydrochloric acid (80+20). The sensitivity is 0.04–0.05 μg of I and 0.10–0.12 μg of II per ml of blood or urine. The two compounds can also be determined by differential pulse polarography, which is especially suitable for urine. The fluorimetric method was applied to the determination of blood levels and the urinary excretion of the drug in man after single oral 500-mg doses.

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 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.

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 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.

Laser-Induced Fluorimetric Analysis of Drugs in Biological Fluids

Laser-induced molecular luminescence is an exciting new frontier in analytical chemistry. The monochromaticity, coherence, and peak power output of pulsed lasers are all desirable characteristics for fluorescence excitation (1). Tunable dye lasers are of particular interest and applicability to pharmaceutical analysis because they are capable of exciting aromatic molecules over the wavelength range of 360–650 nm by the use of fundamental radiation (2), and over the range of 220–360 nm by the use of second harmonic generation (frequency doubling).