Martin R. Hackman

Determination of flurazepam (dalmaneR) and its major metabolits in blood by electron-capture gas-liquid chromatography and in urine by differential pulse polarography

Abstract A sensitive and specific electron-capture gas chromatographic (EC-GLC) assay was developed for thte determination of flurazepam and its major blood metabolites with a sensitivity limit of 5–10 ng/ml of each compound. The major urinary metabolites are determined by differential pulse polarography. The EC-GLC assay was applied to the determination of blood levels in man following single and multiple 30-mg oral doses of Dalmane. The polarographic assay was used to quantitate the major urinary metabolites in several subjects who received a single 90-mg oral dose of the drug.

Determination of clorazepate and its major metabolites in blood and urine by electron capture gas-liquid chromatography.

A sensitive and specific blood level method employing differential extraction was developed for the determination of clorazepate and its N-desmethyldiazepam metabolite by electron capture gas-liquid chromatography (GLC-ECD). The assay requires the initial extraction of N-desmethyldiazepam, the major metabolite, into benzene-methylene chloride (90:10) from the biological sample made alkaline with 0.1 N NaOH. The samples is then acidified with 2 N HCl to decarboxylate clorazepate to N-desmethyldiazepam, which is then extracted into benzene-methylene chloride (90:10) after adjusting the pH to 12.8 with NaOH. The two extracts are evaporated and the residues are dissolved in benzene which contains griseofulvin as the reference standard. These solutions are assayed by GLC-ECD. The overall recovery and sensitivity limit of the assay for clorazepate is 60+/-5% (S.D.) and 4.0 ng/ml blood, respectively, while that for N-desmethyldiazepam is 95+/-5% (S.D.) and 4.0 ng/ml blood, respectively. The urinary excretion of clorazepate was determined by the measurement of the levels of N-desmethyldiazepam and oxazepam, the major urinary metabolites of clorazepate, both prior to and after enzymatic deconjugation. These methods were applied to the measurement of clorazepate and its metabolites in blood and urine following a single 15-mg dose of clorazepate dipotassium.

N-desmethyldiazepam: a new metabolite of chlordiazepoxide in man.

Following administration of chlordiazepoxide HCI to man, N‐desmethyldiazepam, a known metabolite of diazepam (Valium), was identified in plasma. The metabolite was identified on the basis of its thin‐layer chromatographic (TLC) mobility, electron‐capture gas‐chromatographic (EC‐GC) retention time, and mass spectrum relative to authentic N‐desmethyldiazepam. Plasma levels of N‐desmethyldiazepam in subjects receiving both single and chronic doses of chlordiazepoxide were determined by an EC‐GC method with a limit of sensitivity of 10 ng/ml using 2‐ml samples and by a radioimmunoassay procedure which had a limit of sensitivity of 20 ng/ml using a 0.1‐ml sample. Both assay methods gave good agreement for the levels of N‐desmethyldiazepam. In subjects receiving a single 30‐mg oral or intravenous dose of chlordiazepoxide, measurable levels of N‐desmethyldiazepam in plasma (10 to 60 ng/ml) were obtained 24 to 72 hr after administration. In 5 subjects receiving 10 mg of chlordiazepoxide three times a day, steady‐state levels of N‐desmethyldiazepam in plasma were reached after about 1 wk of administration. The mean maximum and minimum steady‐state levels of N‐desmethyldiazepam were 260 and 220 ng/ml of plasma, respectively. Similar steady‐state levels were observed on treatment with 30 mg of chlordiazepoxide over 24 hr.

A differential pulse polarographic examination of the 1,4-benzodiazepines.

Abstract A differential pulse polarographic examination of 1,4-benzodiazepines structurally related to medazepam. chlordiazepoxide and N-desmethyldiazepam is presented. The data are used to correlate polarographic peak potentials (E p ) and Hammett substituent constants for the three series of compounds. Good correlations were found for the reduction of the 4,5-azomethine functional group for the three series of compounds in pH 3 and pH 7 supporting electrolyte. No correlation could be established for the reduction of the 1,2-azomethine and N 4 -oxide in the series of compounds structurally related to chlordiazepoxide. Polarographic data are also presented for two groups of benzodiazepines structurally related to medazepam and N-desmethyldiazepam with heterocyclic substituents in the 5-position.

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (CipralanTM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20). A 10-microns ion-exchange (sulfonate) column was used with acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard. The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10-1000 ng/ml and 50-5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.

Differential pulse amperometric detection of drugs in plasma using a dropping mercury electrode as a high-performance liquid chromatographic detector

High-performance liquid chromatographic separation prior to reductive electrochemical determination at its dropping mercury electrode imparts specificity and sensitivity not attainable by conventional polarographic analysis of drugs and their metabolites. The utility of this novel approach is demonstrated by the analysis of chlordiazepoxide and its N-desmethyl metabolite in plasma which previously required thin-layer chromatographic separation prior to polarographic measurement. A mobile phase of methanol-isopropanol--0.0075 M acetate buffer, pH 3.5 (53:5:42), is used with the detector operated in the differential pulse mode at Ep = -0.820 V vs. Ag/AgCl. The response was linear (r = 0.998) in the concentration range of 0.05--2.0 micrograms/ml plasma for each component. The minimum detectability for each component under these conditions is 5.0 ng injected at a current range of 0.5 microamperemeter full scale. Techniques for oxygen removal and hydrodynamic consideration for the pumping system are presented.

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.

HPLC Analysis of the Major Urinary Metabolite of Flurazepam

Pharmacokinetic parameters of a drug are commonly evaluated by the determination of unchanged drug in blood or plasma. An acceptable alternative [1], especially if a non-invasive method is desired, is to use urinary excretion data for either the unchanged drug or a major metabolite. If the latter represents a significant percentage of the administered dose, its quantitation places fewer constraints on the sensitivity required of the analytical procedure, and on the sample preparation and ‘clean-up’ required prior to analysis. HPLC, either normal-phase (NP) or reverse-phase (RP), is ideally suited to this type of determination [2]. RP conditions have now been developed.