Determination of alpidem and its metabolites in human plasma by high-performance liquid chromatography and fluorimetric detection

A high-performance liquid chromatographic method has been developed for the simultaneous determination of alpidem and its metabolites in human plasma. The method involved a single extraction of the parent drug and metabolites into diethyl ether from alkalinized plasma, evaporation of the organic solution and chromatography of the extracts on a C18 column coupled to a fluorimetric detector. An internal standard was used for the quantitative determination of the compounds. The method was selective for alpidem and three of its metabolites and has a limit of detection of less than 1 ng ml-1 for all the compounds. Since the chromatographic run took more than 20 min, the chromatographic process was fully automated and performed overnight.     

Determination of diazepam and its metabolites by high-performance liquid chromatography and thin-layer chromatography

A sensitive, simple high-performance liquid chromatographic assay, capable of simultaneously measuring diazepam, its active metabolites oxazepam, temazepam and N-desmethyldiazepam and two phenyl hydroxylated metabolites, 4'-hydroxy-N-desmethyldiazepam and 4'-hydroxydiazepam, is described. The assay is easily modified to include separation of additional metabolite(s), e.g. oxazepam glucuronide(s). A thin-layer chromatographic assay, which resolves diazepam, the active metabolites and the two phenyl hydroxylated derivatives in one solvent system, is also reported. Application of these procedures to the quantitation of diazepam and its metabolites was shown, after delivery of diazepam (5 micrograms/ml or 16 microM) at a constant flow-rate (10 ml/min per liver) through the single-pass perfused rat liver preparation. Blood perfusion medium and bile were analysed for parent drug and metabolites before and after enzyme hydrolysis. These assay methods are found to be particularly pertinent and useful in providing a more comprehensive metabolic profile of diazepam metabolism, especially when aromatic hydroxylation pathways predominate.     

Determination of bromazepam in plasma and of its main metabolites in urine by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bromazepam in plasma and of its main metabolites in urine is described. The unchanged drug is extracted from plasma with dichloromethane, using Extrelut 1 extraction tubes. The residue from this extract is subsequently analysed by reversed-phase high-performance liquid chromatography with ultraviolet detection (230 nm). The limit of detection is 6 ng/ml of plasma, using a 1-ml specimen. For the determination of the metabolites, the urine samples are incubated to effect enzymatic deconjugation and are then extracted with dichloromethane. Following two clean-up steps (back extractions), the final residue is analysed on the same reversed-phase system as the plasma samples. The limit of detection for the two metabolites is 200 ng/ml.     

Determination of lansoprazole and its metabolites in plasma by high-performance liquid chromatography using a loop column.

A high-performance liquid chromatographic method for the simultaneous determination of lansoprazole, a new proton pump inhibitor, and its metabolites in human plasma is described. Lansoprazole, its metabolites and an internal standard were extracted with tert.-butyl methyl ether. Samples were injected using an automatic injector via a loop column, and separation was obtained using a reversed-phase column under isocratic conditions. The absorbance was monitored at 285 and 303 nm. The quantification limit was 2 ng/ml for lansoprazole and 3 or 5 ng/ml for the metabolites. No endogenous compounds were found to interfere. The mean overall recovery was between 75 and 95% for lansoprazole and its metabolites. This method is suitable for pharmacokinetic studies.     

Determination of diclofenac and its metabolites in plasma and cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitation of diclofenac and metabolites in plasma and cerebrospinal fluid has been developed. Pirprofen is employed as internal standard. Samples are extracted with C18 solid-phase extraction columns and eluted with methanol. Oxidation potentials for detection were established by constructing voltammograms for each compound. In the concentration range found in human studies, the intra-day coefficients of variation were always less than 6%. The procedure allows the simultaneous determination of diclofenac and its four major metabolites with very low detection limits (less than 1 ng/ml), which were sufficient even for kinetic studies in cerebrospinal fluid.     

Measurement of dothiepin and its major metabolites in plasma by high-performance liquid chromatography.

This paper describes a reversed-phase high-performance liquid chromatographic method which will simultaneously measure dothiepin and its three major metabolites (northiaden, northiaden-S-oxide and dothiepin-S-oxide) in plasma using trimipramine as internal standard. Sample preparation involved a basic extraction using diethyl ether followed by an acid back-extraction. The method we report is linear over the range 50-1000 ng/ml (r = 0.999), for all analytes. Total imprecision is less than 11% (coefficient of variation) and accuracy is greater than 94% (n = 20). Recovery of analytes varied considerably from 51.7% for northiaden-S-oxide to 90.2% for dothiepin-S-oxide.     

Determination of paracetamol and its four major metabolites in mouse plasma by reversed-phase ion-pair high-performance liquid chromatography.

A reversed-phase ion-pair high-performance liquid chromatographic method has been used for the separation of paracetamol and its four major metabolites (glucuronide, sulphate, cysteine and mercapturate conjugates) in mouse plasma samples. An ODS column was used and the mobile phase consisted of an aqueous solution of 0.01 M tetrabutylammonium chloride and 0.01 M Tris buffered to pH 5.0 with phosphoric acid, with methanol as the organic solvent. The gradient elution started with 30% methanol. After a delay of 0.5 min the methanol concentration was increased linearly to 75% over 7.5 min. The column was returned to the initial conditions after a delay of 1 min. A methanol solution of theophylline was added to the mouse plasma sample, centrifuged and immediately injected into the chromatographic system. The advantages of this method include good and rapid separation (last metabolite detected at 6.86 min), well resolved peaks, only a small amount of sample required for assay, adequate precision (no coefficient of variation was greater than 10% for paracetamol metabolites) and a high sensitivity (particularly for unchanged paracetamol and the cysteine conjugate).     

Determination of ibuprofen and its major metabolites in human urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic assay for free and total ibuprofen and its major metabolites in human urine is described. Urine is acidified, drug and metabolites are extracted into hexane-propanol, back-extracted into sodium bicarbonate, neutralized and chromatographed. Ibufenac (4-isobutylphenylacetic acid) and 2-phenylpropionic acid were employed as internal standards. The extraction efficiencies were 94-100% for all compounds. The two metabolites and their internal standard were separated using an isocratic chromatographic system, followed by an abrupt step gradient to a second eluent for separation of ibuprofen and its internal standard with a total run time of 18 min. Detection was by a fixed-wavelength detector (214 nm). Sample-to-sample and day-to-day reproducibility studies yielded coefficients of variability of less than 9% for all compounds. The sensitivity was sufficient to determine 2.5 micrograms/ml free ibuprofen in 100 microliters urine.     

Determination of isoniazid methanesulphonate and its metabolites in rabbit blood by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic method is described for simultaneous determination of isoniazid methanesulphonate (IHMS) and its metabolites, such as isoniazid (INH) and acetylisoniazid (AcINH) in rabbit blood. According to stability studies, IHMS was most stable at pH 3-5. After acidifying the blood to pH 5.0, a suitable amount of acetonitrile was added to the supernatant for extraction and niacinamide served as an internal standard. After evaporation, the residue was reconstituted with phosphate buffer and aliquots of this solution were separated on a reversed-phase phenyl column by a mobile phase consisting of 0.25 mM tetrabutylammonium phosphate as a paired-ion reagent. UV detection was performed at 280 nm. Under these conditions, the between-run coefficients of variation of IHMS, INH and AcINH from 1 to 25 microns/ml were 4.7 +/- 2.5, 5.4 +/- 1.0 and 5.1 +/- 3.1%, respectively. Hence this sensitive, reproducible and accurate method was suitable for pharmacokinetic studies of IHMS.