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.     

Sub-nanogram analysis of yohimbine and related compounds by high-performance liquid chromatography

A sensitive (50 pg/ml) method is described for the analysis of yohimbine in blood by high-performance liquid chromatography with fluorescence detection. The chromatographic behaviour of eserine (employed as internal standard), reserpine, corynanthine, yohimbinic acid, and yohimbine are examined on a series of reversed-phase and normal-phase chromatographic columns with methanol-water mobile phases.     

Determination of piribedil and its basic metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of piribedil and its p-hydroxylated, catechol and N-oxide metabolites in plasma is described. After addition of an internal standard (buspirone), the plasma samples were subjected to a three-step extraction procedure. The final extracts were evaporated to dryness under nitrogen, and the residues were reconstituted in 100 microliters of mobile phase (0.01 M phosphate buffer-acetonitrile, 50:50, v/v) and chromatographed by acetonitrile gradient elution on a C18 reversed-phase column coupled to an ultraviolet detector set at 240 nm. The method was selective for piribedil and its metabolites, and sufficiently sensitive and precise for studies aimed at elucidating the role of the metabolites in the parent drug's pharmacological effects.     

Simultaneous analysis of prednisone, prednisolone and their major hydroxylated metabolites in urine by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the simultaneous determination of prednisone, prednisolone and their major hydroxylated metabolites, viz., 20 beta-hydroxyprednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone, in human urine is presented. The retention times were 6.5, 11.4, 18.1, 24.2, 31.6 and 35.3 min, respectively. The technique employs betamethasone as the internal standard. Samples are extracted with ethyl acetate using a diatomaceous earth extraction column, and the extract was dried and injected onto a silica gel column with ultraviolet detection at 254 nm. The calibration curve is linear within the studied range 50-1500 ng/ml for prednisolone and 50-750 ng ml for the other steroids. The intra-day and inter-day coefficients of variation are less than 10% for prednisone and prednisolone but higher for the metabolites. The assay was used to study the excretion rate profile of each of these steroids in the urine of a normal male subject receiving a 49.3-mg intravenous dose of prednisolone. The results indicate that prednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone may be the major unconjugated metabolites of prednisolone while 20 beta-hydroxyprednisone may be a minor metabolite.     

Determination of piroxicam and its major metabolites in the plasma, urine and bile of humans by high-performance liquid chromatography.

A simple and sensitive liquid chromatographic method with ultraviolet detection is described for the determination of the nonsteroidal anti-inflammatory drug piroxicam and its major metabolites in human plasma, urine and bile. Separation of these components occurs on a reversed phase C10CN column with a mobile phase consisting of acetonitrile-water-sodium dihydrogenphosphate solution. The detection limit of the assay was 50 ng/ml with intra- and inter-assay coefficients of variation for piroxicam of the order of 2 and 5%, respectively. The assay linearity was good (typically r = 0.9999). This method can be readily utilised for clinical pharmacokinetic and mass-balance studies.     

Determination of nizatidine and two of its main metabolites in human serum using high-performance liquid chromatography

A high-performance liquid chromatographic assay has been developed for the determination of nizatidine, a new histamine H2-receptor antagonist, and two of its main metabolites, N-desmethylnizatidine and nizatidine sulphoxide. Drugs were extracted with chloroform-2-propanol (90:10, v/v) from alkalinized samples of serum, using ranitidine as an internal standard. After evaporation of the extraction solvent, the residue was removed and analysed on a LiChrosorb Si60 5-microns column with a mobile phase of acetonitrile-methanol-water-ammonia solution (1000:200:20:5, v/v). The compounds were detected at 320 nm. The lower detection limits were 6-18 ng/ml at a signal-to-noise ratio of 3. This method is simple and specific, and the single-step extraction makes it rapid. It is the first high-performance liquid chromatographic assay to be described for the determination of nizatidine metabolites.     

Determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography.

A method for the identification and determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography was developed. Tannic acid and four phenolic compounds, namely gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid, were successfully extracted from the biological fluids by using ethyl acetate at acidic conditions. Gallic acid, pyrogallol and 4-O-methylgallic acid were found in the sheep urine, gallic acid, 4-O-methylgallic acid and ellagic acid in plasma, and gallic acid and ellagic acid in abomasal fluid after abomasal dosing of tannic acid. Tannic acid was found in the plasma apart from the abomasal fluid into which it was administered. The concentrations of tannic acid, gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid in plasma, abomasal fluid and urine were measured. This method could be applied to measurement of other hydrolysable tannins and their phenolic metabolites in biological materials.     

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.     

Analysis of phenprocoumon and its hydroxylated and conjugated metabolites in human urine by high-performance liquid chromatography after solid-phase extraction

The anticoagulant phenprocoumon is mainly metabolized in humans to hydroxylated metabolites and their glucuronides. A method is described for the determination of phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon, 7-hydroxyphenprocoumon, and their glucuronide and sulphate conjugates in human urine. Reversed-phase high-performance liquid chromatography is performed after selective extraction with disposable quaternary amine columns of untreated, and beta-glucuronidase- or sulphatase-treated urine samples. Urinary excretion data are presented for total, glucuronidated, sulphated and free phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon and 7-hydroxyphenprocoumon in twelve patients after an average daily dosage of 1.3-4.2 mg phenprocoumon.     

Determination of the serum concentration of spironolactone and its metabolites by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic assay is described for the simultaneous determination in serum of the aldosterone antagonist spironolactone and its metabolites 7 alpha-thiomethylspirolactone, 6 beta-hydroxy-7 alpha-thiomethylspirolactone and canrenone. Ultraviolet absorption at 240 nm was used to detect the different compounds after elution on a normal-phase column. Endogenous serum substances did not interfere with the assay. This method provides a convenient tool in pharmacokinetic studies of spironolactone, in contrast to previously reported aspecific fluorimetric assays or time-consuming thin-layer chromatographic analyses of radioactive biological material.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous determination of tanshinone IIA and its three hydroxylated metabolites by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of tanshinone IIA and its three hydroxylated metabolites, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, in a rat liver microsome was developed and fully validated. A single step of liquid-liquid extraction with ethyl acetate was utilized in this method. Chromatographic separation of the sample matrix from the analytes and the internal standard diazepam was performed using a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1-500 ng/mL for all analytes. The intra- and inter-day precisions (RSD %) were within 15% and deviations of the assay accuracies were within 15.0% for all analytes. The analytes proved to be stable during sample storage, preparation and analyses. This validated method was successfully applied to the enzyme kinetic study of tanshinone IIA in liver microsome. The elimination of tanshinone IIA and formation of tanshinone IIB and hydroxytanshinone IIA in the liver microsome all exhibited a sigmoidal kinetics profile. The formation of przewaquinone A shows a typical hyperbolic profile. In addition, this method has now been applied in the analysis of other bio-samples including plasma, urine, bile and feces.     

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.