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).     

Simultaneous determination of trimipramine and its major metabolites by high-performance liquid chromatography

Trimipramine is a tricyclic antidepressant drug often assayed by gas chromatographic or gas chromatographic-mass spectrometry techniques. A high-performance liquid chromatographic method with electrochemical detection is described for the assay of trimipramine and its major metabolites, monodesmethyltrimipramine and 2-hydroxytrimipramine, in plasma. The method is sensitive, accurate and robust and thus suitable for routinely assaying samples following single doses of trimipramine to man. The assay was applied to plasma samples obtained following a single 50-mg dose of trimipramine to healthy volunteers.     

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.     

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 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.     

Simultaneous determination of caffeine and its primary demethylated metabolites in human plasma by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its three primary metabolites (theophylline, theobromine and paraxanthine) in human plasma is described. The four substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 150 mm x 4.6 mm I.D.) by use of the mobile phase methanol-0.1 M NaH2PO4 (30:70, v/v) with a flow-rate of 0.8 ml/min. Absorbance was monitored at 274 nm. The detection limit was 5 ng/ml for theobromine and caffeine and 10 ng/ml for paraxanthine and theophylline. The linearity and reproducibility were sufficient for drug monitoring of caffeine and its primary methylxanthines.     

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.     

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.     

Measurement of epidoxorubicin and its metabolites by high-performance liquid chromatography using an advanced automated sample processor.

A sensitive and rapid method for measuring epidoxorubicin and its six metabolites by high-performance liquid chromatography using an advanced automated sample processor is described. Plasma samples (1 ml) were extracted using C2 cassettes, and reversed-phase chromatography was performed with an Apex II ODS column. The isocratic mobile phase of acetonitrile-0.019 M NaH2PO4 (pH 4.0) had a flow-rate of 1 ml/min and the fluorescence detector an excitation wavelength of 480 nm with an emission at 580 nm. Linear calibration curves were obtained which were reproducible both within-day and day-to-day (coefficients of variation less than 10%). The extraction efficacy of epidoxorubicin was 88% and ranged from 51 to 88% for the metabolites. This method has been successfully applied to measure the plasma levels of these compounds in patients receiving epidoxorubicin over a wide dose range (12-120 mg/m2) and in patients with disturbed liver biochemistry.