Simultaneous high-performance liquid chromatography determination of carbamazepine and five of its metabolites in plasma of epileptic patients.

A high-performance liquid chromatographic method with UV detection for the simultaneous analysis of the antiepileptic drug carbamazepine and five of its metabolites in human plasma has been developed. The analysis was carried out on a reversed-phase column (C8, 150x4.6 mm I.D., 5 microm) using acetonitrile, methanol and a pH 1.9 phosphate buffer as the mobile phase. Under these chromatographic conditions, carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine are baseline separated in less than 18 min. The extraction of the analytes from plasma samples was performed by means of an original solid-phase extraction procedure using Oasis HLB cartridges. The method requires only 250 microl of plasma for one complete analysis. The repeatability (RSD%<2.4), intermediate precision (RSD%<3.5) and extraction yield (84.8-103.0%) were very good for all analytes. The method is suitable for reliable therapeutic drug monitoring of patients undergoing chronic treatment with carbamazepine and for kinetic-metabolic studies of this drug.

A sensitive high-performance liquid chromatographic method using electrochemical detection for the analysis of olanzapine and desmethylolanzapine in plasma of schizophrenic patients using a new solid-phase extraction procedure.

A high-performance liquid chromatographic method with amperometric detection for the analysis of the novel antipsychotic drug olanzapine and its metabolite desmethylolanzapine in human plasma has been developed. The analysis was carried out on a reversed-phase column (C8, 150 x 4.6 mm I.D., 5 microm) using acetonitrile-phosphate buffer, pH 3.8, as the mobile phase. The detection voltage was + 800 mV and the cell and column temperature was 30 degrees C. The flow-rate was 1.2 ml min(-1). Linear responses were obtained between 5 and 150 ng ml(-1), with repeatability <3.3%. A careful pretreatment of the biological samples was implemented by means of solid-phase extraction (SPE) on C8 cartridges. The method requires 500 microl of plasma for one complete analysis. Absolute recovery exceeded 97% for both olanzapine and desmethylolanzapine, and the detection limit was 1 ng ml(-1) for both analytes. Repeatability, intermediate precision and accuracy were satisfactory. This sensitive and selective method has been successfully applied to therapeutic drug monitoring in schizophrenic patients treated with Zyprexa tablets.

Quantitation of olanzapine in tablets by HPLC, CZE, derivative spectrometry and linear voltammetry.

Four analytical methods have been developed for the quality control of pharmaceutical formulations containing the novel antipsychotic drug, olanzapine: high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), derivative spectrometry and linear voltammetry. All methods require only a simple extraction procedure of olanzapine from the tablets before analysis. HPLC with ultraviolet detection at 260 nm is carried out with a C8 column and a mobile phase constituted of acetonitrile and aqueous tetramethylammonium perchlorate. CZE is performed in an uncoated capillary with phosphate buffer, pH 3.0, as the background electrolyte, with UV detection at 214 nm. Spectrophotometry uses the derivative of the spectrum at 298 nm. In linear voltammetric method (LSV) the current intensity of the oxidation wave at +495 mV is measured. All methods gave similar results in terms of precision and accuracy. For HPLC and CZE, repeatability and intermediate precision, expressed by the RSD was better than 1.8%. The accuracy, resulting from recovery experiments, was between 99.9 and 101.1%. Spectrometry and voltammetry gave slightly higher RSD values (up to 2.9%) and a larger variation of the accuracy (the recovery was between 97.8 and 102.6%). However, the requirements for quantitative analysis are fulfilled for all methods.

A rapid LC method for the identification and determination of CNS drugs in pharmaceutical formulations.

Antidepressant, neuroleptic and antiepileptic drugs were identified and determined in pharmaceutical formulations (tablets, capsules and oral solutions) by a rapid high-performance liquid chromatography method. The sample pretreatment consisted of a one-step extraction, filtration and dilution. The chromatographic conditions were: reversed-phase C8 column (150 x 4.6 mm i.d., 5 microm); acetonitrile-tetramethylammonium perchlorate aqueous solution (pH 2.8; 12.6 mM) (45:55, v/v) as the mobile phase; detection wavelength, 230 nm. Calibration curves were linear in the 100-1000 ng ml(-1) range for all tested drugs except for phenobarbital. The repeatability (or intra-day precision), expressed by the relative standard deviation, was better than 2.0%. The accuracy, resulting from recovery studies, was between 98.1 and 101.3%. The amount of drug found agreed with the declared content within the limits specified by United States Pharmacopeia and British Pharmacopeia.

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection.

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92-98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.

Determination of fluoxetine and norfluoxetine in human plasma by high-pressure liquid chromatography with fluorescence detection.

Fluoxetine is an atypical antidepressant drug, which selectively inhibits the neuronal reuptake of serotonin, and is widely used in the treatment of depressive disorders. The aim of this research is the development of an HPLC method with fluorescence detection for the monitoring of fluoxetine plasma levels. The determination requires no more than 250 microl of plasma, which undergo solid phase extraction (SPE), then are injected in the HPLC. For the analytical separation a reversed phase C8 column (150 x 4.6 mm I.D.) was used, while the mobile phase was a mixture of acetonitrile and water containing perchloric acid and tetramethylammonium perchlorate (flow rate: 1 ml min(-1)). The very low levels of analytes in plasma required the employment of a fluorescence detector (lambda(exc) = 230 nm, lambda(em)=290 nm), which also granted a good selectivity. Fluoxetine is revealed as a single peak at a retention time of 9.7 min, while norfluoxetine, the main metabolite of fluoxetine, is revealed at a retention time of 8.1 min. Linearity was obtained over the concentration range 8-200 ng ml(-1) for both substances. The method seems suitable, in accuracy and precision, for the determination of fluoxetine plasma levels of patients; furthermore, it is rapid and sensitive.

HPLC determination of glutathione and other thiols in human mononuclear blood cells.

A simple and sensitive HPLC method is proposed for the determination of glutathione (GSH) in human mononuclear cells, based on the derivatization of the tripeptide with Ellman's reagent. The mobile phase was composed of a mixture of methanol and ammonium formate (10:90 v/v, with a flow rate of 1 mL/min). The stationary phase was a C18 (4.6 microns, 250 x 4 mm) reversed phase column. The detection of GSH was performed at 280 nm, resulting in a neat chromatographic peak at 5.8 min. A calibration curve showed good linearity over the concentration range 3 x 10(-6) - 6 x 10(-5) M, with a satisfactory precision. The method was found to yield a quantitative recovery of glutathione (96%), to be sensitive (down to 30 pmol of glutathione per injection) and to have a high precision (R.S.D.% approximately equal to 2). The proposed HPLC method allows for the separation and quantitation of cysteine and N-acetylcysteine, if present in biological samples. Furthermore, the method allows for the determination of total thiol present in human mononuclear cells.

Analytical methods for the quality control of Prozac capsules.

Some analytical methods (two spectrophotometric and two chromatographic procedures) for the determination of fluoxetine in Prozac capsules are described. All of them are applied to the samples after extracting the drug with a methanol water mixture. The direct and derivative spectrophotometric methods are simple and reliable; the derivative method gives better recovery and lessens interference. Both methods show linearity in the 5-30 microg ml(-1) range of the fluoxetine concentration range. Both HPLC methods (spectrophotometric and spectrofluorimetric detection) use a tetramethylammonium perchlorate buffer-acetonitrile mixture as the mobile phase and a C8 reversed phase column. The UV detection is performed at 226 nm, while the fluorimetric detection is performed by exciting at 230 nm and revealing the emission at 290 nm. The HPLC method with UV detection is more precise, but the procedure with fluorimetric detection is more sensitive.