Cesare Sabbioni

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.

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.

Separation and analysis of glycyrrhizin, 18β-glycyrrhetic acid and 18α-glycyrrhetic acid in liquorice roots by means of capillary zone electrophoresis

Abstract Glycyrrhizin is the main active compound of Glycyrrhiza glabra root extracts; according to recent studies, glycyrrhizin and its aglycon, glycyrrhetic acid, have interesting therapeutic properties. A new capillary electrophoretic method has been developed for the separation and quantification of glycyrrhizin, β-glycyrrhetic acid and its isomer α-glycyrrhetic acid. Separation of the analytes was achieved in less than 3min on a fused silica capillary, by injecting the samples at the short end of the capillary (effective length: 8.5cm). The background electrolyte was composed of pH 10.0 carbonate buffer, methanol and ethylene glycol (80/10/10) and contained 0.4% β-cyclodextrin; indomethacin was used as the internal standard. Diode array detection was used, with quantitative assays carried out at 254nm. Linearity was found over the 5–200 and 2.5–100μgmL−1 concentration ranges for glycyrrhizin and glycyrrhetic acid, respectively. This method has been applied to the determination of the analytes in different matrices (liquorice roots and commercial confectionery products), and to the purity control of β-glycyrrhetic acid obtained from the hydrolysis of glycyrrhizin. When analysing β-glycyrrhetic acid and its epimer in roots, the samples were purified by means of a suitable solid-phase extraction (SPE) procedure with Oasis HLB cartridges, which granted good selectivity, eliminating matrix interference.

Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products

Abstract Glycyrrhizin (1), the main active principle of Glycyrrhiza glabra (liquorice) roots, is extensively used in herbal medicines, in pharmaceutical preparations and confectionery products. A feasible and reliable method which allows the simultaneous analysis of 1 and its aglycone, 18β‐glycyrrhetic acid (2), by means of an isocratic HPLC procedure is described. The system uses a C8 column as the stationary phase, and a mixture of acetonitrile, methanol, water and glacial acetic acid as the mobile phase. Good linearity was found in the concentration ranges 1–50 and 0.05–2.50 µg/mL for 1 and 2, respectively. A simple and rapid sample pre‐treatment, based on the extraction of the two analytes with a mixture of water and ethanol, was developed for the examination of liquorice confectionery products and root samples. The HPLC method was shown to be appropriate, in terms of precision and feasibility, for the quality control of the analytes in these matrices. Copyright

DEVELOPMENT OF AN HPLC METHOD FOR THE TOXICOLOGICAL SCREENING OF CENTRAL NERVOUS SYSTEM DRUGS

A simple and sensitive HPLC (high performance liquid chromatography) method has been developed for the qualitative and quantitative analysis of several CNS (central nervous system) drugs in clinical and forensic toxicology. The leading conditions were studied, namely parameters such as mobile phase pH, organic modifier percentage, and salt concentration. An isocratic HPLC elution, using a mobile phase composed of acetonitrile and pH 2.8 aqueous tetramethylammonium perchlorate and a C8 reversed phase column as the stationary phase, was found to be convenient for the separation of several CNS drugs, and for their detection and quantitation. The identification of the drugs was assured using their relative retention times, together with the peak area ratios at two different wavelengths (230 and 270 nm). A quick pre-treatment of the plasma samples, based on a SPE (solid phase extraction) procedure, with good extraction efficiency and satisfactory selectivity was developed. Under these conditions, a mixture of fifteen CNS drugs (including antipsychotics, antidepressants and antiepileptics) and some selected active metabolites, was well separated for identification and quantitative determination purposes.

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.

Separation of five recently commercialized selective serotonin reuptake inhibitor antidepressants by capillary electrophoresis

Several strategies were applied in order to find a simple electrophoretic method for the investigation of five selective serotonin reuptake inhibitors (SSRI) (citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline). Variation of the pH and of the ionic strength of the background electrolyte (BGE) were studied, but did not enable separation of the analytes. A micellar electrokinetic chromatography (MEKC) strategy for the simultaneous separation of the five SSRIs was developed involving a sodium dodecylsulfate (SDS) MEKC system. The electroosmotic flow (EOF) and the migration of the analytes were determined for separation buffers of several surfactant concentrations and organic modifier percentages. The most favourable MEKC system consisted of 20 mmol/L SDS in a phosphate buffer (pH 7.5) with 30% methanol; the separation was carried out using an uncoated fused-silica capillary, a separation voltage of 25 kV with currents typically less than 40 μA, and spectrophotometric detection at 200 nm. Full separation of the mixture with baseline resolution of all analytes was obtained.

Rapid capillary electrophoretic method for the determination of clozapine and desmethylclozapine in human plasma

A rapid and sensitive high-performance capillary electrophoretic method for the determination of clozapine and its main metabolite desmethylclozapine in human plasma was developed. The separation of the two analytes was carried out in an untreated fused-silica capillary [33 cm (8.5 cm effective length) x 50 microm I.D.] filled with a background electrolyte at pH 2.5 containing beta-cyclodextrin. Baseline separation of clozapine and desmethylclozapine was recorded in less than 3 min. An accurate sample pretreatment by means of solid-phase extraction and subsequent concentration allows for reliable quantitation of clozapine in the plasma of schizophrenic patients under treatment with the drug. The method showed good precision (mean RSD = 4.0%) as well as satisfactory extraction yields (approximately 88%) and a good sensitivity (limit of quantitation = 0.075 microg ml(-1), limit of detection = 0.025 microg ml(-1)).

Quality control of benserazide-levodopa and carbidopa-levodopa tablets by capillary zone electrophoresis

In modern practice, the treatment of Parkinsons disease and syndrome is carried out using pharmaceutical formulations containing a combination of levodopa and a decarboxylation inhibitor (carbidopa or benserazide). Two pharmaceutical formulations were quantified by capillary zone electrophoresis using two procedures which differed only in the kind of background electrolyte used. One procedure used a 25 mM phosphate buffer, pH 2.5, while the second one used a 25 mM borate buffer, pH 8.5. The electrophoretic analysis was carried out using an uncoated fused‐ silica capillary, a separation voltage of 20 kV with currents typically less than 60 μA, and spectrophotometric detection at 205 nm. Calibration curves were performed for levodopa (concentration range 1—100 μg/mL), for carbidopa and benserazide (1—50 μg/mL), and the plots of the peak area versus concentration were found to be linear with a correlation coefficient better than 0.9990. Satisfactory results were obtained when commercial tablets were analyzed in terms of accuracy (98—102%), repeatability (0.6—2.0%), and intermediate precision (1.1—2.6%).

Determination of olanzapine and desmethylolanzapine in the plasma of schizophrenic patients by means of an improved HPLC method with amperometric detection

SummaryAn improved HPLC method with electrochemical detection has been developed for the determination of olanzapine and its main metabolite, desmethylolanzapine, in human plasma. Chromatographic separation and analysis were performed on a C8 reversed-phase column with a mixture of methanol, acetonitrile, and pH 3.7 phosphate buffer as mobile phase; 2-methylolanzapine was used as internal standard. Careful pretreatment of the plasma samples was implemented by means of solid phase extraction (SPE).Response was linearly dependent on concentration and precision was satisfactory over the concentration range 0.5–75.0 ng mL−1 for both analytes. The limit of detection was 0.2 ng mL−1 for both analytes. Application to plasma samples of patients treated with Zyprexa tablets gave good results. Because of its sensitivity and selectivity, and the need for small plasma samples, this method seems to be a useful tool for clinical monitoring.