V. Volterra

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.

Development of Acute Psychotic Disorders and HIV-1 Infection

Objective: To gain more understanding about the relationship between human immunodeficiency virus type 1 (HIV-1) infection and new-onset psychosis, we compared clinical and immunological findings, psychiatric symptoms, global cognitive performance and, when available, computerized tomography (CT) findings between HIV-1-seropositive patients with new-onset psychosis and well-matched nonpsychotic HIV-1-seropositives. Methods: Two groups of subjects: HIV-1-seropositives with new-onset psychosis (n = 12) and HIV-1-seropositives without psychosis (n = 15) were recruited through outpatient departments. Organic Delusional Syndrome and Organic Hallucinosis were clinically diagnosed using DSM-III-R diagnostic criteria. Of the baseline participants, twenty-two participated in the two-year follow-up examination. Results: The prevalence of new-onset psychosis in HIV-1-infected subjects was 3.7 per 100 (95% C.I. = 1.6–5.7). HIV-1-seropositive persons with new-onset psychosis had more frequently a positive past psychiatric history, no antiretroviral therapy, and a lower global cognitive performance than did the nonpsychotic HIV-1-seropositives. CT was positive, showing generalized brain atrophy, in three out of nine patients. Remission of psychotic symptoms was observed only in two HIV-1-seropositive persons with new-onset psychosis. Death occurred in two psychotic HIV-1-seropositives with simple loosely held delusions. Autopsy results showed that cortical sulci and ventricle size were graded as with moderate/severe enlargement. Conclusions: New-onset psychosis in HIV infected patients could raise considerable problems in deciding whether a presentation is organic or functional. An interaction of the disease or of psychologically “having” the disease with the presence of a psychotic reaction should also be considered. Interestingly, a protective effect of antiretroviral therapy for new-onset psychosis is suggested.

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.

Improved HPLC determination of fluoxetine and norfluoxetine in human plasma

SummaryAn HPLC method with fluorescence detection has been developed for the determination of fluoxetine and its main metabolite norfluoxetine in human plasma. Pretreatment of the biological samples by liquid-liquid extraction was used to improve the sensitivity of a previously published SPE procedure. The method uses 200 μL plasma and recovery is good for both analytes. On a C8 column with a mixture of perchlorate buffer and acetonitrile as mobile phase fluoxetine, norfluoxetine and the internal standard (paroxetine) were eluted in less than 9 min, without interference from the biological matrix. Response for both analytes was linearly dependent on concentration over the range 2.5–500 ng mL−1, and repeatability (RSD%) was <4%. The limit of detection was 1 ng mL−1 for both fluoxetines. Application to plasma samples from depressed patients treated with fluoxetine gave good results. There was no interference from other common CNS drugs. This method seems to be a useful tool for clinical monitoring, because it requires small plasma samples and is highly sensitive and highly selective.

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.

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.

Age-dependent changes in the susceptibility to apoptosis of peripheral blood CD4+ and CD8+ T lymphocytes with virgin or memory phenotype.

Susceptibility to apoptosis changes with age and most of the available data on lymphocytes refer to mitogen stimulated cells. We studied this susceptibility in quiescent, purified CD4+ or CD8+ T cells from a group of Italian old people compared with a group of young people. We found that an apoptotic agent such as 2-deoxy-D-ribose (dRib), which acts via glutathione depletion and oxidative stress, was more effective in CD4+ T cells from young donors, while no difference was found in CD8+ T cells. On the contrary, another agent such as TNF-alpha, which acts via receptor engagement, was more effective in CD8+ T cells from old subjects, and no difference was found in CD4+ T cells. When marker of activation-memory were investigated, no difference between young and old subjects was found when dRib was used. Differently, when TNF-alpha was used, memory and activated CD4+ T cells from old donors were less sensitive than younger counterparts, while memory CD8+ T cells from old donors were more sensitive than younger counterparts. This suggests that age-related changes in susceptibility to apoptosis of resting T cells largely depend on the type of the apoptotic stimulus which is used as well as on the memory phenotype of the cells. These results may also account, at least in part, for the deep remodelling of T cell repertoire that occurs during ageing.

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)).

Determination of the Novel Antipsychotic Drug Olanzapine in Human Plasma Using HPLC with Amperometric Detection

SummaryA new sensitive high performance liquid chromatograpic method has been developed for the analysis of the novel antipsychotic drug, olanzapine, in human plasma. Chromatography was performed on a reversed-phase column (C8, 150×4.6 mm i.d., 5 μm) with acetonitrile-phosphate buffer (pH=2.5) as the mobile phase. The detection voltage was +900 mV and the cell and column temperature were 50°C. The flow rate was 1 mL min−1.The method provides a linear response over an olanzapine concentration range of 2–100 ng mL−1. Isolation of olanzapine from plasma was accomplished by a solidphase extraction procedure, using C8 cartridges, which gave high extraction yields and needed a small amount of human plasma (only 0.25 mL). The results obtained analysing plasma samples from patients in therapy with olanzapine were satisfactory in terms of precision and sensitivity, and were compared to those obtained by means of a method based on HPLC with UV detection.