Michele Protti

Monitoring of chronic Cannabis abuse: an LC-MS/MS method for hair analysis.

An advanced analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been developed for the identification and determination in hair of Δ(9)-tetrahydrocannabinol together with its major metabolite 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol. Since the latter is formed endogenously, it allows the assessment of chronic use excluding passive exposure to Cannabis. The sample pre-treatment procedure is based on a feasible incubative extraction followed by a liquid-liquid extraction step. Chromatographic separation was performed using a reversed-phase column and gradient elution with a formic acid/acetonitrile/water mobile phase. The limits of quantitation and of detection were 3pg/mg and 1pg/mg, respectively, for both analytes. The method was successfully applied to the analysis of hair samples from Cannabis abusers; the analyte concentrations found ranged from 55 to 100pg/mg for Δ(9)-tetrahydrocannabinol and from 5 to 10pg/mg for 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol. Accuracy studies also gave satisfactory results (recovery>87%), thus confirming the suitability of the assay for chronic consumption monitoring.

A fast and feasible microextraction by packed sorbent (MEPS) procedure for HPLC analysis of the atypical antipsychotic ziprasidone in human plasma

An original high-performance liquid chromatographic method coupled to microextraction by packed sorbent (MEPS) was developed for the therapeutic drug monitoring (TDM) of psychiatric patients treated with the recent atypical antipsychotic ziprasidone. The chromatographic separation was achieved on a RP C18 column, using an isocratic mobile phase and setting the wavelength at 320nm. The analyte was extracted from human plasma by means of a fast and feasible innovative MEPS procedure, optimised on C2 sorbent and requiring only 100μL of biological sample. A second pre-treatment procedure, based on solid phase extraction (SPE), has been also developed for comparison. The availability of different pre-treatment procedures allows the choice of the one best suiting the specific clinical, economic and scientific needs. The extraction yield values were always higher than 90% and sensitivity was also good, with a limit of quantitation (LOQ) of 1ng/mL. The method was successfully applied to plasma samples from ten subjects undergoing therapy with ziprasidone, thus confirming its suitability for the TDM of psychiatric patients, in order to personalise their pharmacological treatments.

Δ9‐Tetrahydrocannabinol‐induced anti‐inflammatory responses in adolescent mice switch to proinflammatory in adulthood

Marijuana abuse is prominent among adolescents. Although Δ9‐THC, one of its main components, has been demonstrated to modulate immunity in adults, little is known about its impact during adolescence on the immune system and the long‐lasting effects in adulthood. We demonstrate that 10 days of Δ9‐THC treatment induced a similar alteration of macrophage and splenocyte cytokines in adolescent and adult mice. Immediately at the end of chronic Δ9‐THC, a decrease of proinflammatory cytokines IL‐ 1β and TNF‐α and an increase of anti‐inflammatory cytokine IL‐10 production by macrophages were present as protein and mRNA in adolescent and adult mice. In splenocytes, Δ9‐THC modulated Th1/Th2 cytokines skewing toward Th2: IFN‐γ was reduced, and IL‐4 and IL‐10 increased. These effects were lost in adult animals, 47 days after the last administration. In contrast, in adult animals treated as adolescents, a perturbation of immune responses, although in an opposite direction, was present. In adults treated as adolescents, a proinflammatory macrophage phenotype was observed (IL‐1β and TNF‐α were elevated; IL‐10 decreased), and the production of Th cytokines was blunted. IgM titers were also reduced. Corticosterone concentrations indicate a long‐lasting dysregulation of HPA in adolescent mice. We measured blood concentrations of Δ9‐THC and its metabolites, showing that Δ9‐THC plasma levels in our mice are in the order of those achieved in human heavy smokers. Our data demonstrate that Δ9‐THC in adolescent mice triggers immune dysfunctions that last long after the end of abuse, switching the murine immune system to proinflammatory status in adulthood.

Dried blood spot testing: a novel approach for the therapeutic drug monitoring of ziprasidone-treated patients

BACKGROUNDnA novel analytical approach, based on dried blood spot (DBS) testing, has been developed, validated and applied for the first time to the analysis of ziprasidone (ZPR) for the therapeutic drug monitoring (TDM) of schizophrenic patients. DBS represent a more feasible but reliable matrix, alternative to blood and plasma.nnnMETHODSnThe assays were carried out using an HPLC method with native fluorescence. Blood drops were applied to DBS cards and dried by microwaves, an internal standard solution was added to the DBS and 5-mm punches were cut out for analysis. ZPR was extracted from DBS with methanol, giving good extraction yields, precision and selectivity results.nnnRESULTSnThe method was applied with satisfactory results to DBS samples from psychiatric patients to determine ZPR levels for therapy optimization.nnnCONCLUSIONnThis innovative methodology provides reliable and significant TDM information, with important advantages over classical blood sampling in terms of collection, storage and processing.

Quantitative Evaluation of Auraptene and Umbelliferone, Chemopreventive Coumarins in Citrus Fruits, by HPLC-UV-FL-MS

An analytical strategy, based on the development of two HPLC methods with spectrophotometric (UV), spectrofluorometric (FL), and mass spectrometric (MS) detection, has been developed to investigate the presence of and to quantitate two important chemopreventive coumarins, auraptene and umbelliferone, in foodstuffs. The analytes were determined in fruits, and fruit parts, of plants belonging to the Citrus , Poncirus , and Fortunella genera, to test their nutraceutical potential. The method validation has been carried out according to international guidelines, with good results in terms of precision (RSD < 6.9%) and extraction yields (>91%). Application to the quantitative analysis of auraptene and umbelliferone in several kinds of citrus fruits was successful, providing reliable and consistent data. Exploiting three different kinds of detection, the analytical methodology proposed herein has been demonstrated to be sound but versatile, as well as reliable. Performances and results were compared and always found in good agreement among themselves. Thus, this approach is suitable for the identification and simultaneous quantitation of auraptene and umbelliferone in citrus fruits, with the aim of evaluating their nutraceutical potential.

Evaluation of the pharmacokinetics, safety and clinical efficacy of ziprasidone for the treatment of schizophrenia and bipolar disorder

Introduction: Multiple strategies exist for the pharmacological treatment of schizophrenia and related disorders. In the last 20 years, several ‘new’ compounds have been introduced, called ‘atypical antipsychotics’, which have higher efficacy and better tolerability than first-generation neuroleptics. Among them, ziprasidone (ZPR) is currently finding widespread use, and it has also been shown to be active as an augmenter in bipolar disorder therapy. Areas covered: This review aims to provide the latest information on ZPR, an ‘atypical’ agent for the pharmacological therapy of schizophrenia and bipolar disorder. A literature search has been carried out with the keywords ‘ziprasidone’, ‘schizophrenia’, ‘psychosis’, ‘bipolar’, ‘pharmacokinetics’ and ‘clinical trials’. In this process, particular attention has been paid to the drug pharmacokinetic characteristics and its safety in clinical use. Expert opinion: ZPR shares most advantages and disadvantages with other atypical antipsychotics. However, it can be useful for its low tendency to cause metabolic syndrome and hyperprolactinaemia, especially in patients suffering from excess weight, hyperlipidaemia, diabetes or who have suffered from hyperprolactinaemia when using other antipsychotics. However, there are serious doubts as to whether ZPR should be administered to patients suffering from arrhythmias or QTc prolongation, and even more for administration to bipolar patients undergoing polypharmacy with antidepressants.

Front Cover: Enantioseparation and determination of asenapine in biological fluid micromatrices by HPLC with diode array detection

Asenapine is a recent drug approved in the European Union for the treatment of bipolar disorder. An original approach has been developed for asenapine analysis in patients treated with the drug, including miniaturized microsampling procedures, separation and quantitation of drug enantiomers. An original enantioselective method based on high-performance liquid chromatography with diode array detection was developed and applied to the determination of asenapine enantiomer levels in innovative haematic samples: four micromatrices have been tested, two based on dried matrix spots (dried blood spots and dried plasma spots) and two based on volumetric absorptive microsampling (from blood and plasma). Chiral separation was achieved on a cellulose-tris(3,5 dimethylphenylcarbamate) column, with a mobile phase containing bicarbonate buffer and acetonitrile. The method was validated with satisfactory results of linearity and precision on all matrices that showed also a significant performance in terms of stability, feasibility and reliability, when compared to fluid plasma sampling, handling and processing. Among micromatrices, both volumetric absorptive microsampling types were superior to dried matrix spots in terms of data reproducibility and correspondence with plasma levels. The bioanalytical approach proposed herein provides for the first time a chiral high-performance liquid chromatographic method for the determination of asenapine enantiomers, coupled to a very effective microsampling strategy.

Effects of oral d-aspartate on sperm quality in B6N mice

The goal of this study was to investigate the effects of oral administration of d-aspartate (D-Asp) to sexually immature male C57BL/6NTacCnrm (B6N) mice on the inxa0vitro fertilisation (IVF) rate with cryopreserved-thawed spermatozoa and on cryopreserved sperm quality as well as on LH, epitestosterone, and testosterone levels. Males were treated at 7 weeks of age with a dose of 20u202fmM sodium d-aspartate in drinking water for 1.3, 2, 4 or 6 weeks so that they were 8.3, 9, 11 or 13 weeks of age, respectively, at the end of the study. The timepoints for controls were week 0 (start of experiment), 1.3, 2, 4 or 6, whereby mice received no D-Asp. At each timepoint, spermatozoa were cryopreserved for IVF and testes as well as sera were frozen for hormone level measurement. After 2 and 4 weeks of treatment, the IVF rate was significantly higher in the D-Asp group than in the controls (64% vs. 44% and 52% vs. 38%, respectively). Spermatozoa from D-Asp-treated males showed lower morphological abnormalities than their control counterparts. After 2 and especially after 4 weeks of treatment, the hormone levels in sera and testes and the total and progressive motility of the spermatozoa were higher in D-Asp-treated males. Although we did not elucidate the full mechanism leading to an improved IVF rate with spermatozoa from D-Asp-treated males lower morphological abnormalities and increased motility contribute to this observation. Importantly, D-Asp significantly improved the IVF rates as early as 2 weeks after treatment when mice were only 9 weeks old. This implies that sperm can be efficiently cryopreserved from younger males, compared to 13-weeks-old males, which are usually used for sperm cryopreservation. This is of relevance when genetic alterations cause premature death in males as well as high severity levels in older mice and aids in better resource management.

Tutorial: Volumetric absorptive microsampling (VAMS)

Volumetric absorptive microsampling (VAMS) is a recent microsampling technique used to obtain dried specimens of blood and other biological matrices for application to a plethora of bioanalytical purposes. As such, it can be likened to dried blood spot (DBS) technique that has been in wide use for the last 40 years. However, VAMS promises to bring some significant advantages over DBS, related to sampling volume accuracy, haematocrit (HCT) dependence, pre-treatment and automation. Although some aspects still need to be investigated in depth, VAMS is increasingly recognised as a viable alternative to DBS and other dried microsampling techniques. In this tutorial, different aspects of VAMS approach are described and discussed, presenting the procedures adopted and the results obtained by those authors who have developed this kind of analytical workflow in the last few years. Hopefully, this will help other scientists to find new solutions to old and recent problems related to microsampling and to produce new, sound and interesting science in this field.