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Introduction: The argan tree (Argania spinosa) is an endemic species from south-western Morocco. Argan-based preparations have been widely used in Moroccan traditional medicine for their biological properties, as well as for several cosmetic purposes. Whereas kernel, pulp of fruit and trunk have been extensively studied for their nutritional and pharmacological effects, relatively little is known about argan tree leaves., Objective: The main purpose of the present study is to investigate and characterise the bioactive phenolic fractions in both crude and aqueous extracts derived from argan tree leaves., Methodology: A qualitative profile of the antioxidant phenolic compounds in argan leaves was obtained by means of structural hypothesis based on UV spectra and mass spectrometric fragmentation patterns. Moreover, selected phenolics were quantified in argan leaves by using a fully validated method based on liquid chromatography coupled to diode array detection and tandem mass spectrometry (LC-DAD-MS/MS). All the extracts were purified by a fast and reliable microextraction by packed sorbent (MEPS) procedure, before analysing them by LC-MS/MS., Results: Based on retention times, mass spectrometric fragmentation and UV spectra, 13 phenolic compounds were identified or tentatively elucidated from crude and aqueous extracts derived from Argania spinosa leaves, while seven compounds were quantified in both extracts., Conclusion: The obtained results could represent a first step towards a complete characterisation of the argan plant, its bioactive profiling and the valorisation of its by-products as a source of potentially beneficial bioactive molecules., (Copyright © 2015 John Wiley & Sons, Ltd.)

γ-Hydroxybutyric acid (GHB) is a powerful central nervous system depressant, currently used in medicine for the treatment of narcolepsy and alcohol dependence. In recent years, it has gained popularity among illegal club drugs, mainly because of its euphoric effects as well as doping agent and date rape drug. The purpose of the present work was the development of a rapid analytical method for the analysis of GHB in innovative biological matrices, namely dried blood spots (DBSs) and dried urine spots (DUSs). The analytical method is based on capillary zone electrophoresis with indirect UV absorption detection at 210 nm and capillary wall dynamic coating. The background electrolyte is composed of a phosphate buffer containing nicotinic acid (probe for detection) and cetyltrimethylammonium bromide (CTAB, reversal of electroosmosis in wall dynamic coating). The influence of probe and CTAB concentration, together with buffer pH, on migration time and signal response was investigated. Under the optimized conditions, analytical linearity and precision were satisfactory; absolute recovery values were also high (>90 %); the use of dried matrices (DBSs and DUSs) was advantageous as an alternative matrix to classical ones. No interferences were found either from the most common exogenous or from endogenous compounds. This analytical approach can offer a rapid, precise and accurate method for GHB determination in innovative biological samples, which could be important for screening purposes in clinical and forensic toxicology. Graphical Abstract CE method, by combined indirect UV detection and dynamic coating, for GHB determination in DBSs and DUSs.

Background: Skin adverse events associated with D-Penicillamine (DPA) are common and multi-faceted, although the presence of DPA or its metabolites has never been documented in the skin, because of inherent difficulties in determining its tissue levels. Thus, the association between DPA and DPA-related dermatoses has been only hypothesized on the basis of careful history, clinical observation and typical histopathological findings., Objective: To detect DPA in biopsy specimens in a unique case of 25-year-late-onset elastosis perforans serpiginosa and pseudo-pseudoxanthoma elasticum associated with a history of long-term high dose DPA, by applying a recently described analytical method to assess the presence of DPA in skin., Methods: We used a reliable analytical method based on high-performance liquid chromatography coupled with amperometric detection to look for the presence of DPA in skin biopsy specimens., Results: A chromatographic peak corresponding to DPA was evidenced in some affected skin samples collected from the patient., Conclusion: We documented the effective presence and the persistence after 25 years of DPA in the skin of a woman affected by elastotic cutaneous change due to a long-term therapy with DPA. This report provides further evidence of the relationship between DPA deposit in affected skin and clinical manifestation., (© 2014 European Academy of Dermatology and Venereology.)

A new microextraction by packed sorbent (MEPS) procedure coupled to a high-performance liquid chromatographic method has been developed for quantitation of catecholamines (i.e. norepinephrine, epinephrine and dopamine) in innovative biological samples, namely dried plasma and urine spots. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 2.5% methanol and 97.5% aqueous citrate buffer, containing octanesulfonic acid. Coulometric detection was used, setting the first analytical cell at -0.350 V and the second analytical cell at +0.400 V. Dried matrices were purified by means of a fast and feasible MEPS procedure, optimized on C18 sorbent and requiring only a small amount of biological sample. The availability of miniaturized procedures allowed satisfying specific requirements that ought to be considered during pre-treatment intended for catecholamine analysis. The extraction yield values were always higher than 85% and sensitivity was good, with a limit of quantitation of 100 pg mL(-1) for all the analytes. Satisfactory results were also obtained in terms of linearity, precision and accuracy. The method was successfully applied to dried plasma and urine spots derived from two socially diversified groups, namely psychiatric patients and poly-drug abusers, in comparison to healthy volunteers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Many CNS drugs are effective for the treatment of psychiatric disorders. Psychotropic drugs work differently, thus clinical outcomes for many patients may be insufficient. For this reason it could be useful the measurement of drug levels for clinical decision-making. Analytical goals in therapeutic drug monitoring (TDM) should be established by selecting the appropriate biological matrix. The aim of this review is to highlight the usefulness of TDM for antiepileptics, antidepressants and antipsychotics, with a focus on current advances in biosampling. The literature on TDM was reviewed up to March 2015. An overview on the use of alternative biological matrices is provided to address the current issues and advances in the field of biosampling for psychiatric CNS drug TDM.

A rapid and reliable analytical method has been developed to quantify the melatonergic antidepressant agomelatine in three matrices, and namely saliva, plasma and dried blood spots. The method is based on the use of liquid chromatography with fluorimetric detection exploiting the native fluorescence of agomelatine. For saliva and plasma samples an original microextraction by packed sorbent procedure was implemented obtaining satisfactory extraction yield of the analyte (always higher than 89%) and a good clean-up of the matrices. On the contrary, agomelatine was extracted from dried blood spots by suitable solvent microwave-assisted extraction and injected into chromatographic system. Satisfactory results in terms of sensitivity, linearity, precision, selectivity and accuracy were obtained. Thus, the developed method seems to be suitable for therapeutic drug monitoring of depressed patients under agomelatine therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of this paper was to investigate the subjective responses of abstinent heroin users to both neutral and negative stimuli and the related hypothalamus-pituitary-adrenal reactions to emotional experience in relationship to their perception of childhood adverse experiences. Thirty male abstinent heroin dependents were included in the study. Emotional responses and childhood neglect perception were measured utilizing the State-Trait Anxiety Inventory Y-1 and the Child Experience of Care and Abuse Questionnaire. Neutral and unpleasant pictures selected from the International Affective Picture System and the Self-Assessment Manikin procedure have been used to determine ratings of pleasure and arousal. These ratings were compared with normative values obtained from healthy volunteers used as control. Blood samples were collected before and after the experimental sessions to determine both adrenocorticotropic hormone and cortisol plasma levels. Basal anxiety scores, cortisol and adrenocorticotropic hormone levels were higher in abstinent heroin users than in controls. Tests showed that anxiety scores did not change in controls after the vision of neutral slides, whilst they did in abstinent heroin addicts, increasing significantly; and increased less significantly after the unpleasant task, in comparison to controls. Abstinent heroin users showed significantly higher levels of parent antipathy and childhood emotional neglect perception than controls for both the father and the mother. Plasma adrenocorticotropic hormone and cortisol levels did not significantly increase after unpleasant slide set viewing among addicted individuals, because of the significantly higher basal levels characterizing the addicted subjects in comparison with controls. Multiple regression correlation showed a significant relationship between childhood neglect perception, arousal reaction, impaired hypothalamus-pituitary-adrenal axis response and addiction severity. Early adverse experiences seem to affect the entire interaction between hyper-arousal, reduced hormonal response to stress and addiction severity. Our findings, although obtained in a small number of subjects, indicate a significant link between the perception of parental style/care/support during childhood and the ability to cope with stressful emotional stimuli in adulthood and addiction severity., (© 2013.)

The development and validation of a bioanalytical assay for the simultaneous determination of cortisol, cortisone and corticosterone levels in several matrices, such as saliva, plasma, blood and urine samples have been described. The method is based on a rapid test which combines a microextraction by packed sorbent procedure and liquid chromatography-diode array technique. Chromatographic separation of the analytes (cortisol, cortisone and corticosterone) and the internal standard (methylprednisolone) was achieved in less than 10min on a reversed-phase pentafluorophenyl column using a mobile phase composed of phosphate buffer and acetonitrile. The assay was performed after an innovative microextraction procedure by means of C8 sorbent which guaranteed good clean-up of the matrices and satisfactory extraction yield of the analytes. Moreover, the method gave linear results over a range of 5-100ngmL(-1) and showed good selectivity and precision. This method was successfully applied for quantifying corticosteroids in specific matrices derived from some healthy volunteers in comparison to two socially diversified groups, namely former heroin addicts undergoing opioid replacement therapy and poly-drug abusers., (Copyright © 2013 Elsevier B.V. All rights reserved.)

D-penicillamine is a thiol drug mainly used for Wilson's disease, rheumatoid arthritis and cystinuria. Adverse effects during normal use of the drug are frequent and may include skin lesions. To evaluate its toxic effects in clinical cases an original method based on high performance liquid chromatography coupled to amperometric detection in a specific biological matrix such as skin has been developed. The chromatographic analysis of D-penicillamine was carried out on a C18 column using a mixture of acid phosphate buffer and methanol as the mobile phase. Satisfactory sensitivity was obtained by oxidizing the molecule at +0.95 V with respect to an Ag/AgCl reference electrode. A chemical reduction of D-penicillamine-protein disulphide bonds using dithioerythritol combined with microwaves was necessary for the determination of the total amount of D-penicillamine in skin specimens. A further solid-phase extraction procedure on C18 cartridges was implemented for the sample clean-up. The whole analytical procedure was validated: high extraction yield (>91.0%) and satisfactory precision (RSD<6.8%) values were obtained. It was successfully applied to skin samples from a patient who was previously under a long-term, high-dose treatment with the drug and presented serious D-penicillamine-related dermatoses. Thus, the method seems to be suitable for the analysis of D-penicillamine in skin tissues., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A novel test has been developed for the analysis of methadone in dried blood spot specimens from patients undergoing methadone maintenance treatment. An isocratic reversed-phase high-performance liquid chromatography method with coulometric detection has been optimized for the determination of methadone. The clean-up of dried blood spots was performed by means of an original microextraction by packed sorbent procedure after microwave-assisted extraction of the drug with a suitable solvent. Extraction yields were satisfactory, always being higher than 90.0 %. The calibration curve was linear over the 4-500 ng mL(-1) concentration range. The method had satisfactory sensitivity (limit of quantitation of 4 ng mL(-1)), precision (relative standard deviation less than 5.8 %), selectivity and accuracy (recovery greater than 87.0 %). It was successfully applied to dried blood spot samples collected from heroin-addicted patients undergoing methadone maintenance therapy at dosages between 40 and 240 mg day(-1). The statistical analysis (Bland-Altman plot) showed that the results were in good agreement with those found from the analysis of plasma samples obtained from the same patients. Thus, the method has proved to be suitable for the monitoring of methadone by means of dried blood spots.

New-generation antidepressants are a heterogeneous class of drugs used in the treatment of depression and related disorders. This review deals with the first new-generation antidepressant class to enter the pharmaceutical market, i.e., selective serotonin reuptake inhibitors (SSRIs), which are still the most prescribed and widely used ones. Their common characteristics are the comparable clinical efficacy, good tolerability and relative safety in comparison to "first generation antidepressants", i.e. classic tricyclic antidepressants and monoamine oxidase inhibitors. This class of drugs includes fluoxetine, citalopram, paroxetine, sertraline, fluvoxamine and, since 2011, vilazodone. In this review, the main pharmacodynamic and pharmacokinetic properties of the six commercially available SSRIs are described, focusing on side and toxic effects, chemical-clinical correlations, interactions with other drugs, the role of therapeutic drug monitoring (TDM) and related bioanalytical methodologies.

The sublingual combination of buprenorphine and naloxone (Suboxone(®)) and Methadone Maintenance Therapy have been found effective in treating heroin addiction. A new analytical method suitable for the simultaneous determination of buprenorphine, norbuprenorphine, methadone and naloxone in human plasma by means of liquid chromatography with coulometric detection has been developed. The chromatographic separation was achieved with a phosphate buffer-acetonitrile mixture as the mobile phase on a cyano column. The monitoring cell of the coulometric detector was set at an oxidation potential of +0.600 V. A rapid clean-up procedure of the biological samples using a microextraction by packed sorbent technique has been implemented, employing a C8 sorbent inserted into a syringe needle. The extraction yield values were satisfactory for all analytes (>85%). The calibration curves were linear over a range of 0.25-20.0 ng mL(-1) for buprenorphine and norbuprenorphine, 3.0-1000.0 ng mL(-1) for methadone and 0.13-10.0 ng mL(-1) for naloxone. The sensitivity was also high with limits of detection of 0.08 ng mL(-1) for both buprenorphine and norbuprenorphine, 0.9 ng mL(-1) for methadone and 0.04 ng mL(-1) for naloxone. The intraday and interday precision data were always satisfactory. The method was successfully applied to plasma samples obtained from former heroin addicts treated with opioid replacement therapy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Epidemiological and clinical data show frequent associations between adverse childhood experiences (ACEs) and substance abuse susceptibility particularly in adolescents. A large body of evidences suggests that the possible dysregulation of neuroendocrine responses as well as neurotransmitters function induced by childhood traumatic experiences and emotional neglect could constitute one of the essential biological changes implementing substance abuse vulnerability. Moreover, genotype variables and its environment interactions have been associated with an increased risk for early onset substance abuse. In this paper we present several data that support the hypothesis of the involvement of hypothalamus-pituitary-adrenal (HPA) axis in mediating the combined effect of early adverse experiences and gene variants affecting neurotransmission. The presented data also confirm the relationship between basal plasma levels of cortisol and ACTH, on the one hand, and retrospective measures of neglect during childhood on the other hand: the higher the mother and father neglect (CECA-Q) scores are, the higher the plasma levels of the two HPA hormones are. Furthermore, such positive relationship has been proved to be particularly effective and important when associated with the "S" promoter polymorphism of the gene encoding the 5-HTT transporter, both in homozygote and heterozygote individuals., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cocaine dependence is characterized by compulsive drug seeking and high vulnerability to relapse. Overall, cocaine remains one of the most used illicit drugs in the world. Given the difficulty of achieving sustained recovery, pharmacotherapy of cocaine addiction remains one of the most important clinical challenges. Recent advances in neurobiology, brain imaging and clinical trials suggest that certain medications show promise in the treatment of cocaine addiction. The pharmacotherapeutic approaches for cocaine dependence include medications able to target specific subtypes of dopamine receptors, affect different neurotransmitter systems (i.e. noradrenergic, serotonergic, cholinergic, glutamatergic, GABAergic and opioidergic pathways), and modulate neurological processes. The systematic reviews concerning the pharmacological treatment of cocaine dependence appear to indicate controversial findings and inconclusive results. The aim of future studies should be to identify the effective medications matching the specific needs of patients with specific characteristics, abandoning the strategies extended to the entire population of cocaine dependent patients. In the present review we summarize the current pharmacotherapeutic approaches to the treatment of cocaine dependence with a focus on the new patents.

A novel analytical approach has been developed for the determination of clozapine and its metabolites in dried blood spots on filter paper, using a chromatographic method coupled with a microextraction by packed sorbent procedure. The analytes were separated on a RP-C18 column using a mobile phase composed of 20% methanol, 16% acetonitrile and 64% aqueous phosphate buffer. Coulometric detection was used, setting the guard cell at +0.050 V, the first analytical cell at -0.200 V and the second analytical cell at +0.500 V. Clozapine and its metabolites were extracted from dried blood spots with phosphate buffer and, then, a microextraction by packed sorbent procedure for the sample clean-up was implemented obtaining good extraction yields. The calibration curve was linear over the 2.5-1000 ng mL(-1) blood concentration ranges for all the analytes. The method validation gave satisfactory results in terms of sensitivity, precision, selectivity and accuracy. The analytical method was successfully applied to dried blood spots from several psychiatric patients for therapeutic drug monitoring purpose., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A reliable chromatographic method for the determination of soy isoflavones (genistein, daidzein and glycitein) using a coulometric detection has been developed and applied to analyse plasma of postmenopausal women. The chromatographic separation was performed on a C18 reversed phase column with a mobile phase composed of acetonitrile-phosphate buffer mixture. Coulometric detection was carried out at +0.500 V. A careful and rapid solid phase extraction procedure on hydrophilic/lipophilic cartridges was chosen for plasma sample purification with and without hydrolysis obtaining good extraction yield values for all the analytes (>90.0%). The enzymatic hydrolysis step was necessary for the determination of the total amount of soy isoflavones. The limit of quantitation was 0.5 ng mL(-1) for genistein and 0.25 ng mL(-1) for daidzein and glycitein. The method was found to be precise and accurate. Thus, the proposed method is suitable for the analysis of soy isoflavones (free and total amounts) in plasma of postmenopausal women under treatment with the SoymenGN dietary supplement., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

An isocratic high-performance liquid-chromatographic method has been developed for the simultaneous determination of disulfiram and bupropion in human plasma samples. Analyses were carried out on a C(8) reversed-phase column using a mobile phase composed of 50% acetonitrile and 50% aqueous phosphate buffer, containing triethylamine. Diode-array detection was used, operating at a wavelength of 250 nm. For the clean-up of plasma samples, a solid phase extraction procedure, based on C(2) cartridges, was implemented. Extraction yields of the analytes were satisfactory, being always higher than 84%. The calibration curve was linear over the 5-500 ng mL(-1) plasma concentration range for both disulfiram and bupropion. The method showed a high sensitivity (limit of detection of 1.5 ng mL(-1)) and satisfactory precision, selectivity and accuracy. The application to human plasma samples obtained from some alcohol and nicotine abusers also gave good results.

An isocratic high-performance liquid chromatographic method has been developed for the measurement of serotonin, 5-hydroxyindolacetic and homovanillic acids in dried blood spots and in platelet poor and rich plasma samples. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 13% methanol and 87% aqueous citrate buffer, containing octanesulfonic and ethylendiaminotetracetic acids. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at -0.200 V and the second analytical cell at +0.400 V. For the pre-treatment of biological samples a novel solid-phase extraction procedure, based on mixed-mode reversed-phase--strong anion exchange Oasis cartridges, was implemented. Extraction yields of the analytes from all these matrices were satisfactory, being always higher than 89.0%. The calibration curve was linear over the on-column concentration range of 0.1-22.5 ng mL(-1) for serotonin and 5-hydroxyindolacetic acid and of 0.25-22.5 ng mL(-1) for homovanillic acid. The sensitivity was good with a limit of detection of 0.05 ng mL(-1) for serotonin and 5-hydroxyindolacetic acid and 0.12 ng mL(-1) for homovanillic acid. Results were also satisfactory in terms of precision, selectivity and accuracy. The analytical method was successfully applied to human platelet poor and rich plasma samples and to dried blood spots from volunteers and psychiatric patients., (2010. Published by Elsevier B.V.)

A new analytical method, based on the use of liquid chromatography with coulometric detection, has been developed and applied to quantify risperidone and its main active metabolite 9-hydroxyrisperidone in human plasma and saliva. The analytes were separated on a reversed phase C18 column, using a mobile phase composed of acetonitrile (26%) and a pH 6.5 phosphate buffer (74%). Pipamperone was used as the internal standard. A high sensitivity coulometric detection analytical cell containing two flow-through working electrodes was used: electrode 1 was set at +0.500V and electrode 2 at +0.700V. The detector response was linear over a plasma and saliva concentration range of 0.5-50.0ngmL(-1) for risperidone and 0.5-100.0ngmL(-1) for 9-hydroxyrisperidone. The limit of quantitation and the limit of detection for risperidone and 9-hydroxyrisperidone were 0.5ngmL(-1) and 0.17ngmL(-1), respectively. A novel clean-up procedure of biological samples was developed using the microextraction by packed sorbent technique, which gave good extraction yield for both the analytes, with absolute recovery values higher than 90.1%. The intra-day and the inter-day precision results, expressed by relative standard deviation values, were lower than 5.8%. Accuracy and selectivity assays were also satisfactory. The validated method has been successfully applied to the analysis of risperidone and 9-hydroxyrisperidone in plasma and saliva of psychiatric patients undergoing therapy with risperidone.

A rapid and reliable analytical method suitable for the simultaneous determination of the antiepileptic drug, oxcarbazepine and its metabolites in human plasma and saliva by means of liquid chromatography with diode array detection (DAD) has been developed. Oxcarbazepine and its metabolites (10,11-dihydro-10-hydroxycarbamazepine, trans-10,11-dihydro-10,11-dihydroxycarbamazepine and 3-hydroxycarbamazepine) were baseline separated within 6.5 min on a reversed-phase C18 column with a phosphate buffer-acetonitrile-triethylamine mixture as the mobile phase. The DAD detector was set at 240 nm. A sample preparation method for biological samples using a microextraction by packed sorbent technique has been implemented, employing a C18 sorbent inserted into a microvolume syringe and using only a small volume (25 microL) of plasma or saliva. The extraction yield values were satisfactory for all analytes (>86.5%) as well as the precision data, which were always in the low percentage of relative standard deviation values (<4.6%). The method was successfully applied to both plasma and saliva samples drawn from psychiatric and neurological patients undergoing treatment with oxcarbazepine (Tolep) tablets., (Copyright 2009 Elsevier B.V. All rights reserved.)

Orphenadrine is an antimuscarinic agent mainly used for the treatment of parkinsonism and to alleviate the neuroleptic syndrome induced by antipsychotic drugs. A new, rapid analytical method, based on liquid chromatography with diode array detection (DAD), has been developed and applied to the determination of orphenadrine in plasma of schizophrenic patients for therapeutic drug monitoring and toxicological purposes. The chromatographic separation was performed on a pentafluorophenyl reversed phase column with a mobile phase composed of acetonitrile-phosphate buffer mixture. DAD detection was carried out at 220 nm. A careful and rapid solid-phase extraction procedure on cyanopropyl cartridges was chosen for plasma sample purification and pre-concentration obtaining good extraction yield values for the analyte (>96.0%). The assays showed a linear response for orphenadrine (30-1000 ng mL(-1)). The method is also precise and selective. Thus, the method developed seems to be suitable for routine analysis of orphenadrine in psychiatric patients. Moreover, it was applied to plasma samples from a psychotic patient who had tried to poison himself with 1000 mg of orphenadrine and was undergoing polypharmacy.

Bipolar disorder (BD) is a long-term illness with mood swings which are characterized by recurrent episodes of mania/hypomania and depression, with variable interpolations of relatively asymptomatic periods, called euthymic, in which, however, some psychopathological symptoms may persist. Although mood stabilizers, such as lithium, are the first-line treatment for the prevention of new BD episodes, combination therapy has become the standard of care for BD patients. Besides lithium, the use of a mood stabilizer along with an atypical antipsychotic is recommended in many patients. Recently, atypical antipsychotics (quetiapine, olanzapine, risperidone and aripiprazole) and antiepileptic agents (valproate, lamotrigine and oxcarbazepine) are increasingly used as mood stabilizers. To reduce side effects and optimize treatment it is important to perform accurate monitoring of drug blood levels in these patients, who are often treated with multiple drugs. Therapeutic drug monitoring (TDM) is in fact a powerful tool that, starting from clinical-chemical correlation data, allows to tailor-cut treatment to the specific needs of individual patients; hence the need to have reliable analytical methods available for the determination of plasma levels of drugs and their metabolites. Analyses of biological samples are mainly carried out using high-performance liquid chromatography (HPLC) coupled with different detectors, capillary electrophoresis and gas-chromatography. Various procedures are employed to remove biological interferences before analyzing the samples. This review focuses on currently available analytical TDM methods for atypical antipsychotics and antiepileptic agents used in the treatment of patients with bipolar disorder. Advantages and limitations of the various analytical methods will be reviewed and discussed, together with an evaluation of the role of TDM.

The pharmaco-toxicological profile of duloxetine, a novel SNRI antidepressant, is still not completely known; in particular, intoxication cases have been scarcely studied. Here a duloxetine overdose case, in combination with other antidepressants and benzodiazepines, is reported and the chemical-clinical correlations discussed; this is probably the first detailed report of such a case. The patient referred to have ingested nine tablets of Cymbalta (more than 500 mg of duloxetine) and high amounts of four other drugs (venlafaxine, trazodone, sertraline and clonazepam). The patient was dozy and confused and some electrolyte imbalances were found. After gastrolavage, toxicological analyses revealed high plasma levels of duloxetine (384 ng/ml) and low levels of the other supposedly involved drugs. The overdose resulted to be not fatal and the outcome was relatively benign, also thanks to the fast emergency assistance. This case suggests that clinicians should be alerted to the possibility of toxic effects caused by simultaneous overdoses of duloxetine and other antidepressants and that caution should be used when prescribing more than one of these drugs to patients at risk of suicide.

A new analytical method, based on liquid chromatography with coulometric detection, has been developed and applied to the determination of selected phenothiazines (chlorpromazine, promazine, fluphenazine and levomepromazine) in human plasma. The drugs were separated on a Discovery pentafluorophenylpropyl column, using a mobile phase composed of acetonitrile (32%) and a pH 1.9 phosphate buffer (68%). Promethazine was used as the internal standard. Detection was carried out at an oxidation potential of +0.500 V. A novel clean-up procedure was developed by means of solid-phase extraction, using cyanopropyl cartridges, which gave good extraction yield for all the analytes, with absolute recovery values higher than 91.0%. The detector response was linear over a plasma concentration range of 0.5-250.0 ng mL(-1) for chlorpromazine, promazine and levomepromazine and of 0.2-4.0 ng mL(-1) for fluphenazine. Precision results, expressed by the intra-day and the inter-day relative standard deviation values, were good, being lower than 3.9%. Accuracy data were satisfactory as well. The method has been successfully applied to the analysis of drug plasma levels of psychiatric patients undergoing therapy with selected phenothiazines.

Three analytical methods have been developed and compared for the quality control of a new formulation (Soymen GN(R) capsules) containing soy extract and melatonin for the treatment of menopausal symptoms. The first method is based on MEKC with diode-array detection, using a mixture of basic carbonate buffer (95%) and methanol (5%), containing 55 mM SDS, as the BGE. The second method is an HPLC method with UV detection at 260 nm. The third method is an HPLC method coupled to amperometric detection which is carried out at an oxidation potential of +0.8 V. In both HPLC systems, the chromatographic separation is obtained on an RP C18 column using a mixture of ACN and an acidic phosphate buffer (25:75 v/v) as the mobile phase. A feasible pretreatment procedure with a methanol/water mixture has been implemented to achieve the quantitative extraction of the main soy isoflavones and of melatonin from the capsules. The results obtained with the three methods are in good agreement with each other and satisfactory in terms of linearity (r(2) >0.9996), precision (RSD <5.4%) and accuracy (recovery >97%). Thus, each of the three analytical methods seems to be suitable for the simultaneous analysis of the main soy isoflavones and melatonin in the new commercial formulation.

Two methods, based on the use of capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC), respectively, were developed for the analysis of the atypical antipsychotic aripiprazole in plasma of schizophrenic patients for therapeutic drug monitoring purposes. Good analytical performances were obtained with the CE method, using uncoated fused silica capillaries and a background electrolyte composed of 50mM phosphate buffer at pH 2.5. With 20 kV voltage, aripiprazole was detectable at 214 nm within 5 min. The second analytical method, based on HPLC with diode array detection, employed a C8 reversed-phase column and a mixture of a 12.5mM phosphate buffer, pH 3.5, containing triethylamine and acetonitrile as the mobile phase. Aripiprazole was detected at 254 nm and a complete chromatographic run lasted about 10 min. For both analytical methods loxapine was used as the internal standard and the same plasma sample pre-treatment by means of solid-phase extraction on cyano cartridges was carried out, with extraction yield values always higher than 91.3%. Linear responses for aripiprazole were obtained between 70 and 700 ng mL(-1) and precision assays (expressed as relative standard deviation values) were lower than 7.0%. After validation, both methods were successfully applied to human plasma samples drawn from schizophrenic patients undergoing therapy with Abilify tablets. Accuracy was satisfactory, with recovery value higher than 91.0%.

An original method based on the use of high-performance liquid chromatography with both coulometric and diode array detection has been developed for the therapeutic drug monitoring of patients with bipolar disorders being treated with olanzapine and lamotrigine. Chromatographic separation was achieved on a reversed-phase C8 column (150 x 4.6 mm internal diameter, 5 microm) using a mobile phase composed of methanol (27%) and a 50.0 mmol/L, pH 3.5 phosphate buffer (73%). For the analysis of olanzapine and its main metabolite, N-desmethylolanzapine, a coulometric detector was used, with electrode 1 set at -200 mV and electrode 2 at +500 mV. Lamotrigine was determined using a diode array detection at 220 nm. The two detectors were connected in series. For the analysis of biological samples, a clean-up procedure was implemented by means of solid-phase extraction using phenyl cartridges and eluting the analytes with methanol; only a small volume of plasma (150 microL) was needed to analyze both olanzapine and lamotrigine. Linear responses were obtained between 0.1 and 50.0 ng mL(-1) for olanzapine, 0.1 and 25.0 ng mL(-1) for N-desmethylolanzapine, and between 0.25 and 10.0 microg mL(-1) for lamotrigine. The extraction yield values were always higher than 90% for all the analytes, with precision (expressed as relative standard deviation values) lower than 3.4%. The method was applied successfully to some human plasma samples drawn from bipolar patients undergoing combined therapy with the two drugs. Satisfactory values for accuracy were obtained, with mean recovery higher than 91%. Thus, the method appears suitable for the investigation of the chemical-clinical correlations in patients receiving therapy with olanzapine and lamotrigine.

A liquid chromatographic method with diode array detection (DAD) has been developed for the analysis of the antiepileptic agent lamotrigine (LTG) and its metabolites, lamotrigine 2-N-glucuronide and 2-N-methylated in plasma samples. The analytes were separated on a C8 RP column, using a mobile phase composed of methanol and a 0.45 mM, pH 3.5 phosphate buffer containing 0.17% triethylamine (24:76 v/v). Melatonin was used as the internal standard (IS). The DAD detector was set at 220 nm for the detection of all the analytes. A simple protein precipitation with methanol guaranteed high extraction yield values (>90%) and good purification from matrix interference. Good linearity was obtained in the 0.1-15.0 microg/mL range for LTG and lamotrigine 2-N-glucuronide and in the 0.1-2.0 microg/mL range for lamotrigine 2-N-methylated. The analytical method was validated in terms of precision, extraction yield, and accuracy. These assays gave RSD% values for precision always lower than 4.3% and mean accuracy higher than 80%. The method seems to be suitable for the analysis of plasma samples from patients treated with Lamictal.

Background: Attention deficit hyperactivity disorder (ADHD) seems to be a risk condition for substance use disorders, possibly in relationship to common neurobiological changes, underlying both addictive and externalising behaviour susceptibility. Although this vulnerability has been primarily attributed to gene variants, previous studies suggest that also adverse childhood experiences may influence neurotransmission, affecting in particular brain dopamine (DA) system and possibly concurring to the development of behavioural disorders. Therefore, we decided to investigate ADHD symptoms and plasma concentrations of the DA metabolite homovanillic acid (HVA) in abstinent addicted patients, in comparison with healthy control subjects, evaluating whether ADHD scores were related with HVA levels, as expression of DA turnover, and whether HVA values, in turn, were associated with childhood emotional neglect., Methods: Eighty-two abstinent drug dependent patients, and 44 normal controls, matched for age and sex, completed the Wender Utah Rating Scale (WURS), measuring ADHD symptoms, and the Childhood Experience of Care and Abuse Questionnaire (CECA-Q). Blood samples were collected to determine HVA plasma levels., Results: Addicted individuals showed significantly higher ADHD scores and lower HVA levels respect to control subjects. ADHD scores at WURS in addicted patients negatively correlated with plasma HVA values. In turn, plasma HVA levels were inversely associated with childhood neglect measures, reaching statistical significance with "mother-antipathy" and "mother neglect" scores., Conclusions: These findings suggest the possibility that childhood experience of neglect and poor mother-child attachment may have an effect on central dopamine function as an adult, in turn contributing to both ADHD and substance abuse neurobiological vulnerability.

An original HPLC-UV method has been developed for the simultaneous determination of the atypical antipsychotic quetiapine and the geometric isomers of the second-generation antidepressant fluvoxamine. The analytes were separated on a reversed-phase C8 column (150 mm x 4.6mm i.d., 5 microm) using a mobile phase composed of acetonitrile (30%) and a 10.5mM, pH 3.5 phosphate buffer containing 0.12% triethylamine (70%). The flow rate was 1.2 mL min(-1) and the detection wavelength was 245 nm. Sample pretreatment was carried out by an original solid-phase extraction procedure using mixed-mode cation exchange (DSC-MCAX) cartridges; only 300 microL of plasma were needed for one analysis. Citalopram was used as the internal standard. The method was validated in terms of linearity, extraction yield, precision and accuracy. Good linearity was obtained in plasma over the 5.0-160.0 ng mL(-1) concentration range for each fluvoxamine isomer and over the 2.5-400.0 ng mL(-1) concentration range for quetiapine. Extraction yield values were always higher than 93%, with precision (expressed as relative standard deviation values) better than 4.0%. The method was successfully applied to human plasma samples drawn from patients undergoing polypharmacy with the two drugs. Satisfactory accuracy values were obtained, with mean recovery higher than 94%.

A sensitive high-performance liquid chromatographic method has been developed for the determination of homovanillic acid (HVA), the main metabolite of dopamine, in human plasma. Analyses were carried out on a reversed-phase column (C8, 250 mm x 4.6 mm i.d., 5 microm) using a mobile phase composed of 10% methanol and 90% aqueous citrate buffer, containing octanesulfonic acid and EDTA at pH 4.8. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at -0.200 V and the second analytical cell at +0.500 V. A careful solid-phase extraction procedure, based on strong anion exchange (SAX) cartridges (100 mg, 1 mL), was implemented for the pre-treatment of plasma samples. Extraction yield was satisfactory, being the mean value 98.0%. The calibration curve was linear over the concentration range of 0.2-25.0 ng mL(-1) of homovanillic acid. The limit of quantitation (LOQ) was 0.2 ng mL(-1) and the limit of detection (LOD) was 0.1 ng mL(-1). The method was successfully applied to plasma samples from former alcohol, cocaine and heroin addicts. Results were satisfactory in terms of precision and accuracy. Hence, the method is suitable for the determination of homovanillic acid in human plasma.

A sensitive and selective method was developed for the determination of the antipsychotic drug Olanzapine levels in rat brain tissue, based on HPLC with electrochemical detection. The analyses were carried out on a C8 reversed phase column (150 mm x 4.6 mm, 5 microm), using a mobile phase composed of methanol and a phosphate buffer (44.0 mM, pH 3.5), containing triethylamine (21:79, v/v), flowing at 1.2 mL min(-1). A high sensitivity coulometric detection analytical cell containing two flow-through low volume working electrodes was used: electrode 1 was set at +0.350 V and electrode 2 at -0.200 V. Olanzapine, administered to rats in different doses or in different times, was extracted from tissue homogenate of either the whole brain or specific areas (cortex, hyppocampus, nucleus striatum) with a rapid solid phase extraction procedure (SPE) on Oasis HLB cartridges. The method provided a high extraction yield of Olanzapine and internal standard (2-methylolanzapine) from brain tissue homogenate with absolute recovery values higher than 90.0%. The detector response was linear over a concentration range of 0.2-100.0 ng mL(-1) of Olanzapine. The limit of quantification (LOQ) was 0.2 ng mL(-1). Precision results, expressed by the intra-day and the inter-day relative standard deviation values, were satisfactory, better than 4.6%. Accuracy was satisfactory as well. This method proved to be suitable for the analysis of Olanzapine in rat brain tissues and for the study of distribution and pharmacokinetics of Olanzapine in rat brain after a single treatment with the antipsychotic drug.

An HPLC method has been developed for the simultaneous determination of aromatic and terpenic constituents of cloves on a C8 RP column, with the mobile phase consisting of a pH 3.5 phosphate buffer-triethylamine (30%) and acetonitrile (70%); a flow rate of 1.2 mL/min and a diode-array detector were used. Complete separation of all analytes (eugenol (EUG), eugenol acetate (AEUG), beta-caryophyllene, a-humulene and caryophyllene oxide) was achieved within 7 min. Good linearity was found in the range 0.125-40.0 microg/mL for EUG and AEUG and in the range 0.250-20.0 microg/mL for the terpenic compounds. After validation, the method was successfully applied to the analysis of clove oil and clove extract samples. The results obtained indicate good accuracy (recovery percentage mean value corresponding to 99.9%) and satisfactory precision.

A liquid chromatographic method with ultraviolet detection was developed for the analysis of the recent antidepressant sertraline and its main metabolite N-desmethylsertraline in human plasma. The analytes were separated on a C8 reversed phase column, using a mobile phase composed of acetonitrile and a 12.3 mM, pH 3.0 phosphate buffer containing 0.1% triethylamine (35:65, v/v). Clomipramine was used as the Internal Standard. Using a solid phase extraction procedure with C2 cartridges high extraction yields (>94%) and good purification from matrix interference were obtained. Good linearity was obtained in the 7.5-250.0 ng mL(-1) range for sertraline and in the 10-500 ng mL(-1) range for N-desmethylsertraline. The analytical method was validated in terms of precision, extraction yield and accuracy. These assays gave R.S.D.% values for precision always lower than 3.9% and mean accuracy higher than 90%. Thanks to its good selectivity, the method proved to be suitable for the analysis of plasma samples from patients treated with sertraline as either monotherapy or polypharmacy.

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, beta-glycyrrhetic acid and its isomer a-glycyrrhetic acid. Separation of the analytes was achieved in less than 3 min on a fused silica capillary, by injecting the samples at the short end of the capillary (effective length: 8.5 cm). The background electrolyte was composed of pH 10.0 carbonate buffer, methanol and ethylene glycol (80/10/10) and contained 0.4% beta-cyclodextrin; indomethacin was used as the internal standard. Diode array detection was used, with quantitative assays carried out at 254 nm. Linearity was found over the 5-200 and 2.5-100 microg mL(-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 beta-glycyrrhetic acid obtained from the hydrolysis of glycyrrhizin. When analysing beta-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.

A new, rapid analytical method, based on liquid chromatography with diode array detection, has been developed and applied to the determination of risperidone and its main active metabolite 9-hydroxyrisperidone in human plasma. The chromatographic separation was obtained on a C8 (150 x 4.6 mm, 5 microm) column, using a mobile phase composed of acetonitrile (27%) and a pH 3.0 phosphate buffer (73%). A sample clean-up procedure was carried out by using C8 cartridges and eluting the analytes with methanol. The extraction yield was highly satisfactory for both analytes, with average absolute recovery values of about 95%. The experimental conditions permitted the quantitative determination of risperidone and 9-hydroxyrisperidone with high precision (RSD < 3.6%) and satisfactory sensitivity (LOQ = 4 ng mL(-1)). The method was applied to plasma samples from a patient who had tried to poison himself with 150 mg of risperidone, and was undergoing polypharmacy.

There is a need for fast, simple and reliable analytical methods for the analysis of fluoxetine and norfluoxetine in patients who voluntarily or involuntarily have taken an overdose of the drug. A new liquid chromatographic method with diode array detection is presented herein for the determination of fluoxetine and its main active metabolite in human plasma for toxicological purposes. A mobile phase composed of acetonitrile and aqueous tetramethylammonium perchlorate allows to obtain the complete separation of the analytes on a C18 reversed phase column. The fast and accurate sample pre-treatment step is carried out by means of solid-phase extraction using hydrophilic-lipophilic balance cartridges and loading 100 microL of plasma only. This procedure gives satisfactory extraction yield values, as well as good plasma sample purification from matrix interference. Linearity was obtained in the 150-3000 ng/mL range for both analytes. Selectivity with respect to other psychotropic drugs was satisfactory. The method seems to be suitable for the analysis of fluoxetine and its metabolite in human plasma for depressed patients in overdose.

The comparison of two HPLC methods, one with electrochemical detection and the other with coulometric detection, for the simultaneous analysis of catecholamines and 4-hydroxy-3-methoxyphenylethylene glycol (MHPG) in human plasma is presented. The careful pre-treatment of plasma samples is based on an innovative two-step procedure by means of solid-phase extraction (SPE) which uses one single hydrophilic-lipophilic balance cartridge. The extraction yield values found were higher than 85% for epinephrine, norepinephrine and MHPG, and higher than 70% for dopamine. The assays carried out on real plasma samples with the coulometric system gave good results in terms of sensitivity (limits of quantitation: 0.10-0.15 ng ml(-1) for catecholamines, 0.6 ng ml(-1) for MHPG) and selectivity, while interference was sometimes found when using the amperometric system. Precision was also satisfactory, with relative standard deviation values for intermediate precision always lower than 6%. The HPLC method with coulometric detection coupled to a novel SPE procedure is thus suitable for the simultaneous determination of catecholamines and MHPG in plasma of volunteers subjected to experimental stress.

Background: A definite evidence in favour of an association of deep-vein thrombosis (DVT) with lupus anticoagulant (LA) in patients free from systemic lupus erythematosus is still lacking., Methods: In a case-control study, LA was determined in 176 consecutive outpatients who underwent phlebography because of the first episode of clinically suspected DVT of lower limbs. The association between DVT and LA was described using odds ratios (OR)., Results: Contrast venography confirmed the clinical suspicion in 59 patients (33.5%). LA was detected in 5 of the 59 patients with DVT (8.5%), and in none of the 117 subjects with normal venogram (P = 0.007). The OR for having an acute DVT in patients with LA was 10.7 (95% CI: 1.2-94.2)., Conclusions: LA is significantly associated with DVT in symptomatic patients. Further studies are needed to establish the clinical implications of this association.

The role of oral contraceptives as a triggering factor for thrombosis in patients with lupus anticoagulant (LA) and/or anticardiolipin antibodies (ACA) has not yet been established. We describe the cases of three women aged 19, 29 and 48 years who developed venous thrombosis after 16 +/- 3.4 (mean +/- SD) cycles of oral contraceptives. They were all asymptomatic before taking the pill. Two patients subsequently developed venous and/or arterial recurrence of thrombosis. Laboratory studies performed after the diagnosis of thrombosis, showed the presence of LA and elevated levels of ACA (IgG and IgM) in all three patients. None of these patients had autoimmune diseases and therefore appeared to have a primary antiphospholipid antibody syndrome. The three patients belonged to a group of 45 young females who experienced their first thrombotic event while taking the pill. This group had a similar prevalence (8%) for antithrombin deficiency and antiphospholipid antibodies. We surmise that some of the women who developed venous thrombosis while taking the pill might have an undetected primary antiphospholipid syndrome.

Tissue thromboplastin inhibition assay (TTI) is a sensitive test for lupus anticoagulant (LA). We have performed TTI in 12 LA positive patients using a new recombinant human tissue factor (Innovin, IN) and compared it with Thromborel S (TH), a commonly used human placenta thromboplastin. The effect of using two different dilutions of each thromboplastin (1:10 & 1:26) was investigated. A 1:26 dilution discriminated better than the 1:10 and this was more evident for Innovin. The mean value obtained with a 1:26 IN dilution was statistically different from that observed with TH at the same dilution. Furthermore, when PT and TTI ratios were considered, differences were statistically significant and seemed to increase depending on thromboplastin dilutions. When we used IN at 1:26 all LA positive patients had a value > 1.2. Then we compared TTI at a 1:26 dilution with dilute Russell's Viper Venom Time (dRVVT) in 50 consecutive patients with suspected lupus anticoagulant not treated with warfarin or heparin. In these patients the diagnosis of lupus anticoagulant was carried out using dilute APTT mixing studies and a platelet neutralization procedure: four out of 50 patients thus tested were LA positive. When dRVVT or TTI-I 1:26 were used, five patients were positive for lupus anticoagulant. Innovin showed similar sensitivity of dRVVT for detection of lupus anticoagulant. It is likely that higher dilutions of thromboplastins could further improve the specificity of this method.