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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.

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.

Three analytical methods have been developed and compared for the quality control of a new formulation (Soymen GN® capsules) containing soy extract and melatonin for the treatment of menopausal symptoms. The first method is based on micellar electrokinetic chromatography (MEKC) with diode-array detection, using a mixture of basic carbonate buffer (95%) and methanol (5%), containing 55 mM SDS, as the background electrolyte. The second method is a high-performance liquid chromatographic (HPLC) method with ultraviolet 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 a reversed-phase C18 column using a mixture of acetonitrile and an acidic phosphate buffer (25/75, v/v) as the mobile phase. A feasible pre-treatment 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 (r2 > 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.

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 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.

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 norfluoxetine 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 µL 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.

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.

A review with 279 references. 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.

1. Introduction. The dopaminergic and serotoninergic systems are known to be related to several mental illnesses such as schizophrenia, depression and affective disorders. A change in levels of neurotransmitters including dopamine and serotonin (and/or their metabolites) may lead to pathological processes with specific clinical manifestations. The introduction of the atypical antipsychotics acting on multiple neurotransmitters has improved therapeutic results. In particular, clozapine, the parent drug of the atypical antipsychotics, is more effective for reducing symptoms of schizophrenia than the older typical antipsychotics because of its profile of binding to serotonergic as well as dopamine receptors. 2. Materials and Methods. A high-performance liquid chromatographic method has been developed for the simultaneous determination 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 methanol and aqueous citrate buffer. Coulometric detection was used, operating at +0.400 V. For the clean-up of the biological samples a novel solid-phase extraction procedure, based on mixed-mode reversed-phase – strong anion exchange cartridges, was implemented. 3. Results. The analytical method was linear over the on-column concentration range of 0.1 - 22.5 ng/mL for serotonin and 5-hydroxyindolacetic acid and of 0.25 - 22.5 ng/mL for homovanillic acid. Extraction yields of the analytes from all these matrices were satisfactory, being always higher than 89.0 %. Results were satisfactory in terms of sensitivity, precision, selectivity and accuracy. 4. Conclusion. The analytical method was successfully applied to the analysis of serotonin, 5-hydroxyindolacetic and homovanillic acids in human platelet poor and rich plasma samples and to dried blood spots from volunteers and some psychotic patients treated with clozapine. Assays are currently in progress to apply this method to biological fluids from numerous psychiatric patients for studies regarding neuroendocrine responses.

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.

Epidemiological and clinical data show frequent associations between adverse childhood experiences (ACEs) and substance abuse susceptibility particularly in adolescents. A large body of evidences sug- gests 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 essen- tial biological changes implementing substance abuse vulnerability. Moreover, genotype variables and its environment interactions have been associated with an increased risk for early onset sub- stance 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 experi- ences 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

Il coenzima Q10 (Co Q10) è un benzochinone che svolge un ruolo unico nella catena di trasporto degli elettroni nei mitocondri da cui deriva la sua funzione di vettore di energia. È anche definito ubichinone per indicare la sua distribuzione ubiquitaria. Esso è presente nella maggior parte degli organismi aerobi: batteri, piante ed animali. Il Co Q10 può esistere in tre stati di ossidazione: completamente ossidato (ubichinone), semichinone (ubisemiquinone) e completamente ridotto (ubichinolo). La capacità di questa molecola di esistere in una forma completamente ossidata e una forma completamente ridotto le permette di operare nella catena di trasporto degli elettroni e di agire come antiossidante. Oltre che per sintesi endogena, Co Q10 è presente in vari alimenti o integratori alimentari. I carotenoidi costituiscono una classe di più di 600 pigmenti naturali sintetizzati da piante, alghe e batteri fotosintetici. Questi tetraterpenoidi organici sono divisi in due classi chimiche: xantofille (che contengono ossigeno) e caroteni (che sono puramente idrocarburi, e non contengono ossigeno). Negli ultimi anni, i carotenoidi hanno ricevuto attenzione come potenziali composti anti-cancro e anti-invecchiamento. I carotenoidi sono potenti antiossidanti, che proteggono le cellule del corpo dai danni causati dai radicali liberi. In questo lavoro è stato utilizzato un metodo HPLC-DAD per l'analisi simultanea di Co Q10 e alcuni carotenoidi per il controllo qualità di integratori alimentari. L'estrazione degli analiti è stata effettuata per mezzo di acetone. I risultati preliminari sono soddisfacenti ed è in corso la convalida del metodo.

Soy products are gaining interest in the health food industry, because of the increasing scientific findings proving their beneficial effects. Soy contains high amounts of proteins and lipids and many bioactive compounds like phytosterols and phytoestrogens. In particular, scientific research focuses on the last category that contains isoflavones naturally present in soy and soy foods. Many of the health benefits of soy are derived from its main isoflavones, genistein and daidzein. Despite the beneficial effects of isoflavones on health are still controversial, some epidemiological studies suggest that the consumption of soy products could play a role in the prevention of several cancers, menopausal symptoms, coronary heart disease and osteoporosis. Consequently, the industry is increasingly developing products that offer an alternative to traditional soy foods like tofu or soy milk, and precisely isoflavone concentrates and extracts for dietary supplements and new functional foods designed to provide health benefits for the consumer. In order to verify the content of the bioactive compounds of the main isoflavones (and their glicosides) in soy foods and derived dietary supplements, some analytical methods have been developed in our Laboratory of Pharmaco-Toxicological Analysis. Firstly, a capillary electrophoresis technique was selected for its high efficiency to separate numerous compounds in these complex food matrices. The development of a second method based on the use of HPLC in reversed phase mode is in progress.

L’assunzione cronica di sostanze oppiacee (es. eroina) comporta un rischio elevato che si sviluppino dipendenza fisica e psicologica. Attualmente l’approccio farmacologico più utilizzato per il trattamento della dipendenza da oppiacei è rappresentato dalla terapia sostitutiva a base di oppioidi a lunga emivita, quali il metadone, la buprenorfina e l’associazione buprenorfina/naloxone, formulata con l’intento di ridurre il potenziale d’abuso della buprenorfina. Il metabolismo degli oppioidi a lunga emivita è soggetto a una grande variabilità interindividuale che impedisce l’utilizzo dello stesso schema posologico per tutti i pazienti. Un adeguato monitoraggio dei livelli plasmatici di questi farmaci risulta pertanto fondamentale per una corretta impostazione terapeutica da parte dei clinici e offre anche il vantaggio di contribuire ad arginare il fenomeno dell’uso improprio del medicinale da parte dei pazienti. Per questo motivo è stato sviluppato un metodo cromatografico con rilevazione elettrochimica per la simultanea quantificazione di metadone e buprenorfina in plasma umano . La separazione è stata ottenuta su una colonna ciano utilizzando una miscela di acetonitrile e tampone fosfato come fase mobile. Gli analiti sono stati monitorati in ossidazione a + 0.6 V. La matrice plasmatica (100 µl) è stata pretrattata mediante microestrazione in fase solida impaccata (MEPS) con sorbent C8. Il metodo è poi stato applicato a numerosi campioni di pazienti in terapia sostitutiva con metadone e l’associazione buprenorfina/naloxone, consentendo un eventuale adeguamento del regime posologico da parte dei clinici.

I capolini di diverse specie di camomilla (Matricaria recutita, Chamaemelum nobile) sono utilizzati sotto forma di decotti ed infusi in virtù delle loro presunte proprietà terapeutiche. Numerosi studi svolti negli ultimi 20 anni hanno evidenziato la presenza di sostanze farmacologicamente attive di natura sia idrofila che lipofila. Tra i principi attivi idrosolubili l’apigenina (nella forma libera e in quella di O-glucoside) ha mostrato attività antinfiammatoria e spasmolitica (come riportato in diversi lavori scientifici sugli estratti acquosi e idroalcolici di camomilla). Anche i costituenti principali dell’olio essenziale, camazulene, alfa-bisabololo, bisabololo ossido e beta-farnesene risultano avere proprietà antiflogistiche, antinfiammatorie, spasmolitiche, cicatrizzanti, antibatteriche e antiossidanti. L’importanza della camomilla dal punto di vista farmacologico è testimoniata anche dalle monografie presenti nelle Farmacopee Ufficiali di 26 paesi e in quella Europea. Pertanto è indispensabile disporre di metodiche analitiche precise e affidabili per poter quantificare esattamente il contenuto di queste sostanze. Per la separazione e l’analisi dell’apigenina negli estratti di camomilla si è sviluppato un metodo HPLC-DAD in grado di quantificare l’apigenina libera, l’apigenina 7-O-glucoside e l’apigenina totale ottenuta in seguito ad idrolisi. Si utilizza una colonna C18 a fase inversa ed una fase mobile composta da acqua ed acetonitrile. Questa metodica è stata messa a confronto con un metodo elettroforetico (CZE-DAD) che utilizza un tampone borato a pH basico. I risultati, ottenuti in modo indipendente con le due tecniche, sono in buon accordo tra loro (contenuto di apigenina non inferiore a 0,25%). Inoltre si sta sviluppando un metodo originale semplice e veloce, basato sulla elettrocromatografia micellare (MEKC) per la determinazione quantitativa del contenuto di camazulene, alfa-bisabololo, bisabololo ossido e beta-farnesene nell’olio essenziale commerciale (ottenuto per distillazione) e nell’olio estratto dai capolini con solventi organici. I primi risultati, confrontati con quelli ottenuti con la metodica ufficiale GC presente nella Farmacopea Europea, sembrano promettenti.

N-acetylcysteine is a biologically active compound used as a mucolytic agent, as a nephroprotective agent and to treat poisonings, mainly those due to acetaminophen. In poisoning cases, its action is thought to be the increase of cysteine available to form glutathione, one of the most important endogenous detoxifiers in the human body. N-acetylcysteine is also commonly found in nutritional supplements studied for body-builders. However, it can cause severe side effects, such as potential damage to heart and lungs and an "anaphilactoid" reaction including rash, asthma and hypotension. D-penicillamine is another thiolic compound widely used in the treatment of poisonings, due to its chelating properties, and as an immunosuppressant in the therapy of rheumatoid arthritis. It is a dimethyl analogue of cysteine and can cause severe side effects such as aplastic anaemia, toxic myopathy and autoimmune responses . For this reason, it is important to have at disposal reliable analytical methods to monitor the plasma levels of N-acethylcysteine, D-penicillamine and L-cysteine. The proposed method is based on capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). Analyte separation was obtained by using a fused silica capillary and a background electrolyte composed of basic carbonate buffer, applying a 20 kV voltage. Direct hydrodynamic injection of samples was carried out after an original microwave-assisted derivatisation step with iodoacetamidoaminofluorescein (IAF). The method sensitivity allows the determination of N-acetyl-cysteine, D-penicillamine and L-cysteine levels down to 10 ng/mL. Preliminary results are promising and method validation is currently in progress for subsequent application to human plasma samples.

La D-penicillamina (Figura 1), derivato dall'idrolisi dell'antibiotico penicillina, è un farmaco senza attività antimicrobica che mostra una azione di tipo chelante ed antiinfiammatoria. Strutturalmente è correlato all'amminoacido cisteina. Viene utilizzata principalmente nel trattamento della malattia di Wilson, in cui agisce chelando il rame e facilitandone l’escrezione nelle urine; e dell’artrite reumatoide, in cui indirettamente inibisce il rilascio di composti pro-infiammatori. L’assunzione cronica può dare origine a seri effetti collaterali quali sindromi autoimmunitarie simili al lupus, reazioni di ipersensibilità e l’elastosi perforante pruriginosa. È stato messo a punto un metodo analitico basato sull’uso dell’HPLC con detector amperometrico per l’analisi dei livelli di D-penicillamina in plasma. La rivelazione dell’analita è effettuata in ossidazione. Come fase stazionaria viene utilizzata una colonna a fase inversa C18 e come fase mobile una miscela di tampone fosfato e metanolo. Il pretrattamento dei campioni biologici prevede l’utilizzo di una estrazione in fase solida con cartucce C18 che permette una buona purificazione della matrice. Il metodo è attualmente in fase di convalida.

In all oxygen consuming cells, metabolism and oxidative stress generate several intermediates and by products that are collectively known as reactive oxygen species (ROS). ROS are necessary intermediates in the human body, but, due to their strong oxidizing power, they are also involved in the aging process and in the development of many degenerative diseases, including cancer, Alzheimer’s and Parkinson’s. Biological antioxidants are able to dispose of ROS. In recent years, clinical trials and epidemiological studies have shown an inverse relationship between the consumption of fruits and vegetables and degenerative diseases and a direct correlation between antioxidant laden food and good health; one major drawback to these latter studies has been determining the antioxidant capacity of these foods and analyzing their specific molecules. For the identification and quantitation of principal active components of some vegetables and fruits like Rosa Canina hips and Citrus species, a fast analytical method based on capillary electrophoresis coupled to diode array detector (CE-DAD) has been developed. Separation of quercetin, catechin, epicatechin, resveratrol and ascorbic acid was obtained in a single run by using a fused silica capillary and a background electrolyte composed of tetraborate buffer (voltage = 15 kV). The development of HPLC-DAD and GC-FID methods is now in progress for the analysis of carotenoids.

Chamomile capitula (Matricaria recutita) have been used for centuries in teas as relaxing sleep aid but recently many publications report therapeutic properties of lipophilic compounds in this plant. The main active constituents of the chamomile essential oil are chamazulene, trans-β-farnesene, α-bisabolol, bisabolone, bisabolol oxide A and B. In particular, chamazulene is responsible for the deep blue colour of oil and has been found to possess anti-inflammatory and analgesic activity. Antibacterial and antiulcer properties have been proved for αbisabolol from several experimental models. Thus, it is important to have at disposal a reliable method for the determination of these active constituents. An HPLC method has been developed for the analysis of lipophilic compounds in Matricaria oils using a RP-C18 column, a mobile phase consisting of phosphate buffer and acetonitrile and a diode-array detector. Moreover, a TLC procedure has been carried out to identify all the analytes and a silica-column was used for bisabolone and bisabolol oxide B purification from commercial oil. HPLC preliminary results were in good agreement with those obtained from GC analyses, performed according to European Pharmacopoeia.

Introduction: The collection of blood samples on filter papers, known as dried blood spots (DBS), is an established technique for the screening of newborn errors of metabolism. Recently, there is a large evidence for its use in the therapeutic drug monitoring (TDM) field for the quantitative analysis of small molecules. The collection of whole blood as DBSs has many advantages in comparison to conventional plasma sampling: smaller sample volumes, less invasive withdrawal, easier storage and shipment, less requirements for biohazard arrangements. In the last decade numerous analytical methods have been developed in our Laboratory of Pharmaco-Toxicological Analysis for the plasmatic monitoring of several Central Nervous System drugs in collaboration with national and foreign Psychiatric Clinics. Thus, the DBS technique as sample collection could be advisable for TDM purposes of atypical antipsychotics. Methods: Some analytical methods by means of high performance liquid chromatography coupled with both electrochemical and spectrophotometric detectors have been implemented for the analysis of clozapine, olanzapine, risperidone and aripiprazole in DBSs from psychiatric patients. Blood was spotted onto filter papers and allowed to dry. The quantitative assays employed simple solvent extraction of the analytes from the DBS samples, followed by solid-phase extraction (SPE) or micro-SPE procedures for the clean-up of the biological samples. Results: Preliminary results showed good extraction yield values of clozapine and risperidone from DBSs, being always higher than 85.0 %. Results were satisfactory in terms of sensitivity and selectivity. Moreover, preliminary stability data showed that the samples in the DBS form can be stored for a long period of time before the analysis without the need of refrigeration or any particular arrangements. Clozapine and risperidone levels obtained from DBSs were in good agreement with those obtained from plasma analysis [1,2] after multiplying the DBS values by 1.8. Discussion: Assays are currently in progress to validate the methods in terms of linearity, precision and accuracy and to apply them to TDM of numerous patients treated with clozapine, olanzapine, risperidone and aripiprazole.

Il cortisolo ((11β)-11,17,21-triidrossipregn-4-ene-3,20-dione) è un ormone steroideo o glucocorticoide prodotto dalla parte corticale delle ghiandole surrenali in risposta all’ormone adrenocorticotropo ipofisario (ACTH). Viene spesso definito ormone dello stress, perché la sua produzione aumenta in condizioni particolari di grave stress psico-fisico. Il cortisone (17,21-diidrossipregn-4-en-3,11,20-trione) è uno dei numerosi prodotti finali del processo di steroidogenesi. È il precursore inattivo del cortisolo, infatti, è convertito dall’enzima 11β-steroidodeidrogenasi nella forma attiva del cortisolo. Il corticosterone ((11β)-11,21-diidrossipregn-4-ene-3,20-dione) è un ormone che protegge contro lo stress, influenza l'efficienza muscolare e il bilancio elettrolitico. Lo scopo di questo studio è lo sviluppo di un metodo affidabile e semplice per la quantificazione simultanea di cortisolo, cortisone e corticosterone in plasma umano e dried blood spots (DBS). A tal fine è stato utilizzato un sistema HPLC a fase inversa accoppiato ad un detector Diode Array. Inoltre, è stata sviluppata una procedura di microestrazione in fase solida (MEPS) per il clean-up delle matrici biologiche. Sono stati ottenuti buoni risultati in termini di resa di estrazione, linearità e precisione. Ulteriori analisi sono in corso per terminare la convalida del metodo analitico.

Opioid replacement therapy with long-acting opioids is the most effective approach to counteract heroin dependence, a worldwide health and social problem affecting the lives of millions of people. Since its introduction in the 1960s, methadone ((RS)-6-(dimethylamino)-4,4-diphenylheptan-3-one, MTD), a synthetic full µ-opioid receptor agonist, has been the treatment of choice for opioid addiction. However the outcome of the pharmacological therapy with MTD is highly variable depending on different factors, such as individual metabolism. Thus, it is important to accurately determine blood levels of MTD to optimize the dosage in the patient and to monitor possible abuse of the drug. Nowadays, clinical laboratories are constantly confronted with the increasing demand for higher sample throughput, thus shorter analysis times; it is advisable to have at disposal high-sensitivity and low-cost methods to determine blood levels of MTD in heroin addicted patients. A rapid isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) with coulometric detection has been developed for the determination of MTD in dried blood spot (DBS) and human plasma samples. Furthermore the aim of this study was to evaluate the reliability of dried blood spot specimens in the determination of MTD levels in whole blood from heroin addicted patients. Advantages of DBS technique include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. The clean-up of DBS was performed by means of an original MEPS (microextraction by packed sorbent) procedure after a microwave-assisted extraction of the drug with acetonitrile. Promising results were obtained in terms of extraction yield and sensitivity. Assays are in progress in order to completely validate the method.

Introduction: Therapeutic drug monitoring (TDM) of psychiatric patients is advisable, with the aim of reducing side effects and optimizing the therapy, and is a particularly useful tool if one considers the poor compliance of treated patients. Starting from clinical-chemical correlation data, TDM allows therapy personalization according to the specific needs of the patient. This in turn leads to increased patient compliance and to improved drug efficacy, and finally also to lower total treatment expenses, due to the reduction in hospitalisation length and frequency [1]. Methods: Performing the TDM obviously requires suitable analytical methods, consisting in two steps: sample pre-treatment and instrumental analysis. The first step is critical for the removal of biological interferences. The second step is usually carried out by liquid chromatography or capillary electrophoresis, coupled with different detectors, selected on the basis of the drug physico-chemical properties and expected plasma levels. Therefore, the methods and the corresponding techniques should possess adequate sensitivity and selectivity for the particular analytical problem. Results: Psychiatric patients are very often subjected to polypharmacy: for example, with antipsychotic and anxiolytic-hypnotic drugs for the treatment of schizophrenia; or with atypical antipsychotics (quetiapine, olanzapine) and antiepileptic agents (lamotrigine, oxcarbazepine) for bipolar disorder [2]. This fact constitutes a great challenge for both psychiatrists and analytical laboratories. Conclusions: The possibility of unwanted drug interactions is a constant threat during polypharmacy. Drug metabolism can be strongly influenced by the administration of other drugs, leading to possible toxicity, when a reduced metabolism is observed, or to a lack of response to therapy in the opposite case. The presence of numerous drugs and metabolites in the patient plasma makes the analytical problem, as well as the interpretation of the resulting plasma level data, much more complicated. At the same time, it is exactly this kind of challenge that makes it so exciting and interesting to work on plasma monitoring. References: [1] M.A. Raggi, R. Mandrioli, V. Pucci, C. Sabbioni, Advances in therapeutic drug monitoring of atypical antipsychotic drugs. Med. Chem. Rev. 1, 299–316 (2004). [2] A. Musenga, M.A. Saracino, G. Sani, M.A. Raggi, Antipsychotic and antiepileptic drugs in bipolar disorder: the importance of therapeutic drug monitoring. Curr. Med. Chem. 16, 1463–1481 (2009)

Numerosi studi trasversali hanno evidenziato che la terapia sostitutiva con oppioidi a lunga emivita tra i quali la buprenorfina e l’associazione buprenorfina/naloxone può contribuire a migliorare lo stato di salute dei pazienti, oltre che a ridurre l’incidenza di malattie infettive, delle morti e delle attività criminali connesse all’uso di sostanze d’abuso quali l’eroina. Il corretto dosaggio giornaliero per ottenere la stabilizzazione farmacologica e clinica del paziente, controllata con un adeguato monitoraggio dei livelli plasmatici e/o urinari della buprenorfina, sembra avere un ruolo importante non solo per una corretta impostazione terapeutica da parte dei clinici ma anche per arginare il fenomeno dell’uso improprio del farmaco da parte dei pazienti. A tal fine è fondamentale avere a disposizione metodi analitici affidabili in grado di identificare e quantificare la buprenorfina e i suoi metaboliti in diverse matrici biologiche. Tali metodi devono avere la caratteristica di essere sensibili e selettivi nonché accurati e precisi e di essere validati secondo le linee guida internazionali. In questo contributo si vuole dare una panoramica delle proprietà farmaco-tossicologiche della buprenorfina oltre che delle metodiche presenti in letteratura per l’analisi in diverse matrici biologiche, che verranno confrontate criticamente.

Molti studi preclinici ed epidemiologici hanno evidenziato che i fitoestrogeni, sostanze naturali non steroidee contenute nelle piante, potrebbero svolgere un ruolo importante nella prevenzione di diverse malattie ormone-dipendenti come il cancro al seno, i disturbi della menopausa e l'osteoporosi. Negli ultimi anni, sono stati introdotti sul mercato integratori alimentari o formulazioni a base di estratti di soia, per il trattamento dei sintomi della menopausa , in quanto osservazioni epidemiologiche hanno evidenziato una minore incidenza dei disturbi climaterici, dei tumori mammari, uterini e intestinali nelle donne asiatiche, la cui dieta giornaliera è molto ricca in estratti della soia. Questa contiene fitoestrogeni, in particolare gli isoflavoni genisteina e daidzeina, che hanno una conformazione chimica simile a quella dell'estradiolo, il principale ormone sessuale femminile. Si ritiene che gli isoflavoni della soia agiscano attraverso un effetto di modulazione selettiva sui recettori estrogenici (azione ormone-simile), che riducano la produzione di radicali liberi (“radical scavenger”) e che inibiscano la proliferazione di cellule tumorali (azione non ormonale). L’abbondanza di prodotti commerciali a base di derivati della soia determina la necessità di procedere ad un loro accurato controllo di qualità al fine di evidenziare possibili adulterazioni o escludere la possibilità di sovradosaggi. Nel Laboratorio di Analisi Farmaco-Tossicologica sono stati sviluppati alcuni metodi analitici basati sia sulla cromatografia elettrocinetica micellare (MEKC) che sulla cromatografia liquida ad alta prestazione (HPLC) per il controllo di qualità di integratori commerciali contenenti estratti di soia. In particolare, l’analisi elettroforetica di genisteina, daidziena, gliciteina (e loro glicosidi) è stata effettuata, utilizzando un elettrolita supporto (BGE) a pH basico contenente sodio dodecil solfato . Si sta ora mettendo a punto un metodo più sensibile e selettivo basato sull’uso di un HPLC con detector coulombometrico. Inoltre, è in fase di sviluppo anche un metodo semplice e veloce che utilizza un HPLC con detector Diode Array che permette oltre alla quantificazione anche l’identificazione degli analiti attraverso gli spettri di assorbimento UV. I risultati preliminari sembrano essere soddisfacenti; si sta proseguendo con i test per la convalida dei metodi.

Studi epidemiologici e clinici evidenziano la frequente associazione tra le Esperienze Infantili Avverse (EIA) e l’aumento di vulnerabilità allo sviluppo di abuso di sostanze psicotrope. Le diverse forme di EIA sono altresì coinvolte nella predisposizione allo sviluppo di vari disturbi quali l’obesità patologica, i disturbi cardiovascolari, e di altre condizioni di rischio per la salute. A tale azione conseguono stabili perturbazioni a carico del sistema nervoso centrale che esitano in alterazioni sia sul piano neurobiologico che comportamentale. Ad oggi, non sono ancora del tutto noti i meccanismi sottesi all’interazione tra geni ed ambiente responsabili dell’aumento della vulnerabilità allo sviluppo di abuso di sostanze psicotrope. Anche se l’aumento della vulnerabilità all’uso di sostanze psicotrope è stata principalmente attribuita ad alterazioni di tipo genetico, recenti studi hanno evidenziato come le varie forme di EIA siano anche in grado di modificare diverse vie neurotrasmettitoriali cerebrali, ed in particolare quella serotoninergica e catecolaminergica, ed alcuni sistemi neuroendocrini, con conseguente sviluppo di disturbi comportamentali. In particolare, la correlazione inversa evidenziata dalle nostre precedenti ricerche tra le concentrazioni plasmatiche di acido omovanillico (HVA) e le misure di attaccamento disorganizzato in pazienti con dipendenza da sostanze sembra avanzare l’ipotesi già postulata anche da vari autori circa il ruolo degli EIA nell’indurre alterazioni neuroendocrine a lungo termine.

La buprenorfina ((2S)-2-[(-)-(5R,6R,7R,14S)-9α-ciclopropilmetil-4,5-epossi-6,14-etano-3-idrossi-6-metossimorfinan-7-il]-3,3-dimetilbutan-2-olo, BUP) è un agonista parziale dei recettori per gli oppiacei di tipo μ e un antagonista dei recettori k che trova indicazione nel trattamento farmacologico della dipendenza da oppiacei. Al fine di ridurre il potenziale di misuso endovenoso di questo farmaco nei pazienti tossicodipendenti, è stata sviluppata una nuova specialità medicinale, Suboxone®, costituita dall’associazione di BUP con l’antagonista non selettivo dei recettori per gli oppiacei, il naloxone ((5α,17R)-4,5-epossi-3,14-diidrossi-6-oxo-17-(2-propenil)-morfinano, NAL), in un rapporto stechiometrico di 4:1. Allo stato attuale delle conoscenze cliniche risulta essere particolarmente importante ottenere dati di farmacocinetica sia del prodotto di associazione che della sola BUP, al fine di poter comparare su ampia scala di pazienti la biodisponibilità plasmatica dei due farmaci. A questo scopo è stato sviluppato un metodo HPLC con detector coulombometrico che consente la simultanea quantificazione di BUP e NAL nel plasma. La separazione è stata ottenuta su una colonna CN utilizzando una miscela di tampone fosfato e metanolo come fase mobile. La matrice plasmatica è stata purificata mediante procedura di Microestrazione in Fase Solida Impaccata (MEPS). La metodica, la cui ottimizzazione e convalida sono tuttora in corso, ha fornito finora risultati promettenti. Una volta convalidato, il metodo sarà utilizzato per condurre studi di farmacocinetica su un ampio numero di pazienti trattati con Suboxone®.

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.

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.

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 metabolite9-hydroxyrisperidone in human plasma and saliva. The analytes were separatedon 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 +0.500 V and electrode 2 at +0.700 V. The detector response was linear over a plasma and saliva concentration range of 0.5–50.0 ng mL−1 for risperidone and 0.5–100.0 ng mL−1 for 9-hydroxyrisperidone. The limit of quantitation and the limit of detection for risperidone and 9-hydroxyrisperidone were 0.5 ng mL−1 and 0.17 ng mL−1, respectively. A novel clean-up procedure of biological samples was developed using the micro extraction 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.

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.

Il risperidone (4-[2-[4-(6-fluorobenzo[d]isossazol-3-il)-1-piperidil]etil]-3-metil-2,6-diazabiciclo[4.4.0]deca-1,3-dien-5-one) è una antipsicotico atipico utilizzato per il trattamento di patologie psichiatriche, come schizofrenia e disturbo bipolare; viene impiegato anche per curare l'irritabilità di bambini e adolescenti autistici (1). Il risperidone viene metabolizzato dal citocromo P-450 in 9-idrossirisperidone con attività farmacologica paragonabile a quella del risperidone; la somma delle loro concentrazioni forma la frazione antipsicotica attiva (active moiety). Un metodo precedentemente sviluppato nel nostro Laboratorio di Analisi Farmaco-Tossicologica per la determinazione di antipsicotici mediante HPLC-UV permetteva anche l’analisi su plasma del risperidone dopo procedura di estrazione in fase solida (SPE). Lo scopo di questo lavoro è lo sviluppo di un metodo analitico in grado di determinare l’active moiety in “dried blood spots” di pazienti psichiatrici, previa microestrazione in fase solida (MEPS). Il sangue intero raccolto dai pazienti viene fatto essiccare su dischetti di carta da filtro; successivamente gli analiti vengono estratti con un opportuno solvente e l’estratto viene sottoposto a procedura MEPS. L’analisi cromatografica si basa sull’uso di una colonna a fase inversa C18 e di una fase mobile composta da tampone fosfato e acetonitrile. La rivelazione è elettrochimica, di tipo coulombometrico. I primi risultanti sono promettenti e il metodo analitico è in fase di convalida.

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 chemicalclinical 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.

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 50 mM 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 12.5 mM 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%.