A research route in the actions against the abuse of psychotropic substances

The research route of this PhD started from workplace drug testing, an emerging need for safety and hygiene at work. The aim of the work consisted of developing and validating analytical methods: innovative tools for drug addiction monitoring and for illicit drugs testing. Different classes of psychotropic substances are examined in relation to their chemical and pharmacological effects, with a special focus on new psychoactive substances (NPS) and new trends of abuse. The most important biological matrices, suitable for analysis, are studied and the peculiarities of each matrix are highlighted. Analytical methods were developed for drugs determination in biological samples, such as human urine, plasma, oral fluids and hair. All these matrices are equally important for forensic purposes as they give different kind of information about the time of assumption and they show some distinctive features. Sample preparation is a critical step and is a key factor in determining the success of analysis: the task of chemist is to develop the best strategy to ensure that the system, consisting of 3 variables, matrix - target analytes - instrumental equipment, will be solved consistently with the ultimate purpose of the analysis. Therefore in this project appropriate sample preparation techniques have been investigated with special regard to each different matrix. The manual operations associated with these processes are often expensive, labor-intensive and time-consuming and affect the uncertainty of the final analytical data, for these reasons in this work new procedures based on innovative sample preparation were investigated and developed. The goal has been to provide short sample preparation times, automation of the clean-up step and low sample volumes, minimizing the consumption of solvents and chemicals. Development of methods based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was a crucial component of this work. LC–MS is considered to be the benchmark for quantitative/qualitative bioanalysis, providing specificity, sensitivity and speed to the method. Different chromatographic conditions have been evaluated for the optimal separation and detection of the investigated compounds. As a new trend, the application of columns with superficially porous packing materials has been investigated, which allowed fast chromatographic runs and high efficiency. The versatility of the LC-MS/MS has been proven by providing a single analytical method for the determination of 11 drugs of abuse in biological fluids: conventional matrices, such as plasma and urine, and unconventional ones, such as oral fluids. The research route was then directed to hair analysis and for the first time pressurised liquid extraction (PLE) was shown to be effective for the extraction of drugs belonging to different classes from hair samples. Experiments were carried out to ensure correct interpretation of the analytical data by studying the phenomenon of external contamination (a critical point in the analysis of hair for forensic purposes). To improve the methods selectivity, multi reaction monitoring (MRM) acquisition modes were used for quantification of the investigated compounds in both cases. Finally analytical target was extended to NPS: a method for the screening of methylenedioxyamphetamine and piperazine-derived compounds in urine was developed. These substances, characterized by possessing common moieties, are screened using precursor ion and neutral loss scan mode and then quantified in MRM acquisition mode. All the methods developed have been validated according to specific international guidelines to ensure reliable results, unambiguously interpreted and useful for forensic purposes

Pressurized-liquid extraction for determination of illicit drugs in hair by LC-MS-MS

An LC-MS-MS-based procedure for determination in hair of 14 different drugs of abuse belonging to the classes cocaine, amphetamine-like compounds, opiates, and hallucinogens has been developed. A pressurized-liquid extraction procedure was used and proved useful for quantitative recovery of all the analytes tested. This procedure, in conjunction with a simple decontamination step, performed to avoid false-positive samples, enabled the detection of all the analytes with LOQ ranging from 1.8 to 16 pg mg -1 and accuracy varying from 85 to 111 %. The procedure was validated in accordance with the SOFT/AAFS guidelines and seems to be suitable for routine determination of the drugs tested in hair

Analytical approaches for the determination of phytocannabinoids and endocannabinoids in human matrices

Over the last two decades, the role played by phytocannabinoids and endocannabinoids in medicine has gained increasing interest in the scientific community. Upon identification of the plant compound Δ(9) -tetrahydrocannabinol (THC) and of the endogenous substance anandamide (AEA), different methodological approaches and innovative techniques have been developed, in order to evaluate the content of these molecules in various human matrices. In this review, we discuss the analytical methods that are currently used for the identification of phytocannabinoids and endocannabinoids, and we summarize the benefits and limitations of these procedures. Moreover, we provide an overview of the main biological matrices that have been analyzed to date for qualitative detection and quantitative determination of these compounds.Over the last two decades, the role played by phytocannabinoids and endocannabinoids in medicine has gained increasing interest in the scientific community. Upon identification of the plant compound Δ9-tetrahydrocannabinol (THC) and of the endogenous substance anandamide (AEA), different methodological approaches and innovative techniques have been developed, in order to evaluate the content of these molecules in various human matrices. In this review, we discuss the analytical methods that are currently used for the identification of phytocannabinoids and endocannabinoids, and we summarize the benefits and limitations of these procedures. Moreover, we provide an overview of the main biological matrices that have been analyzed to date for qualitative detection and quantitative determination of these compounds

Monitoraggio di droghe d'abuso in fluidi biologici mediante HPLC-MS/MS

L'importanza di poter effettuare analisi multianalita e multiclasse è sempre più evidente in tutti i campi della chimica analitica, in particolare quando sia necessario ottenere dati inequivocabili in tempi brevi. Nel caso della verifica dell'uso di droghe d'abuso da parte di soggetti considerati "a rischio sociale", ovvero alla guida di autoveicoli o con particolari mansioni lavorative, poter unire un campionamento non invasivo, come la raccolta di fluidi orali, con una tecnica analitica potente come l'HPLC-MS/MS, fornisce uno strumento molto utile agli enti di controllo per garantire risposte analitiche spendibili in sedi ufficiali. Lo scopo di questo lavoro è stato, quindi, lo sviluppo di una procedura analitica basata su HPLC-MS/MS per la determinazione di droghe d'abuso appartenenti a diversi classi chimiche (oppiacei, amfetamine, allucinogeni, cocaina, cannabinoidi), insieme ai loro metaboliti in plasma e saliva

Pressurized solvent extraction for the determination of illicit drugs in hair by HPLC-MS/MS

Plasma and urine are the most commonly used matrices for illicit drug testing, but since 1979 there has been an increasing interest in the development of analytical methods in alternative matrices such as hair [1]. Hair has the advantage of a substantially longer detection window (months to years) which enables retrospective investigation of chronic consumption. In addition, hair is a durable and stable matrix difficult to adulteration in which toxic substances are preconcentrated and remain for a long time without significant alterations; furthermore the sampling is not invasive. Indeed hair analysis of illicit drugs has recently been codified in the Italian legislation as a monitoring tool in the field of workplace safety. The critical step is certainly the extraction phase, usually performed by solid liquid extraction for multiclass methods; the main drawback is the long contact time (from 16 to 20 hours) between hair and solvent necessary to have good recoveries. Times are greatly reduced if the extraction is assisted by ultrasonic or increased temperature. Pressurized Liquid Extraction (PLE) was applied to significantly reduce extraction time since there are no PLE applications in this field. So a method based on HPLC-ESI-MS/MS has been developed and validated for multiresidual determination of illicit drugs from hair: amphetamine, methamphetamine, mescaline, MDA, MDMA, MDEA, cocaine, benzoylecgonine, nor-cocaine, ketamine, phencyclidine, diacetylmorphine, morphine, 6-monoacetylmorphine, codeine. PLE led to reduce the analysis time and automate the extraction process, taking into account sample washing to remove external contamination and the clean-up of the extracts. Satisfactory extraction rates have been obtained using water as extracting and 10 minutes as extraction time

Pressurized solvent extraction for the determination of illicit drugs in hair by HPLC-MS/MS

Plasma and urine are the most commonly used matrices for illicit drug testing, but since 1979 there has been an increasing interest in the development of analytical methods in alternative matrices such as hair [1]. Hair has the advantage of a substantially longer detection window (months to years) which enables retrospective investigation of chronic consumption. In addition, hair is a durable and stable matrix difficult to adulteration in which toxic substances are preconcentrated and remain for a long time without significant alterations; furthermore the sampling is not invasive. Indeed hair analysis of illicit drugs has recently been codified in the Italian legislation as a monitoring tool in the field of workplace safety. The critical step is certainly the extraction phase, usually performed by solid liquid extraction for multiclass methods; the main drawback is the long contact time (from 16 to 20 hours) between hair and solvent necessary to have good recoveries. Times are greatly reduced if the extraction is assisted by ultrasonic or increased temperature. Pressurized Liquid Extraction (PLE) was applied to significantly reduce extraction time since there are no PLE applications in this field. So a method based on HPLC-ESI-MS/MS has been developed and validated for multiresidual determination of illicit drugs from hair: amphetamine, methamphetamine, mescaline, MDA, MDMA, MDEA, cocaine, benzoylecgonine, nor-cocaine, ketamine, phencyclidine, diacetylmorphine, morphine, 6-monoacetylmorphine, codeine. PLE led to reduce the analysis time and automate the extraction process, taking into account sample washing to remove external contamination and the clean-up of the extracts. Satisfactory extraction rates have been obtained using water as extracting and 10 minutes as extraction time

Micro-solid phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry for the determination of stimulants, hallucinogens, ketamine and phencyclidine in oral fluids

A confirmatory method for the determination of illicit drugs based on micro-solid phase extraction with modified tips, made of a functionalized fiberglass with apolar chains of octadecylsilane into monolithic structure, has been developed in this study. Drugs belonging to different chemical classes, such as amphetamine, methamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethylamphetamine, cocaine, benzoylecgonine, ketamine, mescaline, phencyclidine and psilocybine were analyzed. The quantitation was performed by liquid chromatography-tandem mass spectrometry and the analytes were detected in positive ionization by means of an electrospray source. The limits of quantification ranged between 0.3 ng mL(-1) for cocaine and 4.9 ng mL(-1) for psilocybine, with coefficients of determination (r(2)) >0.99 for all the analytes as recommended in the guidelines of Society of Forensic Toxicologists-American Association Forensic Sciences. (C) 2010 Elsevier B.V. All rights reserved

Neutral loss and precursor ion scan tandem mass spectrometry for study of activated benzopyrene-DNA adducts

Methodology for detection of activated benzo[a] pyrene (B[a] P)-nucleoside adducts by liquid chromatography-tandem mass spectrometry is reported. Adducts of B[a]P-dihydrodiol epoxide (B[a]PDE) with guanosine and adenosine have been detected for the first time by use of precursor ion scan and neutral loss scan. B[a]P was then activated by use of UV irradiation and some of the products obtained have been identified by taking advantage of the information obtained for B[a]PDE. Photoactivation has also been carried out in the presence of hydrogen peroxide; this resulted in a higher yield of products with increased production of BaP diones. The reactivity of these compounds toward nucleosides has been tested. The proposed method was successfully used for detection of one stable guanosine-B[a]P dione adduct