Monitoring of New Psychoactive Substances in the hair of patients with a history of addiction. Evaluation of the public health impact.

Introduction. The term New Psychoactive Substances (NPS) encompasses a broad category of drugs which have become available on the market in recent years and whose illicit use for recreational purposes has recently exploded. The analysis of NPS usually requires mass spectrometry based techniques. The aim of our study was to define the preva-lence of NPS consumption in patients with a history of drug addiction followed by Public Services for Pathological Addictions, with the purpose of highlighting the effective presence of NPS within the area of Bologna and evaluating their association with classical drugs of abuse (DOA). Materials and methods. Sustained by literature, a multi-analyte UHPLC-MS/MS method for the identification of 127 NPS (phenethylamines, arylcyclohexylamines, synthetic opioids, tryptamines, synthetic cannabinoids, synthetic cathinones, designer benzodiazepines) and 15 classic drugs of abuse (DOA) in hair samples was developed and validated according to International Guidelines [112]. Samples pretreatment consisted of washing steps and overnight incubation at 45°C in an acid mixture of methanol and water. After cooling, supernatant were injected into the chromatographic system coupled with a tandem mass spectrometry detector. Results. Successful validation was achieved for almost all of the compounds. The method met all the required technical parameters. LOQ was set from 4 to 80 pg/mg The developed method was applied to 107 cases (85 males and 22 females) of clinical interest. Out of 85 hair samples resulting positive to classical drugs of abuse, NPS were found in twelve (8 male and 4 female). Conclusion. The present methodology represents an easy, low cost, wide-panel method for the de-tection of 127 NPS and 15 DOA in hair samples. Such multi-analyte methods facilitates the study of the prevalence of drugs abused that will enable the competent control authorities to obtain evi-dence-based reports regarding the critical spread of the threat represented by NPS.

Original analytical methods for the determination of psychoactive compounds in complex matrices

This thesis work aims to develop original analytical methods for the determination of drugs with a potential for abuse, for the analysis of substances used in the pharmacological treatment of drug addiction in biological samples and for the monitoring of potentially toxic compounds added to street drugs. In fact reliable analytical techniques can play an important role in this setting. They can be employed to reveal drug intake, allowing the identification of drug users and to assess drug blood levels, assisting physicians in the management of the treatment. Pharmacological therapy needs to be carefully monitored indeed in order to optimize the dose scheduling according to the specific needs of the patient and to discourage improper use of the medication. In particular, different methods have been developed for the detection of gamma-hydroxybutiric acid (GHB), prescribed for the treatment of alcohol addiction, of glucocorticoids, one of the most abused pharmaceutical class to enhance sport performance and of adulterants, pharmacologically active compounds added to illicit drugs for recreational purposes. All the presented methods are based on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) coupled to various detectors (diode array detector, mass spectrometer). Biological samples pre-treatment was carried out using different extraction techniques, liquid-liquid extraction (LLE) and solid phase extraction (SPE). Different matrices have been considered: human plasma, dried blood spots, human urine, simulated street drugs. These developed analytical methods are individually described and discussed in this thesis work.

Use of bioassays and biomarkers in Daphnia magna to assess the effect of pharmaceutical residuals in freshwater ecosystems

Widespread occurrence of pharmaceuticals residues has been reported in aquatic ecosystems. However, their toxic effects on aquatic biota remain unclear. Generally, the acute toxicity has been assessed in laboratory experiments, while chronic toxicity studies have rarely been performed. Of importance appears also the assessment of mixture effects, since pharmaceuticals never occur in waters alone. The aim of the present work is to evaluate acute and chronic toxic response in the crustacean Daphnia magna exposed to single pharmaceuticals and mixtures. We tested fluoxetine, a SSRI widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization and chronic reproduction tests were performed according to OECD guidelines 202 and 211, respectively. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design with concentrations based on Toxic Units. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. The MixTox statistical method was applied to analyze the experimental results. Results showed a significant deviation from CA model that indicated antagonism between chemicals in both the acute and the chronic mixture tests. The study was integrated assessing the effects of fluoxetine on a battery of biomarkers. We wanted to evaluate the organism biological vulnerability caused by low concentrations of pharmaceutical occurring in the aquatic environment. We assessed the acetylcholinesterase and glutathione s-transferase enzymatic activities and the malondialdehyde production. No treatment induced significant alteration of biomarkers with respect to the control. Biological assays and the MixTox model application proved to be useful tools for pharmaceutical risk assessment. Although promising, the application of biomarkers in Daphnia magna needs further elucidation.