Detection of new psychoactive substances (NPS) use across Europe Sara Castiglioni, Ettore Zuccato, Noelia Salgueiro-González, Emma Gracia Lor Lisbon, 25th October 2017 - LX Addiction

New Psycoactive Substances (NPS) are Identification and Assessment of New Psychoactive Substances: A European Network New Psycoactive Substances (NPS) are substances not controlled by the international drug control conventions, which may pose public health threat NPS have similar structure of drugs under international control such as cannabis, cocaine, heroin, MDMA (“ecstasy”), amphetamine

Identification and Assessment of New Psychoactive Substances: A European Network How many? EMCDDA EU Early Warning System (EWS) were established to identify these substances in the market in a short time and evaluate health risks Cooperation with UNODC, Europol, EMA, WHO EMCDDA–Europol 2016 Annual Report on the implementation of Council Decision 2005/387/JHA

Identification and Assessment of New Psychoactive Substances: A European Network NPS classes EMCDDA–Europol 2016 Annual Report on the implementation of Council Decision 2005/387/JHA

Few information available Identification and Assessment of New Psychoactive Substances: A European Network The route of NPS EMCDDA– New Psycoactive Substances in Europe High number of substances Mostly not controlled Appear very fast in the market Used in specific subpopulations Mixed in unknown cocktails Potential high toxic effects Very difficult to assess prevalence of use Few information available

Wastewater – Based Epidemiology (WBE) Identification and Assessment of New Psychoactive Substances: A European Network Wastewater – Based Epidemiology (WBE) Measurement of excretion residues (biomarker) in urban wastewater Evaluation of consumption or exposure to substances Almost all substances we consume are excreted unchanged or as a mixture of metabolites in urine and/faeces Excreted substances entry in sewer network Enter sewage treatment plants Sampling and analysis untreated wastewater

WBE Advantages and Challenges for NPS Identification and Assessment of New Psychoactive Substances: A European Network WBE Advantages and Challenges for NPS Ability to provide: Challenges: Objective estimates Limited use Lots of substances Low levels in wastewater Near real-time estimates Updated information Human metabolism unknown Difficult identify biomarkers Qualitative and quantitative data Identification of NPS Evaluation of use WBE needs to be tested

Preliminary Investigations Identification and Assessment of New Psychoactive Substances: A European Network Preliminary Investigations Europe González-Mariño et al., ES&T , 2016 Australia Chen et al., Forensic Science International, 2013

Assess the use of New Psycoactive Substances in Europe Identification and Assessment of New Psychoactive Substances: A European Network Aim of the study Identification and Assessment of New Psycoactive Substances in Europe using WBE Select substances (NPS) to be investigated Develop a method to screen the presence of NPS (qualitative analyses) Develop a method to measure NPS (quantitative analyses) Monitor wastewater samples from different countries Assess the use of New Psycoactive Substances in Europe Spatial and Temporal Trends

NPS Selection Sources DATABASE 193 substances Criteria Identification and Assessment of New Psychoactive Substances: A European Network NPS Selection Alerts Reports of new substances (December 2013-March 2016) Sources DATABASE 193 substances phenethylamines, arilcicloexilamine - ketamine analogues, piperidine, tryptamine, synthetic cathinones, synthetic cannabinoids, synthetic opioids, aminorex derivates, natural psychoactive substances, benzodiazepines Alerts (Europe and Italy) + Italy seizure list + Notifications in Italy (intoxications) (October 2013-March 2016) UNODC control list Criteria Frequency of reporting Most recently reported

Samples investigated COST SCORE Monitoring campaign 2016 (March- May) Identification and Assessment of New Psychoactive Substances: A European Network Samples investigated COST SCORE Monitoring campaign 2016 (March- May) Italy Switzerland Portugal Slovakia The Netherlands Slovenia Bulgaria Romania Poland Bosnia and Herzegovina Hungary Serbia Ireland Raw Wastewater Samples 14 European countries Pooled samples from the weekend

Pooled wastewater sample – Solid Phase Extraction Identification and Assessment of New Psychoactive Substances: A European Network Samples analyses Pooled wastewater sample – Solid Phase Extraction 100 mL (pH 7) 100 mL (pH 2) Oasis HLB Oasis MCX 200 µL (10% MeOH) 200 µL (10% MeOH) The second aliquot was extracted with a Oasis MCX cartridge, which is more selective for basic compounds. In both cases the eluates were reconstituted in 200 uL, it means that samples were preconcentrated 500 times. Finally, the extracts were injected in the Orbitrap Qexactive we have. Each vial was injected 2 times, the first time for a screening and the second one for the confirmation of the suspected compounds. Orbitrap Q-Exactive Qualitative Screening No standards available API 5500 QqQ Quantitative Analysis Standards available

Quantitative Analysis - Results Identification and Assessment of New Psychoactive Substances: A European Network Quantitative Analysis - Results Synthetic Cathinones Mephedrone Methcathinone N,N-Dimethylcathinone Ethcathinone Methedrone 4-FMC 3,4-DMMC 4-MEC Buphedrone Pentedrone Methylone Ethylone Butylone Pentylone 1-Naphyrone Naphyrone MDPV α -PVP 4-Cl- α -PPP 19 substances searched 9 substances found in wastewater Phenethylamine PMA PMMA 25-B-NBoMe 25-C-NBoMe 25-I-NBoMe   Ketamine analogues Methoxethamine Tryptamine 5-MeO-MIPT 7 substances searched 1 substance found in wastewater Limits of quantification 0.3 - 2 ng/L

Quantitative Analysis - Results Identification and Assessment of New Psychoactive Substances: A European Network Quantitative Analysis - Results Not found in Bosnia Herzegovina and Ucraine

Quantitative Analysis - Results Identification and Assessment of New Psychoactive Substances: A European Network Quantitative Analysis - Results

Qualitative Analysis – Results Identification and Assessment of New Psychoactive Substances: A European Network Qualitative Analysis – Results (193 substances investigated) Synthetic cathinones 3,4 DmeO-alpha-PVP Poland, Romania Tryptamine α-methyltryptamine The Netherlands Aminorex derivate N-methyaminorex Slovenia   4,4 DMAR Ucraine Phenethylamine NPDPA Serbia, Romania 2-methoxyamphetamine Bosnia Herzegovina 25iP-NBoME Romania Synthetic cannabinoids MDMB-CHMICA Poland 5-fluoropenthyl-3-pyridinoylindole Switzerland, Slovakia Piperidine 2-MeO-diphenidine The UK Criteria for Identification: Accurate Mass Mass Error < 5 ppm Presence of Fragments Standards needed for confirmation

Conclusions WBE was applied successfully to monitor the use of NPS Identification and Assessment of New Psychoactive Substances: A European Network Conclusions WBE was applied successfully to monitor the use of NPS A qualitative screening was performed for about 200 substances A quantitative analysis was done for > 25 substances The levels of NPS were lower than those of the “classical” drugs – lower use ? Differences among countries were assessed WBE is a good complementary tool to evaluate the use of NPS

Paul Griffith, Liesbeth Vandam, Ana Gallegos Identification and Assessment of New Psychoactive Substances: A European Network Acknowledgements Project Partners 1. Mario Negri Institute (coordinator) 2. University Jaume I 3. Faculdade de Farmacia Universidade de Lisboa 4. Instituto Nacional de Medicina Legal e Ciencias Forenses 5. Institute of Health Sciences “Egas Monitz” 6. (Norwegian Institute of Water Research) EMCDDA Paul Griffith, Liesbeth Vandam, Ana Gallegos Project Collaborators (who kindly provided samples) Igor Bodik (Slovakia); Ester Heath (Slovenia); Katarzyna Styszko (Poland); Erik Emke (The Netherlands); Barbara Kasprzyk-Hordern (UK); Lisa Jones (Ireland); Mariya Skobley and Tanya Bodnarchuk (Ucraine); Maja Turk Sekulic and Jelena Radonic (Serbia); Andjelka Petkovic (Serbia); Simone Milanolo (Bosnia Herzegovina); Simion Beldean (Romania); Jelyaz Rangelov and Teodora Todorova (Bulgary); Francesca Pizza (Italy); Francesco Poretti (Switzerland); Miguel Santos (Portugal). Thanks for your attention!