Identification and Assessment of New Psychoactive Substances: A European Network Identification and assessment of new psychoactive substances: a European network - NPS-Euronet (Home/2014/JDRU/AG/DRUG/7086) Félix Hernández Research Institute for Pesticides and Water (IUPA) Laboratory of Pesticide Residue Analysis (LARP) Universitat Jaume I, Castellón, Spain Lisbon, 28th September 2016

Urine samples Provided by Origin Location Sample # INMLFC Identification and Assessment of New Psychoactive Substances: A European Network Urine samples Provided by Origin Location Sample # INMLFC (Mario Joao Dias) Emergency department of central hospitals Lisbon, Porto and Coimbra (Portugal) Total 90 (5 i – 85 p) TICTAC (Trevor Shine) Creamfields Festival 2015* Daresbury (England) Total 31 (31 p) Juvenile detention center Picassent (Valencia, Spain) Total 227 i Castellón de la Plana (Spain) Total 23 i Burjassot (Valencia, Spain) Total 99 i Valencia (Spain) Total 108 i Total 26 i 1st Batch processed (19 samples: 5 i – 14 p) * Confidential information i = individual sample p = pooled sample Lisbon, 28th September 2016

(β-glucuronidase from E.Coli) Identification and Assessment of New Psychoactive Substances: A European Network Urine samples Hydrolysis Without Hydrolysis 1,25 mL urine sample 1 mL urine sample Deglucuronidation (β-glucuronidase from E.Coli) pH 7, 55ºC for 1 hour Centrifuge + 1ml Milli-Q H2O (Dilution (2x)) Centrifuge 20uL extract 20uL LC-QTOF MS LC-QTOF MS Lisbon, 28th September 2016

Wastewater samples Provided by Origin Location Sample # FFUL Mutela Identification and Assessment of New Psychoactive Substances: A European Network Wastewater samples Provided by Origin Location Sample # FFUL (Alvaro Lopes) Mutela Almada (Portugal) 1 p 3 days Alcântara Lisbon (Portugal) USC (José Benito Quintana) Santiago de Compostela Santiago (Spain) CSIC (Cristina Postigo) El Prat Barcelona (Spain) 2 i UJI Castellón Castellón (Spain) Molina de Segura Murcia (Spain) UA (Alexander van Nuijs) Tomorrowland Festival 2015 Belgium * i = individual sample p = pooled sample * To be submitted by Alexander van Nuijs, not sure how many samples Lisbon, 28th September 2016

Wastewater samples Screening Target(1) LC-QTOF MS LC-MS/MS Compounds Identification and Assessment of New Psychoactive Substances: A European Network Wastewater samples Screening Target(1) Compounds N-ethylcathinone MDPV Methylone Butylone Methedrone Mephedrone Naphydrone 25-C-NBOMe 25-I-NBOMe 25-B-NBOMe 25 mL filtered sample +75mL MilliQ H2O (dil x4) 100 mL centrifuged sample (pH 2) + Internal standard Oasis MCX (cationic exchange cartridge) Elution: 5ml MeOH (2% NH3) Oasis HLB (generic cartridge) Elution: 5ml MeOH LC-QTOF MS LC-MS/MS 20uL extract 3uL extract(2) Based on Bade et al, Chemosphere (submitted) Extract will also be injected for screening by LC-QTOF MS Lisbon, 28th September 2016

Urine samples, Norwegian festivals Identification and Assessment of New Psychoactive Substances: A European Network Urine samples, Norwegian festivals Origin Music Style Location Date Sample # Øya Festival* ** Alternative, Electronica, hip hop, indie and rock Oslo 11-13 August 2016 3 Pooled urine Pstereo Festival* Electronica, pop and rock Trondheim 19 August 2016 1 Pooled urine Findings Festival* Electro house and dance pop 20 August 2016 * Confidential information ** Collected by Francesco (MN). These samples will be analyzed also by MN. Lisbon, 28th September 2016

Urine samples, Norwegian festivals Photos + fechas + muestras + festival (tipo) + sample prep in Norway (SPE) is similar to wastewater (previous slide) only sample volume was modified) Identification and Assessment of New Psychoactive Substances: A European Network Urine samples, Norwegian festivals Pooled urine samples Treated as urine Hydrolysis Dilution Treated as WW SPE Oasis HLB SPE Oasis MCX Lisbon, 28th September 2016

Priority list of NPS Information included: Identification and Assessment of New Psychoactive Substances: A European Network Priority list of NPS Compiled by Mario Negri and University Jaume I (To be updated) Information included: Compound name IUPAC name Group (cathinone, cannabinoid, etc) Communication Date CAS number Formula Place Present in the first list (Nov 2013) Additional information Under control (Included in List DPR 309/90) LC Fragments GC Fragments Instrument References Info metabolism References metabolites Standard available (Mario Negri /IUPA) Lisbon, 28th September 2016

Priority list of NPS (MN + UJI) Identification and Assessment of New Psychoactive Substances: A European Network Priority list of NPS (MN + UJI) Group Number # Phenethylamine 37 Arilcicloexilamine - ketamine anal. 3 Piperidine 6 Tryptamine 7 Cathinone 53 Synthetic cannabinoid 71 Synthetic oppioid 9 Aminorex derivate Natural psychoactive substance 4 Benzodiazepine Total # compounds 197 Compounds: Lisbon, 28th September 2016

UNIFI – software package of Waters Identification and Assessment of New Psychoactive Substances: A European Network UNIFI – software package of Waters Good mass error Low RT deviation 5 frag. found by software Bijlsma, L. et al. J. Mass Spectrom. 2011, 46, 865-875 Identification False positive 105.0704 105.0340 m/z 105.03393 Lisbon, 28th September 2016

Lisbon Urines, Positives, Ketamine Identification and Assessment of New Psychoactive Substances: A European Network Lisbon Urines, Positives, Ketamine Also major metabolites found (Tentatively identified): Norketamine Dehydronorketamine Hydroxynorketamine m/z 125.01506 C7H6Cl (-0.7 mDa) m/z 179.06236 C11H12Cl (0.4 mDa) m/z 207.05742 C12H12ClO (0.3mDa) Lisbon, 28th September 2016

Lisbon Urines, Tentative Identification, Mexedrone Identification and Assessment of New Psychoactive Substances: A European Network Lisbon Urines, Tentative Identification, Mexedrone m/z 148.11209 C10H13N (0.01 mDa) m/z 190.12246 C12H16NO (-0.2 mDa) Lisbon, 28th September 2016

Tentatively Identified Other compounds detected: Identification and Assessment of New Psychoactive Substances: A European Network Results NPS, Lisbon Urines (Samples 1-19) Identified Tentatively Identified Other compounds detected: Ketamine Mexedrone MDMA Norketamine Cocaine Dehydronorketamine Benzoylecgonine Hydroxynorketamine 11-nor-9-carboxy-Δ9-THC (THC-COOH) Procaine Desmethylprodine 2-MeO-Ketamine O-Desmethyltramadol JWH-398 MDCPM 5-Me-MDA 2C-YN (phenethylamine analog) Lisbon, 28th September 2016

Further actions Confirm tentative identifications Identification and Assessment of New Psychoactive Substances: A European Network Further actions Confirm tentative identifications Harmonize criteria for tentative identifications Urgent need of reference standards acquisition Complete analysis of urine and wastewater samples Update the priority list of NPS with compounds identified in the samples Lisbon, 28th September 2016

Output – Conferences Wastewater based epidemiology: recent advances Identification and Assessment of New Psychoactive Substances: A European Network Output – Conferences Wastewater based epidemiology: recent advances Lubertus Bijlsma, Pim de Voogt, Sara Castiglioni, Adrian Covaci, Erik Emke, Félix Hernández, Barbara Kasprzyk-Hordern, Christoph Ort, Malcolm Reid, Kevin V. Thomas, Alexander L.N. van Nuijs Oral presentation Lisbon, 28th September 2016

Potential of HRMS (LC-QTOF MS) in wastewater drug testing Identification and Assessment of New Psychoactive Substances: A European Network Output – Conferences Potential of HRMS (LC-QTOF MS) in wastewater drug testing Lubertus Bijlsma Oral presentation Garching / Munich, 2 - 4. November 2016 Lisbon, 28th September 2016

Science of the Total Environment 565 (2016) 984–993 Identification and Assessment of New Psychoactive Substances: A European Network Output – scientific peer-reviewed articles Science of the Total Environment 565 (2016) 984–993 Lubertus Bijlsma acknowledges NPS-Euronet for his post-doctoral fellowship Lisbon, 28th September 2016

Output – scientific peer-reviewed articles Identification and Assessment of New Psychoactive Substances: A European Network Output – scientific peer-reviewed articles Ultra-high performance liquid chromatography-tandem mass spectrometry determination of synthetic cathinones and phenethylamines in influent wastewater Richard Bade, Lubertus Bijlsma, Juan V. Sancho, J. A. Baz-Lomba, Sara Castiglioni, Erika Castrignanò, Ana Causanilles, Emma Gracia-Lor, Barbara Kasprzyk-Hordern, Juliet Kinyua, Ann-Kathrin McCall, Alexander L.N. van Nuijs, Christoph Ort, Benedek G. Plósz, Pedram Ramin, Nikolaos I. Rousis, Yeonsuk Ryu, Kevin V. Thomas, Pim de Voogt, Ettore Zuccato, Felix Hernandez Submitted Chemosphere + Lubertus Bijlsma acknowledges NPS-Euronet for his post-doctoral fellowship Lisbon, 28th September 2016

Identification and Assessment of New Psychoactive Substances: A European Network Identification and assessment of new psychoactive substances: a European network - NPS-Euronet (Home/2014/JDRU/AG/DRUG/7086) Félix Hernández Research Institute for Pesticides and Water (IUPA) Laboratory of Pesticide Residue Analysis (LARP) Universitat Jaume I, Castellón, Spain Lisbon, 28th September 2016