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Wastewater-based epidemiology to monitor synthetic cathinones use in different European countries Iria Gonzalez-Marino, Emma Gracia-Lor, Nikolaos I Rousis, Erika Castrignanò, Kevin V. Thomas, Jose Benito Quintana, Barbara Kasprzyk-Hordern, Ettore Zuccato, and Sara Castiglioni Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02644 • Publication Date (Web): 04 Aug 2016 Downloaded from http://pubs.acs.org on August 7, 2016
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Wastewater-based epidemiology to monitor synthetic cathinones use in
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different European countries
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Iria González-Mariñoa,b,* Emma Gracia-Lora, Nikolaos I. Rousisa, Erika Castrignanòc,
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Kevin V. Thomasd, José Benito Quintanab, Barbara Kasprzyk-Hordernc, Ettore
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Zuccatoa, Sara Castiglionia,*
8 9
a
IRCCS – Istituto di Ricerche Farmacologiche “Mario Negri”, Department of
10
Environmental Health Sciences, Via La Masa 19, 20156, Milan, Italy.
11
b
12
Food Analysis and Research, University of Santiago de Compostela, Constantino
13
Candeira S/N, 15782 – Santiago de Compostela, Spain.
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c
15
United Kingdom
16
d
17
Norway
Department of Analytical Chemistry, Nutrition and Food Sciences, IIAA – Institute for
Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY,
Norwegian Institute for Water Research (NIVA), Gaustadalleen 21, 0349 Oslo,
18 19
*Corresponding authors:
20
Dr. Sara Castiglioni,
21
Head of the Environmental Biomarkers Unit; Department of Environmental Health
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Sciences; IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri"
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Via La Masa 19, 20156 Milan, Italy
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Tel: +39 02 39014776; Fax: +39 02 39014735
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e-mail:
[email protected] 1
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Dr. Iria González-Mariño,
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Department
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Farmacologiche "Mario Negri"
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Via La Masa 19, 20156 Milan, Italy
30
Tel: +39 02 39014518;
31
e-mail:
[email protected] of
Environmental
Health
Sciences;
IRCCS-Istituto
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2
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Ricerche
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Abstract
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Synthetic cathinones are among the most consumed new psychoactive substances
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(NPS), but their increasing number and interchangeable market make difficult to
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estimate the real size of their consumption. Wastewater-based epidemiology (WBE)
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through the analysis of metabolic residues of these substances in urban wastewater can
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provide this information. This study applied WBE for the first time to investigate the
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presence of seventeen synthetic cathinones in four European countries. A method based
41
on solid-phase extraction and liquid chromatography coupled to tandem mass
42
spectrometry was developed, validated and used to quantify the target analytes. Seven
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substances were found, with mephedrone and methcathinone being the most frequently
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detected and none of the analytes being found in Norway. Population normalized loads
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were used to evaluate the pattern of use, which indicated a higher consumption in the
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UK followed by Spain and Italy, in line with the European prevalence data from
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population surveys. In the UK, where an entire week was investigated, an increase of
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the loads was found during the weekend, indicating a preferential use in recreational
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contexts. This study demonstrated that WBE can be a useful additional tool to monitor
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the use of NPS in a population.
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Keywords: synthetic cathinones; wastewater analysis; urban wastewater; mass
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spectrometry; pattern of use.
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INTRODUCTION
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Over the past five years there has been an unprecedented upsurge in the number,
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type and availability of new psychoactive substances (NPS) in Europe. Following
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synthetic cannabinoid receptor agonists, synthetic cathinones are the second largest
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reported group of NPS, as identified by the European Monitoring Centre for Drugs and
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Drug Addiction (EMCDDA) through their Early Warning System.1 There have been 77
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synthetic cathinones reported in Europe since 2005, with 31 new derivatives reported
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for the first time in 2014.2 There has also been a 60-fold increase in the seizure of
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synthetic cathinones between 2008 and 2013, reaching 1.1 tons during 2013.2
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These substances are synthetic derivatives of cathinone, a naturally occurring β-
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keto phenethylamine found in the leaves of Catha edulis.3 Their synthesis started in the
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1920s for therapeutic use, but it was only in the last decade when they appeared on the
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recreational drugs market as legal alternatives (‘legal highs’) to amphetamine, ecstasy or
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cocaine. They are sold as apparently legal drugs under several guises such as ‘plant
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food’ or ‘bath salts’, and new derivatives appear continuously on the market with the
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molecular structure slightly modified to bypass drug legislation.3,4 They retain the
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cathinone bone structure as well as its psychoactive properties, targeting monoamine
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transporters in brain cells to produce stimulatory effects, but they are modified adding
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different substituents to obtain analogs or more potent species. Therefore, as each
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substance becomes banned, a new analog is introduced onto the legal market avoiding
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any potential regulation. Between 2005 and 2010, the most common synthetic cathinone
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on the European market was mephedrone (MEPH),5 but following the introduction of
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EU-level control measures,6 a continuous stream of other synthetic derivatives has
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entered the global legal high market. Due to the growing evolution and sale of novel
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variants with similar effects, consumers’ choices might be randomly influenced by 4
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availability, price and quality; and users also reported having taken unidentified pills or
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powder whose composition they ignore. This makes estimating the level of cathinone
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use extremely difficult and challenging: population surveys can be biased by the limited
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knowledge of users regarding which substance they are consuming. Alternatively, drug
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seizures and forensic analyses provide more reliable information regarding drug
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composition, but they are limited in extension and time and might be not representative
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of a continuously changing market.
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An alternative approach to estimate the level of drug use through the analysis of
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metabolic residues in urban wastewater7-9 can help to overcome some of the
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aforementioned issues. This approach, called wastewater-based epidemiology (WBE),
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has been successfully applied to monitor the use of amphetamine-like stimulants,
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cocaine and cannabis in several countries,10-15 proving its ability to identity geographical
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variations, long-term temporal changes, short-term fluctuations derived from punctual
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events (festivals, holidays, etc) and new drug trends.8,9 In the case of NPS, this approach
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is still in its infancy and presents several challenges,16 such as the very limited
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information available on the pharmacokinetics and metabolism of NPS, that can prevent
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the selection of suitable urinary biomarkers for wastewater monitoring; the low
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prevalence of consumption and the consequent low concentrations expected in
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wastewater; and the lack of information on their stability in this matrix. To date, several
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studies have been conducted worldwide to monitor synthetic cathinones in wastewater,
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but few substances were included and monitored in each study.
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reported
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Methylenedioxypyrovalerone (MDPV), methcathinone (METC), methylone (METL)
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and α-pyrrolidinovalerophenone (α-PVP) have also been found in wastewater from
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Australia;19,20 MDPV in three cities in Finland;22,23 METC and butylone (BUTL) in the
in
wastewater
from
the
UK17,18,
Australia19,20
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MEPH has been
and
Italy21.
3,4-
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UK;17 and METL in Zurich, Switzerland.24 Finally, Borova et al. found low levels of α-
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PVP in wastewater from an island in Greece, Santorini.25 However, to the best of our
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knowledge, there are no systematic studies comparing levels of various cathinones in
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different European countries.
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The aim of this study was to adopt WBE to evaluate the use of a selected panel of
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NPS in the population from different European countries. Since WBE is a potent tool to
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provide objective and updated information on the use of a substance in a population7-10,
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its application for NPS is particularly useful due to the increasing number and highly
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interchangeable market of these drugs that make difficult to estimate their real patterns
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of use. This study is testing WBE as an additional tool to respond to the increasing need
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raised by EMCDDA to establish new methods to monitor the market and the pattern of
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use of NPS2. A sensitive analytical method was developed to quantitatively measure
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seventeen synthetic cathinones in raw wastewater. Target analytes were selected
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according to their availability as reference standards from the Early Warning System in
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Italy. MEPH was added to the list in view of its frequently reported use and it was
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investigated following a previously published method.21 The methods were used to
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analyze samples from eight different cities in four European countries: Italy, Spain, UK
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and Norway.
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EXPERIMENTAL
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Chemicals and reagents
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Seventeen synthetic cathinones belonging to three different chemical families
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were selected as analytes. The groups were: N-alkylated cathinone derivatives,
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containing
only
linear
substituents;
3,4-methylenedioxy-N-alkylated 6
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derivatives, containing a diether ring linked to the benzene ring; and N-pyrrolidine
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cathinone derivatives, which contain two diether-benzene rings (MDPV) or two linked
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benzene rings (1-naphyrone and naphyrone) and also a 5 atoms cycle including the N of
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the amino group (Figure 1). MEPH was acquired from Cerilliant Corporation (Round
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Rock, Texas, USA) as a 0.4 mg mL-1 solution in methanol (CH3OH). METC, N,N-
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dimethylcathinone (DCAT), β-ethyl-methcathinone (pentedrone - PENT), METL,
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ethylone (ETHL), 1-naphyrone (1-NAPH) and naphyrone (NAPH) were purchased from
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LGC (Teddington, UK) as 0.1 mg mL-1 solutions in CH3OH. Ethcathinone (ETHC),
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methedrone (METH), 4-fluoromethcathinone (4-FMC), 3,4-dimethylmethcathinone
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(3,4-DMMC), 4-methylethcathinone (4-MEC), buphedrone (BUPH), BUTL, pentylone
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(PENTL) and methylenedioxypyrovalerone (MDPV) were supplied by Cayman
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Chemicals (Ann Arbor, MI, USA) also as 0.1 mg mL-1 solutions in CH3OH. The
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following
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mephedrone-D3
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methylenedioxymethamphetamine-D5
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methylenedioxyethylamphetamina-D5
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methylbutanamine-D5 (MBDB-D5). They were acquired as 0.1 mg mL-1 solutions in
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CH3OH from Cerilliant Corporation (Round Rock, Texas, USA). Mixed stock solutions,
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containing either the 17 analytes or the 5 deuterated compounds, were prepared in
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CH3OH and stored in the dark at -20 °C up to a maximum of 2 months.
deuterated
compounds
(MEPH-D3),
were
used
as
surrogate/internal
methamphetamine-D9
(METHAMP-D9),
(MDMA-D5), (MDEA-D5)
standards:
and
3,43,4-
1,3-benzodioxolyl-N-
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HPLC-grade CH3OH, acetonitrile (ACN), hydrochloric acid (HCl, 37%) formic
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acid (FA, 98-100%), acetic acid (AA, >99%) and ammonium hydroxide solution
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(NH4OH, 25%) were supplied by Sigma-Aldrich (Steinheim, Germany). Ultrapure
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water was obtained by purifying water in a Milli-Q Gradient A-10 system (Millipore,
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Bedford, MA, USA). 7
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Wastewater sampling
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Composite 24 h raw wastewater samples were collected at the main WWTP of
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each city investigated in this work. Five cities were located in north-central Italy
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(Florence, Bologna, Turin, Perugia and Milan); one city was in the northwest of Spain
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(Santiago de Compostela), one in Norway (Oslo) and one in the south-west of the UK.
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The Italian cities had been previously included in a nation-wide analytical
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campaign and were chosen on the basis of a previous study on MEPH.21 The other three
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European cities were selected for comparison purposes among countries reporting
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differences in the annual number of seizures of cathinones: Norway (50-99), Spain
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(100-499) and UK (>500).2
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Sampling was performed in Italy, Norway and Spain during the weekend (Friday
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to Monday) to evaluate the consumption of synthetic cathinones during the night-life
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time frame, since they are mainly used in recreational contexts and are excreted in urine
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within few hours.4 The city of Milan was sampled throughout the whole Milan Fashion
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Week, a special annual fashion event, in 2014 and 2015; in 2015, the week right after
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this event was also monitored. The city in the south-west of the UK was sampled for
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one week to evaluate the entire weekly profile of cathinones use due to the high
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consumption of these substances in the UK.26
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All WWTPs receive mostly domestic wastewater, and serve a population between
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50,000 and 1300,000 inhabitants (Table S1). 24 h composite samples were collected
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from 9.00 a.m. to 9.00 a.m. of the following day for consecutive days. Sampling was
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performed in volume or time proportional mode, depending on the characteristics of the
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automatic sampler available, and fulfilling the guidelines described by Castiglioni et
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al.27 to obtain non-biased composite samples. They were transferred into polypropylene 8
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bottles and shipped frozen to our laboratory, where they were stored at -20°C to inhibit
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microbial activity until analysis (performed within 15 days).
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Solid-phase extraction
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Sample preparation protocol was adapted from previously published works.21,28
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Briefly, samples were vacuum-filtered, first through glass microfiber filters GF/A 1.6
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µm (Whatman, Kent, U.K.) and subsequently through 0.45 µm nitrocellulose filters
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(Millipore, Bedford, MA, USA); acidified to pH ~2.0 with 37% HCl and spiked with
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labeled surrogate standards (40 ng L-1). 25-50 mL aliquots of each sample were solid-
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phase extracted using mixed reverse-phase cation exchange cartridges Oasis MCX - 150
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mg - 6 mL, (Waters, Milford, MA, USA). Sorbents were vacuum-dried for 10 min and
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analytes were eluted with 2 mL of CH3OH followed by 2 mL of 2% NH4OH in CH3OH.
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Eluates were evaporated to dryness under a gentle stream of nitrogen. Dried extracts
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were redissolved in 100 µL of ultrapure water, centrifuged for 2 min at 2500 rpm, and
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supernatants were transferred into glass inserts for instrumental analysis.
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A procedural SPE blank consisting of 25 or 50 mL of mineral water spiked with
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labeled surrogate standards was processed together with every set of samples to check
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for potential contaminations.
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Liquid chromatography-tandem mass spectrometry analysis
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An Agilent 1200 Series HPLC system with a membrane degasser, a binary high-
203
pressure gradient pump and a refrigerated autosampler kept at +4 °C was employed for
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the chromatographic separation, performed on an XBridgeTM C18 column (100 × 1.0
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mm I.D., particle size 3.5 µm) from Waters (Milford, MA, USA). The performance of
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this column with smaller ID was comparable with the one of 2.1 mm ID, but the 9
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reduced flow rate injected in the instrument source allowed to maintain it cleaner. . The
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column was maintained at room temperature and a dual eluent system consisting of (A)
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0.1% FA in ultrapure water and (B) ACN was employed at a flow rate of 60 µL min-1.
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The elution gradient was as follows: 0 min (2% B), 16 min (50% B), 16.5 min (100%
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B), 20 min (100% B), 20.5 min (2% B), 31 min (2% B). The injection volume was 1
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µL.
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The mass spectrometric analysis was performed with a triple quadruple mass
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spectrometer (Applied Biosystems SCIEX QqQ 5500, Ontario, Canada) equipped with a
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Turbo Ion Spray source. The Ion Source settings were: Ion Spray Voltage (IS) 5400 V;
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Curtain Gas (CUR) 30; Collision Gas (CAD) 7; Source Temperature 400 °C; Ion Source
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Gas 1 (GS1) and Gas 2 (GS2) 40. Mass spectrometric analyses were performed in
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positive mode using the SRM mode under time scheduled conditions (setting a time
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window of 180 s). Scheduled SRM enables to maximize dwell times and, thus,
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sensitivity during acquisition, and to optimize cycle times in order to provide good
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analytical performance for multiple precursor/product ion transitions. Analyses were
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done using the two or three most abundant fragmentation products of the protonated
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pseudo-molecular ions of each analyte and one fragmentation product of each
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deuterated analog. Selected transitions, together with the corresponding optimized
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instrumental parameters, retention times and surrogate/internal standards used for
226
quantification are listed in Table 1.
227 228
Analytes quantification and method validation
229
Analytes were quantified by using surrogate deuterated standards (IS). The most
230
abundant precursor/product ion transition was used as quantifier ion and the area was
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normalized with the proper IS. Five deuterated substances with structures similar to 10
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cathinones were evaluated as potential IS during recovery experiments; for every
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analyte, the labeled compound providing the best value (closer to 100%) was finally
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selected as its IS (Table 1).
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Calibration curves were prepared freshly before each analytical run. The first
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calibration point, containing only the labeled compounds, was used as instrumental
237
blank. Linearity was evaluated between the instrumental quantification limit (IQL) and
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100 µg L-1.
239
Recovery and repeatability of the analytical method were tested in raw wastewater
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(triplicate analysis) by spiking 50 mL aliquots with 100 ng L-1 of each analyte. An
241
additional aliquot was analysed without analyte spiking to correct recovery values for
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the background levels of these compounds in raw wastewater. Two sets of recovery
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tests were set adding IS before and after extraction to calculate, respectively, relative
244
and absolute recoveries. All samples were processed as described before. Method
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repeatability was assessed by calculating relative standard deviations (%RSD).
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IQL values were determined by direct injection of picogram quantities of each
247
substance as the concentrations giving peaks for which the signal-to-noise ratio (S/N)
248
was 10. Limits of quantification of the whole method (LOQ) were estimated in the same
249
way by analysing a wastewater extract (100 µL) spiked with 0.3 ng of each analyte.
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Intra-day precision was assessed in both ultrapure and sewage water by repeated
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injections of a standard mixture and a spiked wastewater extract. Two different
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concentration levels were evaluated: 1 µg L-1 and 10 µg L-1 for standard mixtures in
253
ultrapure water, and 3 µg L-1 and 10 µg L-1 for wastewater. Inter-day precision was
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evaluated only in ultrapure water at 1 µg L-1 and 10 µg L-1.
255 256
Calculation of cathinones loads in real samples
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Concentrations (ng L-1) of analytes detected in real samples were multiplied by
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the corresponding WWTP daily flow rate (L day-1) to obtain their mean loads in
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wastewater (mg day-1). Subsequently, these loads were normalized to the number of
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inhabitants served by the WWTP or to the population equivalents (g day-1 per 1000
261
people). Estimations of consumption could not be back-calculated since there is barely
262
information on the percentage of every cathinone excreted as parental drug in faeces
263
and urine.
264 265
RESULTS AND DISCUSSION
266 267
Chromatographic separation and MS/MS characterization
268
Due to the chemical properties of the amino groups, all cathinones were ionized in
269
positive mode. Ionization was optimised testing two different aqueous phase modifiers:
270
acetic and formic acid. Slightly higher responses were obtained with formic acid, which
271
was finally adopted for chromatographic separation at a concentration of 0.1% (v/v).
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Extracted ion chromatogram (XIC) for the first transition of all the analytes in a
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standard and in a spiked wastewater extract are reported in Figures S1 and S2.
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Confirmation of positives was performed according to the 2002/657/EC29
275
whenever possible, selecting one precursor ion and three product ions per analyte (Table
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1). This allowed us to be as specific as possible, since cathinones are likely to be
277
detected at very low levels and, being small amines, can be affected by a great number
278
of interferences from similar compounds. Conformity of the ion ratio between the
279
recorded transitions and of retention time between samples and standards was checked
280
to be within the maximum tolerances allowed (20%).
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MS/MS spectra for the seventeen investigated cathinones are displayed in Figure
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S3. Main product ions structures were proposed based on expected fragmentation
283
patterns and by comparison with product ions already listed in the literature. Some of
284
them matched (in terms of nominal mass) the fragments acquired by Rossi et al.,30
285
whereas some others had already been reported by Concheiro et al.31 Many products
286
were common to two or more cathinones within every family, as a consequence of their
287
structural similarities.
288 289
Method performance
290
Recoveries were typically higher than 90%, with relative standard deviations
291
(RSD) lower than 15% (Table 2), thus confirming that no losses are occurring during
292
the analytical procedure. The method allowed detection in the low ng L-1 range (0.1-1.6
293
ng L-1) even in a complex matrix such as untreated wastewater, while few pg/injected
294
(0.02-0.33 pg) could be quantified in analytical standards (IQLs) (Table 2). The
295
calibration curve fitted a linear model covering the concentration range in samples with
296
determination coefficients (R2) varying between 0.99940 and 0.99994 (Table S2).
297
Instrumental blanks were below the IQL. Intra-day precision was satisfactory in both
298
ultrapure water (%RSD between 2.2 and 8.5 at 1 µg L-1 and between 1.1 and 6.8 at 10
299
µg L-1) and wastewater extracts (%RSD between 0.7 and 6.4 at 3 µg L-1 and between
300
1.8 and 6.2 at 10 µg L-1). Inter-day %RSD values in ultrapure water ranged from 4.1 to
301
33.3% at 1 µg L-1, and from 5.7 to 31.9 at 10 µg L-1 (Table S2).
302 303
Occurrence of synthetic cathinones in urban wastewater
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Seven synthetic cathinones were detected in the wastewater samples collected
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from seven European cities: MEPH, METC, DCAT, 4-FMC, 4-MEC, ETHL and 13
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MDPV (Table 3, Table S3 and Figure S4), while none of the investigated compounds
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was found in Oslo (Norway).
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In Italy, MEPH was quantified only in the city of Bologna (mean concentration 16
309
ng L-1) and in two of the three samples collected in Turin (mean 1.7 ng L-1). These
310
results were close to those previously reported by Castiglioni et al. in Italy, where
311
MEPH was detected only in Bologna at concentrations up to 24 ng L-1.21 MEPH was not
312
detected neither in Oslo nor in Santiago de Compostela; but it was found at very high
313
concentrations in the UK (mean 110 ng L-1), matching the higher prevalence of use of
314
synthetic cathinones among young people in England (8%) compared to Spain (5%) and
315
Italy (1%).26 Although MEPH was banned in England in 2010 and its purity has
316
decreased in the last years,32 the amount found in wastewater that can be ascribed to
317
consumption is still high. To the best of our knowledge, no information about the
318
prevalence of use of synthetic cathinones in Norway is available, but seizures are lower
319
than in Spain and in the UK2.
320
METC was detected in wastewater from four of the five Italian cities investigated:
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Florence (mean 6.8 ng L-1), Turin (mean 2.6 ng L-1), Perugia (mean 1.2 ng L-1) and
322
Milan (mean 1.1 ng L-1); and in the south-west of the UK (mean 0.4 ng L-1) but only in
323
samples from Sundays and Mondays (Table 3 and Table S3). This substance has already
324
been quantified in Cambridge, UK17 and in Adelaide, Australia.19,20 To the best of our
325
knowledge this is the first time a cathinone other than MEPH has been detected in
326
wastewater in Italy.
327
DCAT, 4-FMC and 4-MEC were sporadically detected in the samples (Table 3),
328
but this study is the first ever to report these substances in wastewater, indicating
329
consumption by the population. DCAT was quantified at a very low concentration (2.1
330
ng L-1) in a sample collected on Friday in Santiago de Compostela (Table S3). 4-FMC 14
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was only detected in the weekend samples (Fri-Sa-Su and Mo) in the UK at levels
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ranging between 4.4 and 25.8 ng L-1 (Table S3). 4-MEC was detected in two weekend
333
samples in Milan at 13.9 and 4.7 ng L-1, respectively, and in another two samples (Sa
334
and Su) in the UK at 4.8 and 2.8 ng L-1 (Table S3).
335
ETHL was found in all of the samples from the UK with a mean concentration of
336
21 ng L-1, and in the samples from Spain (mean 11.6 ng L-1) (Table 3). In the UK, a
337
significant increase was observed during the weekend (Sa-Su-Mo) with concentrations
338
increasing from 5-7 ng L-1 during weekdays to 33-53 ng L-1 during the weekend (Table
339
S3). This study reports, for the first time, the presence of ETHL in wastewater from
340
Spain, while this substance had only been detected, but not quantified, in Belgium and
341
Switzerland.24
342
Finally, MDPV was only found in Milan, both in 2014 and in 2015, but at very
343
low levels (mean 0.8 ng L-1) (Table 3). This compound had already been quantified in
344
Australia19,20 and Finland.22,23
345 346
Use of synthetic cathinones in the cities investigated
347
Population normalized loads (mg day-1 per 1000 people) for each city were plotted
348
to compare the use of synthetic cathinones (Figure 2, Table S4). In the UK (Figure 2A),
349
the highest mean loads were found for MEPH (27 mg day-1 per 1000 people), while
350
lower mean loads were calculated for ETHL (5 mg day-1 per 1000 people) and 4-FMC
351
(2 mg day-1 per 1000 people). Mean loads of METC and 4-MEC were lower than 0.5
352
mg day-1 per 1000 people, and they were found only in few samples, mostly collected
353
during the weekend. In Italy (Figure 2B), the most abundant substance was MEPH (2.6
354
mg day-1 per 1000 people), followed by METC (1 mg day-1 per 1000 people) and 4-
355
MEC and MDPV, with very low average loads (< 0.5 mg day-1 per 1000 people). In 15
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Spain (Figure 2C), the only compound found in more than one sample was ETHL, at
357
4.1 mg day-1 per 1000 people. None of the compounds investigated was found in Oslo,
358
suggesting little or no use. MEPH use had been previously reported in Australia
359
levels similar to Italy, but lower than in the UK. METC had been found at slightly
360
higher loads in Australia19,20 compared to Milan and clearly higher than in the English
361
city. Consumption of MDPV had been estimated in Australia at levels similar20 or
362
slightly higher19 than in Italy and in Finland22,23 at maximum loads around thirty times
363
higher than the loads reported in Milan, Italy (Figure 2B, Table S4).
19,20
at
364
The average sum of the investigated cathinones is ~35 mg day-1 per 1000 people
365
in the UK and 4-4.5 mg day-1 per 1000 people in Italy and Spain, indicating a different
366
pattern of use in these countries. Synthetic cathinones are used as a replacement of
367
classical drugs such as amphetamine, methamphetamine and MDMA (ecstasy).
368
Nevertheless, the loads found in the present study are lower than those found for
369
classical illicit drugs in the same countries.10 We can speculate that the use of synthetic
370
cathinones is actually lower than that of classical illicit drugs, but this difference can
371
also be attributed to the high number of different NPS available on the market.
372
Despite sampling being limited mostly to one city per country and to a limited
373
number of days, the loads can reflect NPS prevalence data in the different countries
374
investigated.26 This suggests that wastewater analysis can be a useful tool to monitor the
375
use of NPS in a population. More extensive studies can be designed to evaluate spatial
376
and geographical differences of use according to specific local, national or international
377
requirements.
378
Back-calculating consumption of these substances is not currently possible due to
379
the lack of available information on human metabolism, excretion and doses of
380
consumption. Further research should be focused on producing this kind of data in order 16
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381
to transform the levels in wastewater into consumption estimates and to use wastewater-
382
based epidemiology to measure and compare cathinones consumption among different
383
populations, as already performed for cocaine and other illicit drugs.7,10 Moreover, this
384
study was focused only on the parent substances, and the amount found in wastewater
385
could also come from drug disposal, thus specific cases (i.e. MEPH in the UK) should
386
be carefully evaluated.
387 388
Weekly pattern of use
389
A UK city and Milan were investigated for one and three weeks, respectively, and the
390
weekly profile of use of the measured substances was evaluated (Figure 3). In the UK,
391
an increase in the loads was found during the weekend for all the compounds measured
392
in higher amounts (MEPH, ETHL and 4-FMC) (Figure 3A). The highest increase was
393
observed for MEPH, with loads increasing from 20 mg day-1 per 1000 people to 60 and
394
40 mg day-1 per 1000 people, respectively, on Saturday and Sunday-Monday. These
395
results highlight the larger weekend use of these substances indicating their use in a
396
recreational context as replacement of some classical drugs such as MDMA.2 In Milan,
397
an increase of METC was observed on Friday (Figure 3B), but due to the very low
398
amounts detected, no clear weekly profile could be observed. Differences in the
399
substances used and the weekly profile (i.e. the day when the highest consumption was
400
found) were observed in the UK and Italy. This may be due to the local market, that can
401
be highly different from country to country, and to the recreational behaviour of
402
consumers, which is also influenced by local habits.
403 404
For the first time, the occurrence of a large number of synthetic cathinones has
405
been evaluated in different European cities and their pattern of use was monitored using 17
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WBE. A limited number of the target substances was found, but different geographical
407
patterns of use could be highlighted and they were in line with European prevalence
408
data available from population surveys26. Although this study included a limited number
409
of cities that cannot be fully representative of an entire nation10, these results can
410
provide an overview of the use of these substances as previously reported for other
411
illicit drugs7,8. This knowledge is very useful to map cathinones and other NPS use in
412
Europe and to monitor their replacement with new molecules in the European market,
413
with the aim to plan prevention strategies and to tackle their spread, with potential
414
important social and public health benefits.
415 416
Acknowledgements
417
This study was supported by Dipartimento Politiche Antidroga (Presidenza del
418
Consiglio dei Ministri, Rome, Project Aqua Drugs) and partially by the European
419
Union’s Seventh Framework Programme for research, technological development and
420
demonstration [grant agreement 317205, the SEWPROF MC ITN project].
421
I. González-Mariño extends her gratitude to the Galician Council of Culture,
422
Education and Universities for her postdoctoral contract (Plan Galego de Investigación,
423
Innovación e Crecemento 2011-2015), project EM2012/055 and “Consolidación” funds,
424
including FEDER/ERDF funding.
425 426
E. Gracia-Lor, E. Castrignanò and N. Rousis acknowledge the SEWPROF MC ITN project for support to their fellowships.
427
T. Rodríguez-Álvarez, I. Racamonde and Viaqua SA are acknowledged for
428
providing access to wastewater samples in Spain, J.A. Baz-Lomba in Norway, R.
429
Mazzini, F. Pizza and W. Bodini in Italy, and Wessex Water in the UK.
430 18
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Supporting Information Available
432
Additional information reporting method validation, mass spectra and results is
433
available free of charge via the Internet at http://pubs.acs.org.
434 435
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Figure Captions
Figure 1. Chemical structures of the selected synthetic cathinones.
550
Figure 2. Normalized loads (mg day-1 per 1000 people) of the synthetic cathinones detected in wastewater in UK (A), Italy (B) and Spain (C).
Figure 3. Weekly profiles of use of the substances measured in the UK (A) and Milan (B).
555
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1. Retention time (RT), internal standard (IS) and individual SRM parameters (Collision Energy (CE) and Cell Exit Potential (C
e seventeen analytes investigated.
ompound
RT min
IS
[M+H]+ Formula
MEPH METC DCAT ETHC METH 4-FMC 4-DMMC 4-MEC BUPH PENT METL ETHL BUTL PENTL 1-NAPH NAPH MDPV MEPH D3 THAMP D9 MDMA D5 MDEA D5 MBDB D5
12.8 6.8 7.1 7.4 8.2 7.4 10.5 9.5 8.3 9.8 7.4 8.0 8.7 10.2 13.7 14.2 10.9 8.9 8.1 8.4 9.1 9.7
MEPH-D3 METHAMP-D9 METHAMP-D9 METHAMP-D9 MEPH-D3 METHAMP-D9 MEPH-D3 MEPH-D3 METHAMP-D9 METHAMP-D9 MEPH-D3 MBDB-D5 MDEA-D5 MDMA-D5 METHAMP-D9 METHAMP-D9 MEPH-D3 — — — — —
C11H16NO C10H14NO C11H16NO C11H16NO C11H16NO2 C10H13FNO C12H18NO C12H18NO C11H16NO C12H18NO C11H14NO3 C12H16NO3 C12H16NO3 C13H18NO3 C19H24NO C19H24NO C16H22NO3 C11H13D3NO C10H7D9N C11H11D5NO2 C12H13D5NO2 C12H13D5NO2
[M+H]+ m/z 178.1 164.1 178.1 178.1 194.1 182.1 192.1 192.1 178.1 192.1 208.2 222.2 222.2 236.2 282.2 282.2 276.2 181 159.1 199.1 213.1 213.1
Product 1 m/z 145.1 131.1 105.1 130.1 161.1 149.1 159.1 145.1 131.1 132.1 160.1 174.1 174.1 188.1 141.1 141.1 126.1 148 93.1 107.1 163.1 136.1
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CE 28 26 27 40 27 28 28 27 29 24 23 25 23 24 36 34 36 28 28 32 18 27
Product 2 CXP 15 12 12 10 12 10 12 10 10 10 13 10 14 14 14 12 14 15 12 14 14 14
m/z
CE
119 130.1 133.1 132.1 146.1 148.1 158.1 146.1 130.1 91.1 132.1 146.1 131.1 175.1 126.1 211.2 135.1 — — — — —
28 40 20 23 37 43 43 24 43 32 36 36 46 29 32 26 36 — — — — —
Produ CXP
m/z
CE
15 12 12 10 12 14 10 10 11 10 14 12 11 13 14 14 15 — — — — —
— 72.1 131.1 118.1 103.1 144.1 131.1 91.1 130.1 117.1 91.1 191.1 131.1 155.1 126.1 175.1 — — — — —
—
— — — — —
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Table 2. Absolute and relative recoveries (mean and relative standard deviation – RSD%), instrumental quantification limits (IQL) and limits of quantification (LOQ) of 560
the analytical method in raw wastewater. Compound MEPH METC DCAT ETHC METH 4-FMC 3,4-DMMC 4-MEC BUPH PENT METL ETHL BUTL PENTL 1-NAPH NAPH MDPV
Relative Recovery % %RSD 104.3 10.8 140.6 8.4 115.6 10.3 78.7 10.8 84.8 12.6 105.0 8.6 102.3 9.3 99.2 16.0 84.8 7.9 82.2 13.0 85.0 2.4 85.4 9.6 110.1 5.6 114.9 16.3 123.6 11.5 75.7 17.4
Absolute Recovery % %RSD 104.6 5.4 107.1 12.9 132.3 2.4 113.6 8.9 71.1 13.5 84.4 16.8 99.2 6.6 102.9 11.4 96.6 10.0 84.7 10.2 75.5 16.0 91.0 13.1 82.5 8.2 96.7 7.4 88.9 2.7 98.1 0.9 71.6 4.9
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IQL (pg/injected) 0.33
LOQ (ng L-1) 1.23
0.07 0.07 0.06 0.09 0.07 0.02 0.06 0.14 0.10 0.03 0.04 0.07 0.03 0.13 0.06 0.03
0.67 0.86 0.87 0.62 0.63 0.32 0.64 0.72 1.57 0.46 0.59 0.58 0.54 0.26 0.11 0.36
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3. Concentrations (mean ± SD) of the cathinones detected in urban wastewater in eight European cities. Means were calculat
LOQ/2 when the values were below LOQ.
Location y Florence Bologna Turin Perugia Milan way Oslo in Santiago the Compostela K SW of the UK
MEPH
METC 6.8 ± 1.2
DCAT
4-FMC
4-MEC
ETHL
MDPV
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2.6 ± 0.6 1.2 ± 1.0 1.1 ± 0.5
—
—
0.9 ± 3.1
—
0.8 ± 0.5
—
—
—
—
—
—
—
—
—
0.8 ± 1.1
—
—
11.6 ± 0.7
—
110.2 ± 55.8
0.4 ± 0.5
—
7.9 ± 9.7
1.2 ± 1.9
20.7 ± 19.2
—
—
16.2 ± 9.8 1.7 ± 1.0 —
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