Wastewater-Based Epidemiology To Monitor Synthetic Cathinones

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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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Environmental Science & Technology

169x136mm (96 x 96 DPI)

ACS Paragon Plus Environment


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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,*

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a

IRCCS – Istituto di Ricerche Farmacologiche “Mario Negri”, Department of

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Environmental Health Sciences, Via La Masa 19, 20156, Milan, Italy.

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b

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Food Analysis and Research, University of Santiago de Compostela, Constantino

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Candeira S/N, 15782 – Santiago de Compostela, Spain.

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c

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United Kingdom

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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,

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*Corresponding authors:

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Dr. Sara Castiglioni,

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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

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Tel: +39 02 39014518;

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e-mail: [email protected]

of

Environmental

Health

Sciences;

IRCCS-Istituto

32

2


di

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

37

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

5


17-25

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


cathinone


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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



182

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.

200 201

Liquid chromatography-tandem mass spectrometry analysis

202

An Agilent 1200 Series HPLC system with a membrane degasser, a binary high-

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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.

213

The mass spectrometric analysis was performed with a triple quadruple mass

214

spectrometer (Applied Biosystems SCIEX QqQ 5500, Ontario, Canada) equipped with a

215

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

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Analytes were quantified by using surrogate deuterated standards (IS). The most

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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



232

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

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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

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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

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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

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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

11


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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

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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).

272

Extracted ion chromatogram (XIC) for the first transition of all the analytes in a

273

standard and in a spiked wastewater extract are reported in Figures S1 and S2.

274

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%).

12



281

MS/MS spectra for the seventeen investigated cathinones are displayed in Figure

282

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

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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

305

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

307

was found in Oslo (Norway).

308

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:

321

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



331

was only detected in the weekend samples (Fri-Sa-Su and Mo) in the UK at levels

332

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


Page 16 of 32

Page 17 of 32


356

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



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


Page 18 of 32

Page 19 of 32


406

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



431

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

19


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436 437

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24



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

25


Page 26 of 32

Page 27 of 32


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

26


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 — — — — —

—

— — — — —


Page 28 of 32

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

27


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

Page 29 of 32


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 —

28



e 1.

29


Page 30 of 32

Page 31 of 32


e 2.

30



e 3.

31


Page 32 of 32

Wastewater-Based Epidemiology To Monitor Synthetic Cathinones

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