Wastewater-Based Epidemiology To Monitor ... - ACS Publications

Subscriber access provided by Northern Illinois University

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Environmental Science & Technology

169x136mm (96 x 96 DPI)

ACS Paragon Plus Environment


1 2

Wastewater-based epidemiology to monitor synthetic cathinones use in

3

different European countries

4 5

Iria González-Mariñoa,b,* Emma Gracia-Lora, Nikolaos I. Rousisa, Erika Castrignanòc,

6

Kevin V. Thomasd, José Benito Quintanab, Barbara Kasprzyk-Hordernc, Ettore

7

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.

14

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

22

Sciences; IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri"

23

Via La Masa 19, 20156 Milan, Italy

24

Tel: +39 02 39014776; Fax: +39 02 39014735

25

e-mail: [email protected] 1


Page 2 of 32

Page 3 of 32


26

Dr. Iria González-Mariño,

27

Department

28

Farmacologiche "Mario Negri"

29

Via La Masa 19, 20156 Milan, Italy

30

Tel: +39 02 39014518;

31

e-mail: [email protected]

of

Environmental

Health

Sciences;

IRCCS-Istituto

32

2


di

Ricerche


33 34

Abstract

35

Synthetic cathinones are among the most consumed new psychoactive substances

36

(NPS), but their increasing number and interchangeable market make difficult to

37

estimate the real size of their consumption. Wastewater-based epidemiology (WBE)

38

through the analysis of metabolic residues of these substances in urban wastewater can

39

provide this information. This study applied WBE for the first time to investigate the

40

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

43

substances were found, with mephedrone and methcathinone being the most frequently

44

detected and none of the analytes being found in Norway. Population normalized loads

45

were used to evaluate the pattern of use, which indicated a higher consumption in the

46

UK followed by Spain and Italy, in line with the European prevalence data from

47

population surveys. In the UK, where an entire week was investigated, an increase of

48

the loads was found during the weekend, indicating a preferential use in recreational

49

contexts. This study demonstrated that WBE can be a useful additional tool to monitor

50

the use of NPS in a population.

51 52

Keywords: synthetic cathinones; wastewater analysis; urban wastewater; mass

53

spectrometry; pattern of use.

54 55

3


Page 4 of 32

Page 5 of 32


56 57

INTRODUCTION

58

Over the past five years there has been an unprecedented upsurge in the number,

59

type and availability of new psychoactive substances (NPS) in Europe. Following

60

synthetic cannabinoid receptor agonists, synthetic cathinones are the second largest

61

reported group of NPS, as identified by the European Monitoring Centre for Drugs and

62

Drug Addiction (EMCDDA) through their Early Warning System.1 There have been 77

63

synthetic cathinones reported in Europe since 2005, with 31 new derivatives reported

64

for the first time in 2014.2 There has also been a 60-fold increase in the seizure of

65

synthetic cathinones between 2008 and 2013, reaching 1.1 tons during 2013.2

66

These substances are synthetic derivatives of cathinone, a naturally occurring β-

67

keto phenethylamine found in the leaves of Catha edulis.3 Their synthesis started in the

68

1920s for therapeutic use, but it was only in the last decade when they appeared on the

69

recreational drugs market as legal alternatives (‘legal highs’) to amphetamine, ecstasy or

70

cocaine. They are sold as apparently legal drugs under several guises such as ‘plant

71

food’ or ‘bath salts’, and new derivatives appear continuously on the market with the

72

molecular structure slightly modified to bypass drug legislation.3,4 They retain the

73

cathinone bone structure as well as its psychoactive properties, targeting monoamine

74

transporters in brain cells to produce stimulatory effects, but they are modified adding

75

different substituents to obtain analogs or more potent species. Therefore, as each

76

substance becomes banned, a new analog is introduced onto the legal market avoiding

77

any potential regulation. Between 2005 and 2010, the most common synthetic cathinone

78

on the European market was mephedrone (MEPH),5 but following the introduction of

79

EU-level control measures,6 a continuous stream of other synthetic derivatives has

80

entered the global legal high market. Due to the growing evolution and sale of novel

81

variants with similar effects, consumers’ choices might be randomly influenced by 4



Page 6 of 32

82

availability, price and quality; and users also reported having taken unidentified pills or

83

powder whose composition they ignore. This makes estimating the level of cathinone

84

use extremely difficult and challenging: population surveys can be biased by the limited

85

knowledge of users regarding which substance they are consuming. Alternatively, drug

86

seizures and forensic analyses provide more reliable information regarding drug

87

composition, but they are limited in extension and time and might be not representative

88

of a continuously changing market.

89

An alternative approach to estimate the level of drug use through the analysis of

90

metabolic residues in urban wastewater7-9 can help to overcome some of the

91

aforementioned issues. This approach, called wastewater-based epidemiology (WBE),

92

has been successfully applied to monitor the use of amphetamine-like stimulants,

93

cocaine and cannabis in several countries,10-15 proving its ability to identity geographical

94

variations, long-term temporal changes, short-term fluctuations derived from punctual

95

events (festivals, holidays, etc) and new drug trends.8,9 In the case of NPS, this approach

96

is still in its infancy and presents several challenges,16 such as the very limited

97

information available on the pharmacokinetics and metabolism of NPS, that can prevent

98

the selection of suitable urinary biomarkers for wastewater monitoring; the low

99

prevalence of consumption and the consequent low concentrations expected in

100

wastewater; and the lack of information on their stability in this matrix. To date, several

101

studies have been conducted worldwide to monitor synthetic cathinones in wastewater,

102

but few substances were included and monitored in each study.

103

reported

104

Methylenedioxypyrovalerone (MDPV), methcathinone (METC), methylone (METL)

105

and α-pyrrolidinovalerophenone (α-PVP) have also been found in wastewater from

106

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-

Page 7 of 32


107

UK;17 and METL in Zurich, Switzerland.24 Finally, Borova et al. found low levels of α-

108

PVP in wastewater from an island in Greece, Santorini.25 However, to the best of our

109

knowledge, there are no systematic studies comparing levels of various cathinones in

110

different European countries.

111

The aim of this study was to adopt WBE to evaluate the use of a selected panel of

112

NPS in the population from different European countries. Since WBE is a potent tool to

113

provide objective and updated information on the use of a substance in a population7-10,

114

its application for NPS is particularly useful due to the increasing number and highly

115

interchangeable market of these drugs that make difficult to estimate their real patterns

116

of use. This study is testing WBE as an additional tool to respond to the increasing need

117

raised by EMCDDA to establish new methods to monitor the market and the pattern of

118

use of NPS2. A sensitive analytical method was developed to quantitatively measure

119

seventeen synthetic cathinones in raw wastewater. Target analytes were selected

120

according to their availability as reference standards from the Early Warning System in

121

Italy. MEPH was added to the list in view of its frequently reported use and it was

122

investigated following a previously published method.21 The methods were used to

123

analyze samples from eight different cities in four European countries: Italy, Spain, UK

124

and Norway.

125 126

EXPERIMENTAL

127 128

Chemicals and reagents

129

Seventeen synthetic cathinones belonging to three different chemical families

130

were selected as analytes. The groups were: N-alkylated cathinone derivatives,

131

containing

only

linear

substituents;

3,4-methylenedioxy-N-alkylated 6


cathinone


Page 8 of 32

132

derivatives, containing a diether ring linked to the benzene ring; and N-pyrrolidine

133

cathinone derivatives, which contain two diether-benzene rings (MDPV) or two linked

134

benzene rings (1-naphyrone and naphyrone) and also a 5 atoms cycle including the N of

135

the amino group (Figure 1). MEPH was acquired from Cerilliant Corporation (Round

136

Rock, Texas, USA) as a 0.4 mg mL-1 solution in methanol (CH3OH). METC, N,N-

137

dimethylcathinone (DCAT), β-ethyl-methcathinone (pentedrone - PENT), METL,

138

ethylone (ETHL), 1-naphyrone (1-NAPH) and naphyrone (NAPH) were purchased from

139

LGC (Teddington, UK) as 0.1 mg mL-1 solutions in CH3OH. Ethcathinone (ETHC),

140

methedrone (METH), 4-fluoromethcathinone (4-FMC), 3,4-dimethylmethcathinone

141

(3,4-DMMC), 4-methylethcathinone (4-MEC), buphedrone (BUPH), BUTL, pentylone

142

(PENTL) and methylenedioxypyrovalerone (MDPV) were supplied by Cayman

143

Chemicals (Ann Arbor, MI, USA) also as 0.1 mg mL-1 solutions in CH3OH. The

144

following

145

mephedrone-D3

146

methylenedioxymethamphetamine-D5

147

methylenedioxyethylamphetamina-D5

148

methylbutanamine-D5 (MBDB-D5). They were acquired as 0.1 mg mL-1 solutions in

149

CH3OH from Cerilliant Corporation (Round Rock, Texas, USA). Mixed stock solutions,

150

containing either the 17 analytes or the 5 deuterated compounds, were prepared in

151

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-

152

HPLC-grade CH3OH, acetonitrile (ACN), hydrochloric acid (HCl, 37%) formic

153

acid (FA, 98-100%), acetic acid (AA, >99%) and ammonium hydroxide solution

154

(NH4OH, 25%) were supplied by Sigma-Aldrich (Steinheim, Germany). Ultrapure

155

water was obtained by purifying water in a Milli-Q Gradient A-10 system (Millipore,

156

Bedford, MA, USA). 7


Page 9 of 32


157 158

Wastewater sampling

159

Composite 24 h raw wastewater samples were collected at the main WWTP of

160

each city investigated in this work. Five cities were located in north-central Italy

161

(Florence, Bologna, Turin, Perugia and Milan); one city was in the northwest of Spain

162

(Santiago de Compostela), one in Norway (Oslo) and one in the south-west of the UK.

163

The Italian cities had been previously included in a nation-wide analytical

164

campaign and were chosen on the basis of a previous study on MEPH.21 The other three

165

European cities were selected for comparison purposes among countries reporting

166

differences in the annual number of seizures of cathinones: Norway (50-99), Spain

167

(100-499) and UK (>500).2

168

Sampling was performed in Italy, Norway and Spain during the weekend (Friday

169

to Monday) to evaluate the consumption of synthetic cathinones during the night-life

170

time frame, since they are mainly used in recreational contexts and are excreted in urine

171

within few hours.4 The city of Milan was sampled throughout the whole Milan Fashion

172

Week, a special annual fashion event, in 2014 and 2015; in 2015, the week right after

173

this event was also monitored. The city in the south-west of the UK was sampled for

174

one week to evaluate the entire weekly profile of cathinones use due to the high

175

consumption of these substances in the UK.26

176

All WWTPs receive mostly domestic wastewater, and serve a population between

177

50,000 and 1300,000 inhabitants (Table S1). 24 h composite samples were collected

178

from 9.00 a.m. to 9.00 a.m. of the following day for consecutive days. Sampling was

179

performed in volume or time proportional mode, depending on the characteristics of the

180

automatic sampler available, and fulfilling the guidelines described by Castiglioni et

181

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

183

microbial activity until analysis (performed within 15 days).

184 185

Solid-phase extraction

186

Sample preparation protocol was adapted from previously published works.21,28

187

Briefly, samples were vacuum-filtered, first through glass microfiber filters GF/A 1.6

188

µm (Whatman, Kent, U.K.) and subsequently through 0.45 µm nitrocellulose filters

189

(Millipore, Bedford, MA, USA); acidified to pH ~2.0 with 37% HCl and spiked with

190

labeled surrogate standards (40 ng L-1). 25-50 mL aliquots of each sample were solid-

191

phase extracted using mixed reverse-phase cation exchange cartridges Oasis MCX - 150

192

mg - 6 mL, (Waters, Milford, MA, USA). Sorbents were vacuum-dried for 10 min and

193

analytes were eluted with 2 mL of CH3OH followed by 2 mL of 2% NH4OH in CH3OH.

194

Eluates were evaporated to dryness under a gentle stream of nitrogen. Dried extracts

195

were redissolved in 100 µL of ultrapure water, centrifuged for 2 min at 2500 rpm, and

196

supernatants were transferred into glass inserts for instrumental analysis.

197

A procedural SPE blank consisting of 25 or 50 mL of mineral water spiked with

198

labeled surrogate standards was processed together with every set of samples to check

199

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-

203

pressure gradient pump and a refrigerated autosampler kept at +4 °C was employed for

204

the chromatographic separation, performed on an XBridgeTM C18 column (100 × 1.0

205

mm I.D., particle size 3.5 µm) from Waters (Milford, MA, USA). The performance of

206

this column with smaller ID was comparable with the one of 2.1 mm ID, but the 9


Page 10 of 32

Page 11 of 32


207

reduced flow rate injected in the instrument source allowed to maintain it cleaner. . The

208

column was maintained at room temperature and a dual eluent system consisting of (A)

209

0.1% FA in ultrapure water and (B) ACN was employed at a flow rate of 60 µL min-1.

210

The elution gradient was as follows: 0 min (2% B), 16 min (50% B), 16.5 min (100%

211

B), 20 min (100% B), 20.5 min (2% B), 31 min (2% B). The injection volume was 1

212

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;

216

Curtain Gas (CUR) 30; Collision Gas (CAD) 7; Source Temperature 400 °C; Ion Source

217

Gas 1 (GS1) and Gas 2 (GS2) 40. Mass spectrometric analyses were performed in

218

positive mode using the SRM mode under time scheduled conditions (setting a time

219

window of 180 s). Scheduled SRM enables to maximize dwell times and, thus,

220

sensitivity during acquisition, and to optimize cycle times in order to provide good

221

analytical performance for multiple precursor/product ion transitions. Analyses were

222

done using the two or three most abundant fragmentation products of the protonated

223

pseudo-molecular ions of each analyte and one fragmentation product of each

224

deuterated analog. Selected transitions, together with the corresponding optimized

225

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

231

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

233

analyte, the labeled compound providing the best value (closer to 100%) was finally

234

selected as its IS (Table 1).

235

Calibration curves were prepared freshly before each analytical run. The first

236

calibration point, containing only the labeled compounds, was used as instrumental

237

blank. Linearity was evaluated between the instrumental quantification limit (IQL) and

238

100 µg L-1.

239

Recovery and repeatability of the analytical method were tested in raw wastewater

240

(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

242

the background levels of these compounds in raw wastewater. Two sets of recovery

243

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

245

repeatability was assessed by calculating relative standard deviations (%RSD).

246

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.

250

Intra-day precision was assessed in both ultrapure and sewage water by repeated

251

injections of a standard mixture and a spiked wastewater extract. Two different

252

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

254

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


Page 12 of 32

Page 13 of 32


257

Concentrations (ng L-1) of analytes detected in real samples were multiplied by

258

the corresponding WWTP daily flow rate (L day-1) to obtain their mean loads in

259

wastewater (mg day-1). Subsequently, these loads were normalized to the number of

260

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).

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

276

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

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

304

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


Page 14 of 32

Page 15 of 32


306

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


Page 20 of 32

Page 21 of 32


436 437

References

438

(1) European Monitoring Centre for Drugs and Drug Addiction, The EU Early warning

439

System: http://www.emcdda.europa.eu/themes/new-drugs/early-warning. Accessed on

440

April 2015.

441 442

(2) New psychoactive substances in Europe. An update from the EU Early Warning

443

System; European Monitoring Centre for Drugs and Drug Addiction, Publications

444

Office of the European Union: Luxembourg, 2015.

445

(3) Valente, M. J.; Guedes de Pinho, P.; Bastos, M. L.; Carvalho, F.; Carvalho, M. Khat

446

and synthetic cathinones: a review. Arch. Toxicol. 2014, 88, 15–45.

447

(4) Prosser, J. M.; Nelson, L. S. The Toxicology of Bath Salts: A Review of Synthetic

448

Cathinones. J. Med. Toxicol. 2012, 8, 33–42.

449

(5) Perspectives on drugs. Injection of synthetic cathinones; European Monitoring

450

Centre for Drugs and Drug Addiction, Publications Office of the European Union:

451

Luxembourg, 2015.

452

(6) New psychoactive substances in Europe. Innovative legal responses; European

453

Monitoring Centre for Drugs and Drug Addiction, Publications Office of the European

454

Union, Luxembourg, 2015.

455

(7) Zuccato, E.; Chiabrando, C.; Castiglioni, S.; Bagnati, R.; Fanelli, R. Estimating

456

community drug abuse by wastewater analysis. Environ. Health Perspect. 2008, 116(8),

457

1027–32.

20



458

(8) van Nuijs, A. L.; Castiglioni, S.; Tarcomnicu, I.; Postigo, C.; López de Alda, M.;

459

Neels, H.; Zuccato, E.; Barceló, D. Illicit drug consumption estimations derived from

460

wastewater analysis: a critical review. Sci. Total Environ. 2011, 409, 3564–3577.

461

(9) Castiglioni, S.; Thomas, K. V.; Kasprzyk-Hordern, B.; Vandamd, L.; Griffiths, P.;

462

Testing wastewater to detect illicit drugs: State of the art, potential and research needs.

463

Sci. Total Environ. 2014, 487, 613–620.

464

(10) Ort, C.; van Nuijs, A. L. N.; Berset, J. D.; Bijlsma, L.; Castiglioni, S.; Covaci, A.;

465

de Voogt, P.; Emke, E.; Fatta-Kassinos, D.; Grifﬁths, P.; Hernández, F.; González-

466

Mariño, I.; Grabic, R.; Kasprzyk-Hordern, B.; Mastroianni, N.; Meierjohann, A.; Nefau,

467

T.; Östman, M.; Picó, Y.; Racamonde, I.; Reid, M.; Slobodnik, J.; Terzic, S.;

468

Thomaidis, N.; Thomas, K. V. Spatial differences and temporal changes in illicit drug

469

use in Europe quantiﬁed by wastewater analysis. Addiction 2014, 109, 1338–1352.

470

(11) Subedi, B.; Kannan, K. Mass Loading and Removal of Select Illicit Drugs in Two

471

Wastewater Treatment Plants in New York State and Estimation of Illicit Drug Usage in

472

Communities through Wastewater Analysis. Environ. Sci. Technol. 2014, 48,

473

6661−6670.

474

(12) Yin Lai, F.; Bruno, R.; Hall, W.; Gartner, C.; Ort, C.; Kirkbride, P.; Prichard, J.;

475

Thai, P. K.; Carter, S.; Mueller, J.F. Profiles of illicit drug use during annual key

476

holiday and control periods in Australia: wastewater analysis in an urban, a semi-rural

477

and a vacation area. Addiction 2012, 108, 556–565.

478

(13) Khan, U.; van Nuijs, A. L. N.; Li, J.; Maho, W.; Dua, P.; Li, K.; Houa, L.; Zhang,

479

J.; Meng, X.; Li, X.; Covaci, A. Application of a sewage-based approach to assess the

21


Page 22 of 32

Page 23 of 32


480

use of ten illicit drugs in four Chinese megacities. Sci. Total Environ. 2014, 487, 710–

481

721.

482

(14) Banta-Green, C. J.; Field, J. A.; Chiaia, A. C.; Sudakin, D. L.; Power, L.; de

483

Montigny, L. The spatial epidemiology of cocaine, methamphetamine and 3,4-

484

methylenedioxymethamphetamine (MDMA) use: a demonstration using a population

485

measure of community drug load derived from municipal wastewater. Addiction 2009,

486

104(11), 1874–1880.

487

(15) Metcalfe, C.; Tindale, K.; Li, H.; Rodayan, A.; Yargeau, V. Illicit drugs in

488

Canadian municipal wastewater and estimates of community drug use. Environ

489

Pollut. 2010, 158(10), 3179–85

490

(16) Reid, M. J.; Baz-Lomba, J. A.; Ryu, Y.; Thomas, K. V. Using biomarkers in

491

wastewater to monitor community drug use: A conceptual approach for dealing with

492

new psychoactive substances. Sci. Total Environ. 2014, 487, 651–658.

493

(17) Mwenesongole, E. M.; Gautam, L.; Hall, S. W.; Waterhouse, J. W.; Cole, M. D.

494

Simultaneous detection of controlled substances in wastewater. Anal. Methods 2013, 5,

495

3248–3254.

496

(18) Castrignanò, E.; Lubben, A.; Kasprzyk-Hordern, B. Enantiomeric profiling of

497

chiral drug biomarkers in wastewater with the usage of chiral liquid chromatography

498

coupled with tandem mass spectrometry. J. Chromatogr. A 2016, 1438, 84–99.

499

(19) Chen, C.; Kostakis, C.; Irvine, R. J.; White, J. A. Increases in use of novel

500

synthetic stimulant are not directly linked to decreased use of 3,4-methylenedioxy-N-

501

methylamphetamine (MDMA). Forensic Sci. Int. 2013, 231, 278–283. 22



Page 24 of 32

502

(20) Tscharke, B. J., Chen, C., Gerber, J. P., White, J. M. Temporal trends in drug use in

503

Adelaide, South Australia by wastewater analysis. Sci Total Environ. 2016, 565, 384-

504

391

505

(21) Castiglioni, S.; Borsotti, A.; Senta, I.; Zuccato, E. Wastewater Analysis to Monitor

506

Spatial and Temporal Patterns of Use of Two Synthetic Recreational Drugs, Ketamine

507

and Mephedrone, in Italy. Environ. Sci. Technol. 2015, 49(9), 5563–5570.

508

(22) Vuori, E.; Happonen, M.; Gergov, M.; Nenonen, T.; Järvinen, A.; Ketola, R. A.;

509

Vahala, R. Wastewater analysis reveals regional variability in exposure to abused drugs

510

and opioids in Finland. Sci. Total Environ. 2014, 487, 688–695.

511

(23) Kankaanpää, A.; Ariniemi, K.; Heinonen, M.; Kuoppasalmi, K.; Gunnar, T. Use of

512

illicit stimulant drugs in Finland: A wastewater study in ten major cities. Sci. Total

513

Environ. 2014, 487, 696–702.

514

(24) Kinyua, J.; Covaci, A.; Maho, W.; McCall, A. K.; Neels, H.; van Nuijs, A. L. N.

515

Sewage-based epidemiology in monitoring the use of new psychoactive substances:

516

Validation and application of an analytical method using LC-MS/MS. Drug Test. Anal.

517

2015, 7(9), 812–818.

518

(25) Borova, V. L.; Gago-Ferrero, P.; Pistos, C.; Thomaidis, N. S. Multi-residue

519

determination of 10 selected new psychoactive substances in wastewater samples by

520

liquid chromatography–tandem mass spectrometry. Talanta 2015, 144, 592–603.

521

(26)

522

http://www.emcdda.europa.eu/attachements.cfm/att_138330_EN_No5_Eurobarometer_

523

Final.pdf

Flash

Eurobarometer

23


available

at:

Page 25 of 32


524

(27) Castiglioni, S.; Bijlsma, L.; Covaci, A.; Emke, E.; Hernández, F.; Reid, M.; Ort,

525

C.; Thomas, K. V.; van Nuijs, A. L. N.; de Voogt, P.; Zuccato, E.; Evaluation of

526

Uncertainties Associated with the Determination of Community Drug Use through the

527

Measurement of Sewage Drug Biomarkers. Environ. Sci. Technol. 2013; 47, 1452–

528

1460.

529

(28) Castiglioni, S.; Zuccato, E.; Crisci, E.; Chiabrando, C.; Fanelli, R.; Bagnati, R.

530

Identification and Measurement of Illicit Drugs and Their Metabolites in Urban

531

Wastewater by Liquid Chromatography−Tandem Mass Spectrometry. Anal. Chem.

532

2006, 78(24), 8421–8429.

533

(29) Commission Decision 2002/657/EC of 12 August 2002 implementing Council

534

Directive 96/23/EC; European Commission 2002.

535

(30) Strano Rossi, S.; Odoardi, S.; Gregori, A.; Peluso, G.; Ripani, L.; Ortar, G.;

536

Serpelloni, G.; Romolo, F. S. An analytical approach to the forensic identification of

537

different classes of new psychoactive substances (NPSs) in seized materials. Rapid

538

Commun. Mass Spectrom. 2014, 28, 1904–1916.

539

(31) Concheiro, M.; Anizan, S.; Ellefsen, K.; Huestis, M. A. Simultaneous

540

quantification of 28 synthetic cathinones and metabolites in urine by liquid

541

chromatography-high resolution mass spectrometry, Anal. Bioanal. Chem. 2013,

542

405(29), 9437–9448.

543

(32) Miserez, B.; Ayrton, O.; Ramsey, J. Analysis of purity and cutting agents in street

544

mephedrone samples from South Wales, Forensic Toxicol. 2014, 32, 305–310.

545

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 ... - ACS Publications

Recommend Documents