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Ultra-high performance liquid chromatography coupled to ion mobility separation and high-resolution mass spectrometry instruments have proven very valuable for screening of emerging contaminants in the aquatic environment. However, when applying suspect or nontarget approaches (

The COVID-19 pandemic spread rapidly worldwide in the year 2020, which was initially restrained by drastic mobility restrictions. In this work, we investigated the use of illicit drugs (amphetamine, methamphetamine, ecstasy, cocaine and cannabis), and licit substances of abuse (alcohol and tobacco) during the earlier months (March–July 2020) of the pandemic restrictions in four Spanish (Bilbao and its metropolitan area, Vitoria-Gasteiz, Castellón and Santiago de Compostela) and two Portuguese (Porto and Vila do Conde) locations by wastewater-based epidemiology (WBE). The results show that no methamphetamine was detected in any of the locations monitored, while amphetamine use was only detectable in the two locations from the Basque Country (Bilbao and its metropolitan area and Vitoria-Gasteiz), with high estimated average usage rates (700–930 mg day−1 1000 inhabitant−1). The remaining substances were detected in all the investigated catchment areas. In general, no remarkable changes were found in population normalized loads compared to former years, except for cocaine (i.e. its main metabolite, benzoylecgonine). For this drug, a notable decrease in use was discernible in Castellón, while its usage in Porto and Santiago de Compostela seemed to continue in a rising trend, already initiated in former years. Furthermore, two events of ecstasy (3,4-methylenedioxymethamphetamine, MDMA) dumping in the sewage network were confirmed by enantiomeric analysis, one in Santiago de Compostela just prior the lockdown and the second one in the Bilbao and its metropolitan area in July after relieving the more stringent measures. The latter could also be associated with a police intervention. The comparison of WBE with (web) survey data, which do not provide information at a local level, points towards contradictory conclusions for some of the substances, thereby highlighting the need for stable WBE networks capable of near real-time monitoring drug use This research was funded by the Spanish Agencia Estatal de Investigación – MCIN/AEI/10.13039/501100011033 (CTM2016-81935-REDT, PID2020-117686RB-C32 and PID2020-117686RB-C31), Consellería de Cultura de Galicia, Educación e Universidades (ED481A-2020/258 and ED431C 2021/06), Delegación del Gobierno para el Plan Nacional Sobre Drogas (2020I009), Instituto de Salud Carlos III-Next Generation EU (RD21/0009/0012 – RIAPAd Network), Generalitat Valenciana (Research Group of Excellence Prometeo 2019/040), Fundación Vital (Vitoria-Gasteiz) and Ayuntamiento de Vitoria-Gasteiz SI

New psychoactive substances (NPS), often designed as (legal) substitutes to conventional illicit drugs, are constantly emerging in the drug market and being commercialized in different ways and forms. Their use continues to cause public health problems and is therefore of major concern in many countries. Monitoring NPS use, however, is arduous and different sources of information are required to get more insight of the prevalence and diffusion of NPS use. The determination of NPS in pooled urine and wastewater has shown great potential, adding a different and complementary light on this issue. However, it also presents analytical challenges and limitations that must be taken into account such as the complexity of the matrices, the high sensitivity and selectivity required in the analytical methods as a consequence of the low analyte concentrations as well as the rapid transience of NPS on the drug market creating a scenario with constantly moving analytical targets. Analytical investigation of NPS in pooled urine and wastewater is based on liquid chromatography hyphenated to mass spectrometry and can follow different strategies: target, suspect and non-target analysis. This work aims to discuss the advantages and disadvantages of the different data acquisition workflows and data exploration approaches in mass spectrometry, but also pays attention to new developments such as ion mobility and the use of in-silico prediction tools to improve the identification capabilities in high-complex samples. This tutorial gives an insight into this emerging topic of current concern, and describes the experience gathered within different collaborations and projects supported by key research articles and illustrative practical examples. Refereed/Peer-reviewed

New psychoactive substances (NPS) are compounds designed to mimic both licit and illicit drugs, and these substances are being discovered each year through forensic toxicology, drug enforcement agencies, and health authorities. However, there is limited information surrounding their international popularity. In this work, influent wastewater samples (n = 144) were collected from 25 sites in 10 countries: Australia, Belgium, Canada, China, Fiji, Italy, New Zealand, Republic of Korea, Spain, and the United States over the 2020-2021 New Year period. All samples were extracted in the country of origin then shipped and analyzed centrally at the University of South Australia using validated liquid chromatography-mass spectrometry methods. This study focused on 28 NPS stimulants, with 11 detected. The emerging substances eutylone and 3-methylmethcathinone (3-MMC) were detected most frequently and with the highest mass loads, indicating international popularity. Interestingly, the "older"generation stimulants, para-methoxyamphetamine (PMA), methylone, and mephedrone, were also detected. From the sites monitored in this work, areas in New Zealand had the highest loads of NPS stimulant consumption. Results here show that wastewater analysis can elucidate the dynamic nature of the NPS market, providing near real-time information on changing consumption patterns whose information can be used to minimize public risk. Refereed/Peer-reviewed

The number of open access databases containing experimental and predicted collision cross section (CCS) values is rising and leads to their increased use for compound identification. However, the reproducibility of reference values with different instrumental designs and the comparison between predicted and experimental CCS values is still under evaluation. This study compared experimental CCS values of 56 small molecules (Contaminants of Emerging Concern) acquired by both drift tube (DT) and travelling wave (TW) ion mobility mass spectrometry (IM-MS). The TWIM-MS included two instrumental designs (Synapt G2 and VION). The experimental TWCCSN2 values obtained by the TWIM-MS systems showed absolute percent errors (APEs) < 2% in comparison to experimental DTIMS data, indicating a good correlation between the datasets. Furthermore, TWCCSN2 values of [M − H]- ions presented the lowest APEs. An influence of the compound class on APEs was observed. The applicability of prediction models based on artificial neural networks (ANN) and multivariate adaptive regression splines (MARS), both built using TWIM-MS data, was investigated for the first time for the prediction of DTCCSN2 values. For [M+H]+ and [M − H]- ions, the 95th percentile confidence intervals of observed APEs were comparable to values reported for both models indicating a good applicability for DTIMS predictions. For the prediction of DTCCSN2 values of [M+Na]+ ions, the MARS based model provided the best results with 73.9% of the ions showing APEs below the threshold reported for [M+Na]+. Finally, recommendations for database transfer and applications of prediction models for future DTIMS studies are made. L. Belova acknowledges funding through a Research Foundation Flanders (FWO) fellowship (11G1821N). L. Bijlsma acknowledges his fellowship funded by “la Caixa” Foundation. The project that gave rise to these results also received the support of a fellowship from “la Caixa” Foundation (ID 10 0 010434). The fellowship code is LCF/BQ/PR21/11840012. Jesse Sterckx is acknowledged for helping with the measurements on the Synapt G2 system. This work received financial support also from the University Jaume I (UJI-B2020-19). The graphical abstract was created with BioRender.com, license no 2641–5211.

The COVID-19 outbreak has forced countries to introduce severe restrictive measures to contain its spread. In particular, physical distancing and restriction of movement have had important consequences on human behaviour and potentially also on illicit drug use and supply. These changes can be associated with additional risks for users, in particular due to reduced access to prevention and harm reduction activities. Furthermore, there have been limitations in the amount of data about drug use which can be collected due to restrictions. To goal of this study was to obtain information about potential changes in illicit drug use impacted by COVID-19 restrictions. Wastewater samples were collected in seven cities in the Netherlands, Belgium, Spain and Italy at the beginning of lockdowns (March-May 2020). Using previously established and validated methods, levels of amphetamine (AMP), methamphetamine (METH), MDMA, benzoylecgonine (BE, the main metabolite of cocaine) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH, main metabolite of tetrahydrocannabinol (THC)) were measured and compared with findings from previous years. Important differences in levels of consumed drugs were observed across the considered countries. Whilst for some substances and locations, marked decreases in consumption could be observed (e.g., 50% decrease in MDMA levels compared to previous years). In some cases, similar or even higher levels compared to previous years could be found. Changes in weekly patterns were also observed, however these were not clearly defined for all locations and/or substances. Findings confirm that the current situation is highly heterogeneous and that it remains very difficult to explain and/or predict the effect that the present pandemic has on illicit drug use and availability. However, given the current difficulty in obtaining data due to restrictions, wastewater analysis can provide relevant information about the situation at the local level, which would be hard to obtain otherwise.

Background Fresh water bodies represent less than 1% of overall amount of water on earth and ensuring their quality and sustainability is pivotal. Although several campaigns have been performed to monitor the occurrence of micropollutants by means of chemical analysis, this might not cover the whole set of chemicals present in the sample nor the potential toxic effects of mixtures of natural and anthropogenic chemicals. In this sense, by selecting relevant toxicity endpoints when performing in vitro bioanalysis, effect-based methodologies can be of help to perform a comprehensive assessment of water quality and reveal biological activities relevant to adverse health effects. However, no prior bioanalytical study was performed in wetland water samples from the Spanish Mediterranean coastline. Methods Eleven samples from relevant water bodies from the Spanish Mediterranean coastline were collected to monitor water quality on 8 toxicity endpoints. Aryl hydrocarbon receptor (AhR), androgenicity (AR+ and AR−), estrogenicity (ER+ and ER−), oxidative stress response (Nrf2) and vitamin D receptor (VDR+ and VDR−) reporter gene assays were evaluated. Results AhR was the reporter gene assay showing a more frequent response over the set of samples (activated by 9 out of 11 samples), with TCDD-eq in the range 7.7–22.2 pM. For AR, ER and VDR assays sporadic activations were observed. Moreover, no activity was observed on the Nrf2 reporter gene assay. Wastewater and street runaway streams from Valencia could be responsible for enhanced activities in one of the water inputs in the Natural Park ‘L’Albufera’. Conclusions Water quality of relevant wetlands from the Spanish Mediterranean coastline has been evaluated. The utilization of a panel of 5 different bioassays to cover for different toxicity endpoints has demonstrated to be a good tool to assess water quality.

Wastewater-based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population-normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011-17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data.Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017.Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries.Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of ΔBenzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North-Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge.The analysis of wastewater to quantify drug loads provides near real-time drug use estimates that globally correspond to prevalence and seizure data.

Ion mobility separation (IMS) coupled to high resolution mass spectrometry (IMS-HRMS) is a promising technique for (non-)target/suspect analysis of micropollutants in complex matrices. IMS separates ionized compounds based on their charge, shape and size facilitating the removal of co-eluting isomeric/isobaric species. Additionally, IMS data can be translated into collision cross-section (CCS) values, which can be used to increase the identification reliability. However, IMS-HRMS for the screening of contaminants of emerging concern (CECs) have been scarcely explored. In this study, the role of IMS-HRMS for the identification of CECs in complex matrices is highlighted, with emphasis on when and with which purpose is of use. The utilization of IMS can result in much cleaner mass spectra, which considerably facilitates data interpretation and the obtaining of reliable identifications. Furthermore, the robustness of IMS measurements across matrices permits the use of CCS as an additional relevant parameter during the identification step even when reference standards are not available. Moreover, an effect on the number of true and false identifications could be demonstrated by including IMS restrictions within the identification workflow. Data shown in this work is of special interest for environmental researchers dealing with the detection of CECs with state-of-the-art IMS-HRMS instruments., Authors acknowledge the financial support by the Swedish Research Council (FORMAS) in “Effect-directed analysis as a tool towards a non-toxic environment - identification of mixture effects and toxicity drivers in water (DANTE)” project (2018–02256). A.C., F.H., F.L., E.P., J.V.S. and L.B. from University Jaume I acknowledge the financial support of Spanish Ministry of Science, Innovation and Universities (RTI 2018-097417-B-100), of Generalitat Valenciana (Research Group of Excellence Prometeo 2019/040) and of University Jaume I of Castellón, Spain (project UJI-B2018-55 and project UJI-B2020-19). A.C. acknowledges the Spanish Ministry of Economy and Competiveness for his predoctoral grant (BES-2016-076914).

Amphetamine (AMP), methamphetamine (MAMP) and 3,4-methylenedioxymethamphetamine (MDMA) occur in wastewater not only as a result of illicit consumption, but also, in some cases, from prescription drug use or by direct drug disposal into the sewage system. Enantiomeric profiling of these chiral drugs could give more insight into the origin of their occurrence. In this manuscript, a new analytical methodology for the enantiomeric analysis of amphetamine-like substances in wastewater has been developed. The method consists of a solid-phase extraction (SPE) followed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-MS/MS), which showed low quantification limits in the 2.4–5.5 ng L−1 range. The LC-MS/MS method was first applied to characterize a total of 38 solid street drug samples anonymously provided by consumers. The results of these analysis showed that AMP and MDMA trafficked into Spain are synthesized as racemate, while MAMP is exclusively produced as the S(+)-enantiomer. Then, the analytical method was employed to analyse urban wastewater samples collected from the wastewater treatment plants (WWTPs) of five different cities in 2018 and 2019. Consumption estimated through normalized population loads in wastewater showed an increased pattern of AMP use in the Basque Country. Furthermore, the enantiomeric profiling of wastewater samples was contrasted to lisdexamfetamine (LIS) and selegiline (SEL) prescription figures, two pharmaceuticals which metabolize to S(+)-AMP, and to R(-)-AMP and R(-)-MAMP, respectively. From this analysis, and considering uncertainties derived from metabolism and adherence to treatment, it was concluded that LIS is a relevant source of AMP in those cases with low wastewater loads, i.e. up to a maximum of 60% of AMP detected in wastewater in some samples could originate from LIS prescription, while SEL does not represent a significant source of AMP nor MAMP. Finally, removal efficiencies could be evaluated for the WWTP (serving ca. 860,000 inhabitants) with higher AMP influent concentrations. The removal of AMP was satisfactory with rates higher than 99%, whereas MDMA showed an average removal of approximately 60%, accompanied by an enrichment of R(-)-MDMA This study was supported by MINECO/MICINN/AEI projects (CTM2016-81935-REDT, CTM2017-84763-C3-2-R, CTM2017-84763-C3-1-R, CTM2017-84763-C3-3-R, PID2020-117686RB-C32, PID2020-117686RB-C31, PID2020-117686RB-C33), Galician Council of Culture, Education and Universities (ED481D-2017/003, ED431C-2017/36, ED481A-2020/258 and ED431C 2021/06), cofounded by FEDER/ERDF. UJI authors acknowledge the financial support of Generalitat Valenciana (Excellence Research Group, Prometeo 2019/040). Alberto Celma acknowledges the Spanish Ministry of Economy and Competitiveness for his predoctoral Grant (BES-2016-076914). Vanessa Gutmann acknowledges the support of the ERASMUS+ program. SI

Compendi d'articles The amount of organic micropollutants (OMP) currently being released to the aquatic environment is incommensurable. Thus, different analytical strategies were developed over the recent years to monitor the occurrence of OMP in environmental samples. In this thesis, different tools have been applied to refine target, suspect and non-target screening strategies for the monitoring of the aquatic environment with special emphasis on the hyphenation of ion mobility separation (IMS) to high resolution mass spectrometry. Additionally, the development of refinement tools for suspect screening strategies such as retention time indexing or in silico prediction of IMS data, as well as the implementation of effect-directed analysis for a comprehensive overview of the quality of water bodies have also been explored. Finally, the monitoring of new psychoactive substances in complex matrices such as influent wastewater and pooled urine samples is evaluated, with special aid of the previously developed tools. La cantidad de microcontaminantes orgánicos (OMP) emitidos al medio ambiente acuático en la actualidad es incalculable. Por ello, se han desarrollado distintas estrategias analíticas para monitorizar la incidencia de OMP en muestras ambientales. En esta tesis se han aplicado diferentes herramientas para refinar estrategias de cribado dirigidas, de sospechosos y no dirigidas para la monitorización del medio ambiente acuático con especial énfasis en el acoplamiento de la separación por movilidad iónica (IMS) con espectrometría de masas de alta resolución. Adicionalmente, se ha explorado el desarrollo de herramientas de mejora para los cribados de sospechosos como, por ejemplo, indexación de tiempo de retención o predicción computacional de datos de IMS, así como la implementación de análisis basados en efecto (EDA) para una visión completa de la calidad de los cuerpos acuáticos. Finalmente, se ha evaluado la monitorización de Nuevas Sustancias Psicoactivas (NPS) en muestras complejas tales como aguas residuales y orina. Programa de Doctorat en Ciències

Data obtained from wastewater analysis can provide rapid and complementary insights in illicit drug consumption at community level. Within Europe, Spain is an important country of transit of both cocaine and cannabis. The quantity of seized drugs and prevalence of their use rank Spain at the top of Europe. Hence, the implementation of a wastewater monitoring program at national level would help to get better understanding of spatial differences and trends in use of illicit drugs. In this study, a national wastewater campaign was performed for the first time to get more insight on the consumption of illicit drugs within Spain. The 13 Spanish cities monitored cover approximately 6 million inhabitants (12.8% of the Spanish population). Untreated wastewater samples were analyzed for urinary biomarkers of amphetamine, methamphetamine, MDMA, cocaine, and cannabis. In addition, weekend samples were monitored for 17 new psychoactive substances. Cannabis and cocaine are the most consumed drugs in Spain, but geographical variations showed, for instance, comparatively higher levels of methamphetamine in Barcelona and amphetamine in Bilbao, with about 1-fold higher consumption of these two substances in such metropolitan areas. For amphetamine, an enantiomeric profiling was performed in order to assure the results were due to consumption and not to illegal dumping of production residues. Furthermore, different correction factors for the excretion of cannabis were used to compare consumption estimations. All wastewater results were compared with previously reported data, national seizure data and general population survey data, were a reasonable agreement was found. Daily and yearly drug consumption were extrapolated to the entire Spanish population with due precautions because of the uncertainty associated. These data was further used to estimate the retail drug market, where for instance cocaine illicit consumption alone was calculated to contribute to 0.2–0.5% of the Spanish gross domestic product (ca. 3000–6000 million Euro/year)., This study was supported by MINECO/AEI projects (CTM2016-81935-REDT, CTM2017-84763-C3-2-R, CTM2017-84763-C3-1-R, CTM2017-84763-C3-3-R, RTI2018-097158-B-C31 and CTM2017-90890-REDT), MICINN project (CEX2018-000794-S), Galician Council of Culture, Education and Universities (ED481D-2017/003, ED431C-2017/36 and ED481A-2020/258), cofounded by FEDER/ERDF. The UJI and CIDE (CSIC-UV-GV) authors acknowledge the financial support of Generalitat Valenciana (Excellence Research Group, Prometeo 2019/040 and Prometeo 2018/150, respectively). IDAEA-CSIC authors acknowledge the financial support of Generalitat de Catalunya (Exp. Num. SA-2018-780 and Consolidated Research Group 2017 SGR 01404) and the Spanish Ministry of Science and Innovation (Project CEX2018-000794-S). Alberto Celma acknowledges the Spanish Ministry of Economy and Competitiveness for his predoctoral Grant (BES-2016-076914). Elena Pitarch acknowledge the financial support of University Jaume I of Castellón, Spain (project UJI-B2018-55). Andreu Rico is supported by a postdoctoral grant provided by the Spanish Ministry of Science, Innovation and University (IJCI–2017–33465).

Currently, the most powerful approach to monitor organic micropollutants (OMPs) in environmental samples is the combination of target, suspect, and nontarget screening strategies using high-resolution mass spectrometry (HRMS). However, the high complexity of sample matrices and the huge number of OMPs potentially present in samples at low concentrations pose an analytical challenge. Ion mobility separation (IMS) combined with HRMS instruments (IMS−HRMS) introduces an additional analytical dimension, providing extra information, which facilitates the identification of OMPs. The collision cross-section (CCS) value provided by IMS is unaffected by the matrix or chromatographic separation. Consequently, the creation of CCS databases and the inclusion of ion mobility within identification criteria are of high interest for an enhanced and robust screening strategy. In this work, a CCS library for IMS−HRMS, which is online and freely available, was developed for 556 OMPs in both positive and negative ionization modes using electrospray ionization. The inclusion of ion mobility data in widely adopted confidence levels for identification in environmental reporting is discussed. Illustrative examples of OMPs found in environmental samples are presented to highlight the potential of IMS−HRMS and to demonstrate the additional value of CCS data in various screening strategies.

Parent and modified mycotoxin analysis remains a challenge because of their chemical diversity, the presence of isomeric forms, and the lack of analytical standards. The creation and application of a collision cross section (CCS) database for mycotoxins may bring new opportunities to overcome these analytical challenges. However, it is still an open question whether common CCS databases can be used independently from the instrument type and ion mobility mass spectrometry (IM-MS) technologies, which utilize different methodologies for determining the gas-phase mobility. Here, we demonstrated the reproducibility of CCS measurements for mycotoxins in an interlaboratory study (average RSD 0.14% ± 0.079) and across different traveling wave IM-MS (TWIMS) systems commercially available (ΔCCS% < 2). The separation in the drift time dimension of critical pairs of isomers for modified mycotoxins was also achieved. In addition, the comparison of measured and predicted CCS values, including regulated and emerging mycotoxins, was addressed.

Wastewater-based epidemiology (WBE) has become a very useful tool to monitor a population's drug consumption or exposure to environmental and food contaminants. In this work, WBE has been applied to estimate tobacco consumption in seven Spanish regions. To this end, 24 h composite wastewater samples were taken daily for one week in 17 wastewater treatment plants, covering altogether a population of ca. 6 million inhabitants. The samples were treated by enzymatic deconjugation and the wastewater content of two human-specific nicotine metabolites (namely, cotinine and trans-3′-hydroxycotinine) was measured to estimate the daily consumption of nicotine. The population-weighted average nicotine consumption in the seven analyzed regions was 2.2 g/(day∙1000 inh.), without any daily pattern. This average estimated nicotine consumption value agreed with the value derived from official tobacco sales data. Differences in consumption among the seven studied regions were found, being Galicia, the region with the lowest rate, and the Basque Country and Catalonia those with the highest rates. However, no conclusive correlation was found between those values and the prevalence data taken from two different national surveys, nor sociodemographic and health data. This study demonstrates that this tool can complement other indicators in order to accurately assess tobacco consumption rates at regional and national levels and provides the most extensive application of the approach in the Spanish territory., Financial support This study was supported by MINECO/AEI projects (CTM2016-81935-REDT, CTM2017-84763-C3-2-R, CTM2017-84763-C3-1-R, CTM2017-84763-C3-3-R), MICINN project (CEX2018-000794-S), Galician Council of Culture, Education and Universities (ED481D 2017/003 and ED431C2017/36), cofunded by FEDER/ERDF.

Background and aims Wastewater‐based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population‐normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4‐methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011–17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data. Design Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017. Setting and Participants Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries. Measurements Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of Δ9‐tetrahydrocannabinol (11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol) were measured in wastewater using liquid chromatography–tandem mass spectrometry. Daily mass loads (mg/day) were normalized to catchment population (mg/1000 people/day) and converted to the number of combined doses consumed per day. Spatial differences were assessed world‐wide, and temporal trends were discerned at European level by comparing 2011–13 drug loads versus 2014–17 loads. Findings Benzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North–Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge. Conclusions The analysis of wastewater to quantify drug loads provides near real‐time drug use estimates that globally correspond to prevalence and seizure data., This study was supported by the following countries, institutions and projects Australia: Thyne Reid foundation; Canada: Natural Sciences and Engineering Research Council of Canada (NSERC); Iceland: The Icelandic Research Fund (Grant number 163049–051); Slovenia: Slovene Research Agency (funding Project L1–9191); Spain: MINECO/AEI projects (CTM2014–56628‐C3–2‐R, CTQ2015–65603‐P, CTM2016–81935‐REDT, CTM2017–84763‐C3–2‐R), Galician Council of Culture, Education and Universities (ED481D 2017/003); UK: Environmental Sustainability Knowledge Transfer Network, Engineering and Physical Sciences Research Council and Thermo Fisher Scientific (CASE industrial scholarship for K. Munro, Ref.: EP/J502029/1). European Monitoring Centre for Drugs and Drug Addiction (EMCDDA); EU International Training Network SEWPROF (Marie Curie‐FP7‐PEOPLE, grant number 317205); COST Action ES1307 supported by COST (European Cooperation in Science and Technology); WATCH (Wastewater Analysis of Traces of illicit drug‐related Chemicals for law enforcement and public Health DG migration and Home affairs ‐ HOME/2015/ISFP/PR/DRUG/0062). Sampling support and provision of catchment information Austria: Klemens Geiger and Michael Schlapp; Belgium: Jonathan Phariseau, Bart Coene, Luc Van Os, Peter Nys, Dieter Lemaire, Jean‐François Mougel, Patrick Vantroyen and Karel Claes; Croatia: Marin Ganjto; Czech Republic: Jiri Stara and Robert Hrich Brněnské; France: Véronique Bremont; Italy: Roberto Mazzini and Francesca Pizza (Milan), Francesco Avolio (Bologna), Fabrizio Moratto and Sergio Ghezzi (Gorizia), Rosanna Brienza and Simona Panariello (Potenza), Pier Paolo Abis and Antonia Attanasio (Bari), Antonella Cicala (Palermo); Norway: Pia Ryrfors; Portugal: João Goulão, José Martins, Pedro Alvaro and Fátima Paixão; Slovakia: Silvia Antalová and Jozef Tichy; Spain: Begoña Martínez López, Cristian Mesa, Santiago Querol Rodriguez, Fernando Llavador, Enrique Albors and Gloria Fayos; the Netherlands: Alex Veltman, Peter Theijssen, Peter van Dijk, Stefan Wijers, Mark Stevens, Ferry de Wilde. Analytical support Australia: Maulik Ghetia; Cyprus: Popi Karaolia; Greece: Nikoilaos Raikos; Spain: Ana Maria Botero‐Coy, Clara Boix, Alberto Celma, Jorge Pitarch, Inés Racamonde and Eddie Fonseca; Italy: Emma Gracia‐Lor, Nikolaos I. Rousis and Noelia Salgueiro‐Gonzalez. Data repository and data screening Eawag: Harald von Waldow; EMCDDA: Renate Hochwieser, Liesbeth Vandam, João Matias and Federica Mathis.

Wastewater-based epidemiology (WBE) can be a useful tool to face some of the existing challenges in monitoring the use of new psychoactive substances (NPS), as it can provide objective and updated information. This Europe-wide study aimed to verify the suitability of WBE for investigating the use of NPS. Selected NPS were monitored in urban wastewater by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The main classical illicit drugs were monitored in the same samples to compare their levels with those of NPS. Raw composite wastewater samples were collected in 2016 and 2017 in 14 European countries (22 cities) following best practice sampling protocols. Methcathinone was most frequent (>65% of the cities), followed by mephedrone (>25% of the cities), and only mephedrone, methcathinone and methylone were found in both years. This study depicts the use of NPS in Europe, confirming that it is much lower than the use of classical drugs. WBE proved able to assess the qualitative and quantitative spatial and temporal profiles of NPS use. The results show the changeable nature of the NPS market and the importance of large WBE monitoring campaigns for selected priority NPS. WBE is valuable for complementing epidemiological studies to follow rapidly changing profiles of use of drugs. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4396]

Wastewater-based epidemiology (WBE) can give valuable light on the extent and actual use of new psychoactive substances (NPS). In this work, a fully validated methodology for the simultaneous determination of illicit drugs and NPS in wastewater by solid-phase extraction followed by UHPLC-MS/MS has been developed. The small sample volume (5 mL) required for analysis is of high interest, especially when performing large sampling campaigns involving many locations of different geographical origin, as it has been performed in the past. The method was applied to wastewater samples from different European locations and permitted the simultaneous monitoring of conventional drugs and NPS. Cocaine, amphetamine, MDMA, methamphetamine and ketamine were found in all wastewater samples, and several NPS (dipentylone, butylone, mephedrone, methedrone and methylone) were observed in some of the samples monitored. It is noteworthy that dipentylone was detected in wastewater for the very first time. Furthermore, a detailed comparison of micro liquid chromatography (μLC) and UHPLC, both coupled to tandem mass spectrometry, in terms of sensitivity and reproducibility has been made for the first time in the application field of WBE. An average increase factor of 14 (mass normalized data) was observed in sensitivity for μLC-MS/MS. The overall method performance was also compared (un-normalized data), and an average increase sensitivity factor of 4.5 was observed for μLC-MS/MS. However, large deviations in retention time (up to 0.4 min) affected the reproducibility and robustness of the μLC-MS/MS method when it was applied to wastewater analysis. Although in this work μLC-MS/MS was strongly influenced by the amount of matrix loaded in the separation device, its enhanced sensitivity and promotion of green chemistry (faster analysis time and less solvent consumption) allow to expect improved future applications, especially when analytes are present at very low concentrations.

New psychoactive substances (NPSs) are compounds that produce similar effects to those induced by illicit drugs (IDs), such as cocaine, cannabis, and amphetamines, but are not strictly regulated by international conventions. The consumption of NPSs is a growing public health problem in many communities. However, there is little knowledge regarding the extent and actual use of these new substances. Monitoring NPS use is arduous, and therefore, different sources of information need to be used to get more insight of the prevalence and diffusion of NPS use. Analysis of pooled urine and wastewater (WW) shows strong potential, giving a different and complementary light on this issue, although presents some limitations and challenges that must be taken into account. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is one of the most powerful approaches for screening a large number of NPSs because of the accurate-mass full-spectrum acquisition measurements. By using a comprehensive and updated NPS database, LC-HRMS is flexible enough to confront the ever-changing NPS market. In this “current opinion”, we give our point of view on the usefulness of pooled urine and WW analysis and on the potential application of wastewater-based epidemiology as source of information for NPS use, explaining the main bottlenecks and future perspectives in this emerging research field.

The number of new psychoactive substances (NPS) on the recreational drug market has increased rapidly in the last years, creating serious challenges for public health agencies and law enforcement authorities. Epidemiological surveys and forensic analyses to monitor the consumption of these substances face some limitations for investigating their use on a large scale in a shifting market. The aim of this work was to develop a comprehensive and flexible screening approach for assessing the presence of NPS in urban wastewater by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Almost 200 substances were selected as “priority NPS” among those most frequently and recently reported by the Early Warning Systems (EWS) of different agencies and were included in the screening. Wastewater samples were collected from several cities all over Europe in 2016 and 2017, extracted using different solid-phase cartridges and analysed by LC-HRMS. The screening workflow comprised two successive analytical steps and compounds were identified and confirmed following specific criteria from the current guidelines. Thirteen NPS were identified at different confidence levels by using analytical standards or information from libraries and literature, and about half of them were phenethylamines. As far as we know, this is the first time that four of them (i.e. 3,4-dimethoxy-α-pyrrolidinovalerophenone, para-methoxyamphetamine, 2-phenethylamine and α – methyltryptamine) have been found in urban wastewater. The proposed screening approach was successfully applied in the largest NPS European wastewater monitoring, providing an innovative and easily adapted procedure for investigating NPS. In the light of current challenges and specific future research issues, this approach may complement epidemiological information and help in establishing measures for public health protection.

The consumption of psychoactive substances is considered a growing probleminmany communities. Moreover, new psychoactive substances (NPS) designed as (legal) substitutes to traditional illicit drugs are relatively easily available to the public through e-commerce and retail shops, but there is little knowledge regarding the extent and actual use of these substances. This study aims to gain new and complementary information on NPS and traditional illicit drug use at sixmusic festivals across Europe by investigatingwastewater and pooled urine. Samples were collected, between 2015 and 2018, at six music festivals across Europe with approximately 465.000 attendees. Wastewater samples were also collected during a period not coinciding with festivals. A wide-scope screening for 197 NPS, six illicit drugs and known metabolites was applied using different chromatographymass spectrometric strategies. Several illicit drugs and in total 21 different NPS, mainly synthetic cathinones, phenethylamines and tryptamines, were identified in the samples. Ketamine and the traditional illicit drugs, such as amphetamine-type stimulants, cannabis and cocaine were most abundant and/or frequently detected in the samples collected, suggesting a higher use compared to NPS. The analyses of urine and wastewater is quick and a high number of attendeesmay be monitored anonymously by analysing only a fewsampleswhich allows identifying the local profiles of use of different drugswithin awide panel of psychoactive substances. This approach contributes to the development of an efficient surveillance system which can provide timely insight in the trends of NPS and illicit drugs use. (C) 2020 Elsevier B.V. All rights reserved.

The accuracy and sensitivity of high resolution mass spectrometry (HRMS) enables the identification of candidate compounds with the use of mass spectrometric databases among other tools. However, retention time (RT) data in identification workflows has been sparingly used since it could be strongly affected by matrix or chromatographic performance. Retention Time Interpolation scaling (RTi) strategies can provide a more robust and valuable information than RT, gaining more confidence in the identification of candidate compounds in comparison to an analytical standard. Up to our knowledge, no RTi has been developed for LC-HRMS systems providing information when acquiring in either positive or negative ionization modes. In this work, an RTi strategy was developed by means of the use of 16 isotopically labelled reference standards, which can be spiked into a real sample without resulting in possible false positives or negatives. For testing the RTi performance, a mixture of several reference standards, emulating suspect analytes, were used. RTi values for these compounds were calculated both in solvent and spiked in a real matrix to assess the effect of either chromatographic parameters or matrix in different scenarios. It has been demonstrated that the variation of injection volume, chromatographic gradient and initial percentage of organic solvent injected does not considerably affect RTi calculation. Column aging and solid support of the stationary phase of the column, however, showed strong effects on the elution of several test compounds. Yet, RTi permitted the correction of elution shifts of most compounds. Furthermore, RTi was tested in 47 different matrices from food, biological, animal feeding and environmental origin. The application of RTi in both positive and negative ionization modes showed in general satisfactory results for most matrices studied. The RTi developed can be used in future LC-HRMS screening analysis giving an additional parameter, which facilitates tedious processing tasks and gain more confidence in the identification of (non)-suspect analytes.

New psychoactive substances (NPS) have increased in use and popularity worldwide. Wastewater analysis has been successfully applied to evaluate illicit drugs use within a population. However, for NPS, such approach may be limited due to low doses of NPS combined with their ever‐changing composition and usage. The dynamic nature of the NPS market means use may be opportunistic, infrequent and with few users. Hence, the use of complementary information sources is recommended to improve the knowledge on NPS consumption. The aim of this study was to investigate the changing landscape of NPS use on a community scale by combining wastewater analysis and forensic toxicology. Forensic analysis provided specific information on NPS prevalence in post‐mortem blood samples in Adelaide, South Australia over five years, while wastewater analysis showed community use over the same period. A qualitative liquid chromatography‐high resolution mass spectrometry method was initially used to screen the wastewater samples. A total of 24 NPS were found: six in wastewater only, 13 in forensic post mortem toxicology samples only and five in both. As these results showed the presence of NPS, a targeted method was subsequently employed to quantify levels of these NPS in wastewater. Temporal trends were found in wastewater with distinct tendencies for synthetic cathinones visible over the period studied. Refereed/Peer-reviewed

The use of collision cross-section (CCS) values obtained by ion mobility high-resolution mass spectrometry has added a third dimension (alongside retention time and exact mass) to aid in the identification of compounds. However, its utility is limited by the number of experimental CCS values currently available. This work demonstrates the potential of artificial neural networks (ANNs) for the prediction of CCS values of pesticides. The predictor, based on eight software-chosen molecular descriptors, was optimized using CCS values of 205 small molecules and validated using a set of 131 pesticides. The relative error was within 6% for 95% of all CCS values for protonated molecules, resulting in a median relative error less than 2%. In order to demonstrate the potential of CCS prediction, the strategy was applied to spinach samples. It notably improved the confidence in the tentative identification of suspect and nontarget pesticides. Refereed/Peer-reviewed