Workflow to facilitate the detection of new psychoactive substances and drugs of abuse in influent urban wastewater.

The complexity around the dynamic markets for new psychoactive substances (NPS) forces researchers to develop and apply innovative analytical strategies to detect and identify them in influent urban wastewater. In this work a comprehensive suspect screening workflow following liquid chromatography - high resolution mass spectrometry analysis was established utilising the open-source InSpectra data processing platform and the HighResNPS library. In total, 278 urban influent wastewater samples from 47 sites in 16 countries were collected to investigate the presence of NPS and other drugs of abuse. A total of 50 compounds were detected in samples from at least one site. Most compounds found were prescription drugs such as gabapentin (detection frequency 79%), codeine (40%) and pregabalin (15%). However, cocaine was the most found illicit drug (83%), in all countries where samples were collected apart from the Republic of Korea and China. Eight NPS were also identified with this protocol: 3-methylmethcathinone 11%), eutylone (6%), etizolam (2%), 3-chloromethcathinone (4%), mitragynine (6%), phenibut (2%), 25I-NBOH (2%) and trimethoxyamphetamine (2%). The latter three have not previously been reported in municipal wastewater samples. The workflow employed allowed the prioritisation of features to be further investigated, reducing processing time and gaining in confidence in their identification.

Three years of wastewater surveillance for new psychoactive substances from 16 countries.

The proliferation of new psychoactive substances (NPS) over recent years has made their surveillance complex. The analysis of raw municipal influent wastewater can allow a broader insight into community consumption patterns of NPS. This study examines data from an international wastewater surveillance program that collected and analysed influent wastewater samples from up to 47 sites in 16 countries between 2019 and 2022. Influent wastewater samples were collected over the New Year period and analysed using validated liquid chromatography - mass spectrometry methods. Over the three years, a total of 18 NPS were found in at least one site. Synthetic cathinones were the most found class followed by phenethylamines and designer benzodiazepines. Furthermore, two ketamine analogues, one plant based NPS (mitragynine) and methiopropamine were also quantified across the three years. This work demonstrates that NPS are used across different continents and countries with the use of some more evident in particular regions. For example, mitragynine has highest mass loads in sites in the United States, while eutylone and 3-methylmethcathinone increased considerably in New Zealand and in several European countries, respectively. Moreover, 2F-deschloroketamine, an analogue of ketamine, has emerged more recently and could be quantified in several sites, including one in China, where it is considered as one of the drugs of most concern. Finally, some NPS were detected in specific regions during the initial sampling campaigns and spread to additional sites by the third campaign. Hence, wastewater surveillance can provide an insight into temporal and spatial trends of NPS use.

Illicit drug use in Reykjavik by wastewater-based epidemiology.

Estimation of illicit drug use on a community level by wastewater-based epidemiology (WBE) is both an objective and reliable way to establish near real-time results. Wastewater samples were collected at eleven timepoints in Reykjavik from 2017 to 2020. The use of commonly abused illicit drugs in Iceland (amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), cocaine, and cannabis) was estimated. Solid phase extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry was used for analysis. Estimated amphetamine and methamphetamine use showed signs of an increase from 2017 to 2020 with amphetamine being the dominant stimulant on the market. MDMA use remained stable from 2017 to 2020. Results showed a large increase in cocaine use from 2017 to 2019 but interestingly, a marked decrease in 2020 during the COVID-19 pandemic. Cannabis use was stable from 2017 to 2019 but showed signs of an increase during the pandemic in 2020. Results by WBE corresponded with data based on two other indicators of drug use, seizure data and driving under the influence cases. Both temporal and spatial trends in illicit drug use were successfully estimated by using WBE, complimenting other indicators which provided a comprehensive picture of drug abuse in Reykjavik.

Analysis of stimulant drugs in the wastewater of five Nordic capitals.

Wastewater-based epidemiology is an efficient way to assess illicit drug use, complementing currently used methods retrieved from different data sources. The aim of this study is to compare stimulant drug use in five Nordic capital cities that include for the first time wastewater samples from Torshavn in the Faroe Islands. Currently there are no published reports that compare stimulant drug use in these Nordic capitals. All wastewater samples were analyzed using solid phase extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry. The results were compared with data published by the European Monitoring Centre for Drugs and Drug Addiction based on illicit drugs in wastewater from over 50 European cities. Confirming previous reports, the results showed high amphetamine loads compared with other European countries. Very little apparent abuse of stimulant drugs was detected in Torshavn. Methamphetamine loads were the highest from Helsinki of the Nordic countries, indicating substantial fluctuations in the availability of the drug compared with previous studies. Methamphetamine loads from Oslo confirmed that the use continues to be high. Estimated cocaine use was found to be in the lower range compared with other cities in the southern and western part of Europe. Ecstasy and cocaine showed clear variations between weekdays and weekends, indicating recreational use. This study further demonstrates geographical trends in the stimulant drug market in five Nordic capitals, which enables a better comparison with other areas of the continent.

A high-throughput solid-phase microextraction and post-loop mixing large volume injection method for water samples.

This article presents a novel approach for the analysis of 13 drugs in wastewater for use in wastewater-based epidemiology (WBE) studies. Sample preparation remains one of the principal bottlenecks in modern high-throughput analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The proposed methodology is based on the micro-extraction of small volumes (1 ml) of wastewater using a HLB 96-well microplate and both large volume injection (LVI) and post-loop mixing injection (PLM). With this configuration, the limits of quantification (LOQ) were below the reported environmental concentrations of the target compounds in wastewater. Furthermore, both the complexity of collecting, transporting and storing the wastewater sample, sample preparation time, cost and amount of solvent used are all diminished, enhancing the suitability of this methodology for future WBE studies. A new workflow is also proposed in order to create a virtual specimen library bank for WBE by using high-resolution mass spectrometry (HRMS). The method was validated and the limits of quantification were between 0.2 and 6.3 ng L-1. The relative standard deviations (RSD) for a standard mixture at 200 ng L-1 (n = 6) was between 3.4 and 14.4% while the recoveries for the 13 drug target residues (DTR) were between 92 and 110%. The developed and validated method was finally successfully applied to 10 wastewater samples collected from Oslo, Norway.