In vitro biotransformation of 3-methylmethcathinone (3-MMC) through incubation with human liver microsomes and cytosol and application to in vivo samples.

Synthetic cathinones are the second largest group of new psychoactive substances (NPS) monitored by the European Monitoring Centre for Drugs and Drug Addiction. Although 3-methylmethcathinone (3-MMC, C11H15NO) is legally banned in many countries, it is readily available for purchase online and on the street. Due to the scarcity of information regarding the pharmacokinetic and toxicological profile of 3-MMC, understanding its biotransformation pathways is crucial in determining its potential toxicity in humans and in the development of analytical methods for screening of human matrices. To gain more insight, Phase I and Phase II in vitro biotransformation of 3-MMC was investigated using human liver microsomes and human liver cytosol. Suspect and non-target screening approaches were employed to identify metabolites. To confirm in vitro results in an in vivo setting, human matrices (i.e., plasma, urine, saliva and hair) positive for 3-MMC (n=31) were screened. In total three biotransformation products were identified in vitro: C11H15NO2 (a hydroxylated derivate), C11H17NO (a keto-reduced derivate) and C10H13NO (an N-desmethyl derivate). All three were confirmed as human metabolites in respectively 16 %, 52 % and 42 % of the analysed human samples. In total, 61 % of the analysed samples were positive for at least one of the three metabolites. Interestingly, three urine samples were positive for all three metabolites. The presence of 3-MMC in saliva and hair indicates its potential applicability in specific settings, e.g., roadside testing or chronic consumption analysis. To our knowledge, C11H17NO was not detected before in vivo. Although some of these metabolites have been previously suggested in vitro or in a single post mortem case report, a wide in vivo confirmation including the screening of four different human matrices was performed for the first time. These metabolites could serve as potential human biomarkers to monitor human 3-MMC consumption effectively.

Current state and future perspectives on de facto population markers for normalization in wastewater-based epidemiology: A systematic literature review.

Wastewater-based epidemiology (WBE) and wastewater surveillance have become a valuable complementary data source to collect information on community-wide exposure through the measurement of human biomarkers in influent wastewater (IWW). In WBE, normalization of data with the de facto population that corresponds to a wastewater sample is crucial for a correct interpretation of spatio-temporal trends in exposure and consumption patterns. However, knowledge gaps remain in identifying and validating suitable de facto population biomarkers (PBs) for refinement of WBE back-estimations. WBE studies that apply de facto PBs (including hydrochemical parameters, utility consumption data sources, endo- and exogenous chemicals, biological biomarkers and signalling records) for relative trend analysis and absolute population size estimation were systematically reviewed from three databases (PubMed, Web of Science, SCOPUS) according to the PRISMA guidelines. We included in this review 81 publications that accounted for daily variations in population sizes by applying de facto population normalization. To date, a wide range of PBs have been proposed for de facto population normalization, complicating the comparability of normalized measurements across WBE studies. Additionally, the validation of potential PBs is complicated by the absence of an ideal external validator, magnifying the overall uncertainty for population normalization in WBE. Therefore, this review proposes a conceptual tier-based cross-validation approach for identifying and validating de facto PBs to guide their integration for i) relative trend analysis, and ii) absolute population size estimation. Furthermore, this review also provides a detailed evaluation of the uncertainty observed when comparing different de jure and de facto population estimation approaches. This study shows that their percentual differences can range up to ±200 %, with some exceptions showing even larger variations. This review underscores the need for collaboration among WBE researchers to further streamline the application of de facto population normalization and to evaluate the robustness of different PBs in different socio-demographic communities.

Retrospective spatiotemporal study of antidepressants in Slovenian wastewaters.

This study utilizes wastewater-based epidemiology (WBE) to evaluate spatiotemporal changes in the consumption of antidepressants before and during the COVID-19 pandemic in Slovenia. Composite 24-h influent wastewater samples (n = 210) were collected from six wastewater treatment plants between summer 2019 and spring 2021. The samples were extracted using 96-well solid-phase extraction and analysed by liquid chromatography-tandem mass spectrometry. The measured concentrations of target antidepressant biomarkers were then converted to population-normalised mass loads (PNMLs), taking into account flow rate and catchment population. Ten biomarkers, including amitriptyline, bupropion, bupropion-OH, citalopram, norcitalopram, normirtazapine, venlafaxine, O-desmethylvenlafaxine, trazodone, and moclobemide, were above the lower limit of quantification and were included in the spatiotemporal temporal assessment. The highest PNMLs were detected for O-desmethylvenlafaxine (mean ± SD: 82.1 ± 21.2 mg/day/1000 inhabitants) and venlafaxine (38.0 ± 10.6 mg/day/1000 inhabitants), followed by citalopram (27.0 ± 10.7 mg/day/1000 inhabitants). In addition, the mean metabolite/parent compound ratios were comparable with other WBE studies indicating consumption rather than direct disposal. Overall, the results indicated significant spatiotemporal variations depending on the location, and the PNMLs of most biomarkers increased during the first wave of the COVID-19 pandemic (spring of 2020). However, no clear spatial patterns were revealed related to the pandemic.

Do the lockdown-imposed changes in a wastewater treatment plant catchment's socio-demographics impact longitudinal temporal trends in psychoactive pharmaceutical use?

Wastewater-based epidemiology (WBE) includes the analysis of human metabolic biomarkers of xenobiotics in influent wastewater. WBE complements existing drug utilization approaches and provides objective, spatio-temporal information on the consumption of pharmaceuticals in the general population. This approach was applied to 24-h composite influent wastewater samples from Leuven, Belgium. Daily samples were analysed from September 2019 to December 2019 (n = 76), and on three days of the week (Monday, Wednesday, Saturday) from January 2020 to April 2022 (n = 367). Sample analysis consisted of 96-well solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry. Measured concentrations of 21 biomarkers for antidepressant and opioid use were converted to population-normalized mass loads (PNML) by considering the flow rate and catchment population. To capture population movements, mobile phone data was used. Amitriptyline, hydroxy-bupropion, norcitalopram, citalopram, normirtazapine, trazodone, O-desmethylvenlafaxine, codeine, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), methadone, morphine, O-desmethyltramadol, and tramadol were included in the temporal assessment since concentrations were above the lower limit of quantification. The PNML of most biomarkers increased (with 3-119 %) throughout the sampling period. The population disruption during the COVID-19 pandemic led to a major change in the socio-demographics of the catchment area, resulting in temporal differences in the PNML of the different biomarkers. As such, higher PNML were observed during the different lockdown phases, which were characterized by the outflow of university students and a decreasing commuting in and out the catchment area. The effects of the fluctuating socio-demographics of the catchment population were further evidenced by the different week-weekend pattern of PNMLs over the course of the sampling campaign. Mean parent/metabolite ratios (i.e., citalopram/norcitalopram, tramadol/O-desmethyltramadol, venlafaxine/O-desmethylvenlafaxine, and methadone/EDDP) remained relatively stable throughout the entire sampling campaign (RSD% below 25 % for all ratios, except for methadone/EDDP) and therefore were not affected by this population change.

Optimization, validation and application of a high-throughput 96-well elution protocol for the quantification of psychoactive substances in influent wastewater.

Wastewater-based epidemiology (WBE) is based on the analysis of human metabolic excretion products (biomarkers) of xenobiotics in wastewater, to gain information about various lifestyles and health aspects of a population in an evidence-based manner. Due to the complex wastewater matrix and trace level occurrence of human biomarkers in the sewage network, it is crucial to have sensitive analytical procedures available. Additionally, to improve the value of WBE as a complementary epidemiological source, there is increasing pressure on the analysis of more compounds, more locations and more samples. A high-throughput method based on 96-well Oasis MCX solid-phase extraction (SPE), requiring less influent wastewater (2 mL), was developed in accordance with the European Medicines Agency guidelines. Validation was successful for 28 parent drugs and metabolites of antidepressants, opioids and drugs of abuse. The selection of biomarkers and quantification limit was chosen to be relevant for WBE and was predominantly 10 ng/L or below. The final method was successfully applied to 24-h composite samples of October 2019 (n = 27), obtained from an urban wastewater treatment plant in Leuven (Belgium).

Evaluating the impact of COVID-19 countermeasures on alcohol consumption through wastewater-based epidemiology: A case study in Belgium.

Wastewater-based epidemiology (WBE) is a complementary approach to monitor alcohol consumption in the general population. This method measures concentrations of xenobiotic biomarkers (e.g., ethyl sulphate) in influent wastewater (IWW) and converts these to population-normalized mass loads (PNML, in g/day/1000 inhabitants) by multiplying with the flow rate and dividing by the catchment population. The aims of this case study were to: (i) investigate temporal trends in alcohol use during the COVID-19 pandemic; and (ii) measure the effect of policy measures on alcohol consumption. Daily 24-h composite IWW samples (n = 735) were collected in the wastewater treatment plant of the university city of Leuven (Belgium) starting from September 2019 to September 2021. This is the first study that investigates alcohol use through WBE for a continuous period of two years on a daily basis. Mobile phone data was used to accurately capture population fluxes in the catchment area. Data was evaluated using a time series based statistical framework to graphically and quantitatively assess temporal differences in the measured PNML. Different WBE studies observed temporal changes in alcohol use during the COVID-19 pandemic. In this study, the PNML of ethyl sulphate decreased during the first lockdown phase, potentially indicating that less alcohol was consumed at the Leuven area during home confinement. Contrastingly, alcohol use increased after the re-opening of the catering industry. Additionally, a decrease in alcohol use was observed during the exam periods at the University of Leuven and an increase during the holiday periods. The present study shows the potential of WBE to rapidly assess the impact of some policy measures on alcohol consumption in Belgium. This study also indicates that WBE could be employed as a complementary data source to fill in some of the current knowledge gaps linked to lifestyle behavior.

Temporal monitoring of stimulants during the COVID-19 pandemic in Belgium through the analysis of influent wastewater.

BACKGROUND AND AIMS: Wastewater-based epidemiology (WBE) is a complementary epidemiological data source to monitor stimulant consumption. The aims were to: (i) study intra- and inter-year temporal changes in stimulant use in Belgium during the first wave of the COVID-19 pandemic; and (ii) evaluate the effect of COVID-19 restrictive measures on stimulant consumption. METHODS: The study population corresponded to the catchments of four wastewater treatment plants corresponding with four Belgian cities (i.e., Antwerp-Zuid, Boom, Brussels, Leuven). Daily 24-h composite influent wastewater samples collected over one week in September 2019 and March through June 2020 during the first wave of the COVID-19 pandemic were analyzed for biomarkers of amphetamine, cocaine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Measured concentrations were converted to population-normalized mass loads by considering the daily flow rate and the catchment population size. Mobile network data was used to accurately capture population movements in the different catchment areas. Temporal changes were assessed with multiple linear regression models, and the effect of the COVID-19 interventions on stimulant consumption were investigated. RESULTS: An increase in amphetamine use was observed in three cities during governmental restrictions, with highest consumption predominantly during lockdown. Similarly, cocaine consumption was higher after the pandemic started, with highest consumption noted during the lockdown period in Boom and Leuven. Consumption of MDMA was similar in Antwerp-Zuid, Brussels and Leuven throughout the entire sampled period. In Boom, the highest consumption was observed during the full lockdown period. CONCLUSIONS: The present study shows the potential of WBE to assess the impact of stringent lockdown measures on stimulant use in Belgium. This paper shows that strong restrictive measures did not have a profound effect on stimulant consumption.

Analytical method for the simultaneous determination of a broad range of opioids in influent wastewater: Optimization, validation and applicability to monitor consumption patterns.

Wastewater-based epidemiology (WBE) employs the analysis of human metabolic biomarkers in influent wastewater (IWW) to estimate community-wide exposure to xenobiotics (e.g. prescription opioids). The low ng/L range of concentrations of these biomarkers and the complex matrix composition pose bioanalytical challenges related to sample preparation and detection/quantification. Therefore, a sensitive analytical method for the detection and analysis of 19 opioid biomarkers was optimized and validated according to the European Medicines Agency guidelines. Oasis HLB cartridges were used for sample concentration and an Atlantis T3 column with gradient elution resulted in sufficient separation of the analytes. Absolute recoveries (RE) were highly reproducible and ranged between 50 and 93% with the exception of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The lower limit of quantification (LLOQ) ranged between 1 and 100 ng/L and was based on the analyte concentrations found in IWW. Process efficiency was acceptable for all biomarkers for which an isotope-labelled deuterated analogue was available. All biomarkers showed high benchtop stability with the exception of buprenorphine, EDDP, fentanyl and normorphine. Apart from buprenorphine and hydrocodone, all analytes under investigation were detected at least once above LLOQ levels in five locations in Belgium. The highest population-normalized mass loads were found for tramadol, O-desmethyltramadol and codeine. The proposed methodology was able to evaluate spatial differences in opioid use.