Detecting Drugs and Psychoactive Substances in Influent Urban Wastewater: An Interview with Richard Bade

Wastewater analysis serves as a surveillance tool for various markers, including lifestyle, exposure, population size, and health indicators. Traditional methods focus on predefined analytes, but face challenges with dynamic substances like new psychoactive substances (NPS). Liquid chromatography-high resolution mass spectrometry (LC–HRMS) has emerged as a solution, offering full-spectrum data screened against databases for compound confirmation. Despite lacking analytical standards, LC–HRMS identifies compounds based on accurate mass, fragment ions, and retention time predictions. Previous studies in Europe and Australia utilized LC–HRMS with in-house or HighResNPS databases. Richard Bade of theQueensland Alliance for Environmental Health Sciences (QAEHS) within The University of Queensland (Australia) and colleagues have recently published a paper (1) outlining the development and application of a new workflow utilizing the open-source platform InSpectra, aiming to complement targeted analysis and identify geographical spatial trends in wastewater samples across multiple countries. Bade spoke to LCGC International about their research paper and the work that inspired it.

How prevalent a problem is psychoactive substances and drugs of abuse in urban wastewaters, and what specific challenges arise compared to other possible contaminants? Is the sewer system a typical way to dispose of unused drugs or a way of tracking the drugs that are being used?

New psychoactive substances (NPS) and drugs of abuse are typically found at quite low levels in wastewater, which pose specific analytical challenges compared to more prevalent contaminants. This necessitates the use of preconcentration methods, such as solid phase extraction (SPE) to ensure that they can be detected. Through the analysis of specific biomarkers in wastewater, it is possible to determine the consumption of these drugs within a community. Many of the compounds measured in our study were parent compounds, as there is limited pharmacokinetic data around metabolism of NPS. The detection of these parent compounds could also indicate direct disposal, but additional analyses would be necessary to confirm.

In your paper, you mentioned that previous wastewater analysis was conducted using liquid chromatography–tandem mass spectrometry (LC–MS/MS), but limitations of that technique have led to using liquid chromatography-high resolution mass spectrometry (LC–HRMS) instead. Briefly summarize these limitations, and why LC–HRMS is a better choice.

Although LC–MS/MS instrumentation are generally more sensitive than LC–HRMS, targeted methods can be limited in terms of the number of analytes that can be analyzed in a single method. Conversely, the use of LC–HRMS takes advantage of high resolution, full-spectrum data, which can be screened against databases to confirm the presence of predefined compounds. In our work, we had a database of greater than 2000 compounds. While LC–HRMS has been used for similar approaches, our workflow was predominantly automated, which greatly reduces data processing time.

Other than using a different analysis technique than LC–MS, does your work differ from what has been previously done by yourself or others?

This was the first time that a web-based, open-source/access data processing platform (InSpectra) had been utilized for this type of work. We also made the code available in our paper for researchers in other groups or fields to utilize or refine.

Wastewaters were sampled from 47 sites in 16 different countries. Were there any particularly interesting findings that stand out from a geographical or sociological perspective?

While most of the drugs of abuse and pharmaceuticals results were like previous work (cocaine and its metabolite were found in most sites, and methamphetamine in sites from Australia, New Zealand, United States, and parts of Eastern Europe), there were some interesting geographical spatial trends from the NPS. For example, 3-methylmethcathinone was only seen in sites in Europe, eutylone only in sites in New Zealand and mitragynine only in sites from the United States. While these trends had been seen in previous work, it was great that we were able to confirm them with our workflow. One particularly interesting finding is that of an isomer of trimethoxyamphetamine, which was seen in one site in the United States. Although we did not have a reference standard to confirm its identity, through fragment ion matching, we narrowed the potential isomer to either 2,3,6- or 3,4,5-trimethoxyamphetamine. Interestingly, trimethoxyamphetmine was found by NPS Discovery in a site in the United States in late 2022, at the time the sampling was carried out in this work.

Are there any other findings you’d like to share?

In total, 50 compounds were found in at least one site. More than half were identified at Level 1 confidence (that is to say, confirmed with a reference standard), while 22 were at level 2 confidence (library matching and fragmentation matching) and a further two at level 3, due to the possibility of several isomers.

Were there any limitations or challenges you encountered in your work, even after using the refined LC–HRMS technique?

We used quite a high intensity threshold for our work, and therefore it is possible that some NPS or drugs present at lower levels were not found using this method.

What best practices that can you recommend in this type of analysis for both instrument parameters and data analysis?

By including the software code in our work, other researchers can adapt the specific thresholds/parameters for their own instruments and matrices. Our thresholds were quite generous for intensity and retention time, which potentially resulted in false negatives (compounds may have been present but outside our thresholds). It is therefore recommended for other researchers wanting to use this workflow to understand their own data and incorporate suitable threshold levels.

Can you please summarize the feedback that you have received from others regarding this work?

Our paper has only recently been published but so far the feedback has only been positive.

Do you imagine these analytical techniques to be adaptable to other industries or applications?

Absolutely. This workflow is suitable for any qualitative screening work.

What are the next steps in this research?

One of the areas that we’re working on is investigating metabolism of NPS and their applicability for wastewater analysis. As this workflow is very flexible, we hope to utilize this workflow to screen for metabolites in wastewater.

Reference

(1) Bade, R.; van Herwerden, D.; Rousis, N.; Adhikari, S.; Allen, D.; Baduel, C. et al. Workflow to Facilitate the Detection of New Psychoactive Substances and Drugs of Abuse in Influent Urban Wastewater. J. Hazard. Mater. 2024, 469. DOI: 10.1016/j.jhazmat.2024.133955

Dr. Richard Bade is an Australian Research Council Discovery Early Career Researcher (DECRA) Fellow and Senior Research Fellow at the Queensland Alliance for Environmental Health Sciences (QAEHS) within The University of Queensland. Originally from New Zealand, he completed his PhD at the University Jaume I, Castellon, Spain in 2016 before moving to the University of South Australia in 2017 and QAEHS in 2021. He is interested in understanding links between environmental and community health using wastewater analysis. His DECRA project focuses on detecting NPS in wastewater. These methods are being applied locally, nationally, and internationally to provide insights to governments and agencies on their prevalence.