Tox Chat Box: Decoding forensic toxicology with LC-MS/MS

Dr. Richard Bade is a senior research fellow at the Queensland Alliance for Environmental Health Sciences at the University of Queensland. His research focuses on the development of quantitative and qualitative methods for the determination of illicit drugs in wastewater, with a special focus on novel psychoactive substances (NPS). 

Pierre Negri: Welcome to the 10th episode of Tox Chat Box SCIEX Vodcast series, where we discuss the latest trends and applications in forensic toxicology.

In today's episode, we're talking about the use of mass spectrometry for wastewater analysis and how this methodology can be used to detect drugs of abuse and assess regional drug consumption.

My guest today is Dr. Richard Bade. Richard is a senior research fellow at the Queensland Alliance for Environmental Health Sciences, which is affiliated with the University of Queensland in Australia. His research primarily focuses on the development of quantitative and qualitative methods for the determination of illicit drugs in wastewater, with a special focus on novel psychoactive substances.

Hi, Richard.

How are you doing?

Richard Bade: Hey, I'm doing great. Many thanks for inviting me to be part of this Tox Chat Box.

P.N.: So, Richard, you've been a research fellow at the Queensland Alliance for Environmental Health Sciences for a couple of years now. You've been very busy developing mass spectrometry-based methods to screen, identify, and quantify drugs of abuse, including NPS in wastewater. So I would say from a toxicologist's standpoint, looking for the presence of these substances in this matrix is quite unusual, right?

Can you tell us a little bit more about why scientists like you with an environmental background are interested in these substances in wastewater? Maybe talk about some of the benefits of wastewater-based drug surveillance.

R.B.: Yeah. Thanks for the question. I mean, wastewater-based epidemiology is really about what's happening within the community. So of course, it came really to the forefront a few years ago during the Covid pandemic, when wastewater analysis was used to understand where Covid was within a community, which variants were around. Drug surveillance through wastewater-based geology has been around for probably more than a decade or so.

Initially, looking at the more prevalent traditional drugs like cocaine, methamphetamine, those sorts of substances, and more recently, looking at the novel psychoactive substances. So it gives us a really great idea of what's happening within a community. It's not the be-all and end-all for drug surveillance. Of course. It's a perfect sort of complementary data source with other sources.

For example, all the data that forensic toxicologists and forensic chemists can also identify.

P.N.: That's great. Thank you for sharing that. So, the first thing that comes to my mind when thinking about wastewater analysis is that, you know, being able to accurately quantify drugs in wastewater must be very challenging, right? So first of all, you have very low concentrations of drugs due to their infrequent consumption, and then obviously, subsequent dilution in the wastewater stream.

Can you talk about some of the challenges of detecting low levels of drugs and NPS in wastewater, and about some of the methodologies that your lab has implemented to overcome these issues?

R.B.: Yeah. Of course. As you mentioned, the dilution factors and even the trace levels, at least common, are used within a community, making it really quite difficult to find some of these substances in wastewater. Especially the novel psychoactive substances. Some of the illicit drugs are, of course, at much higher levels. And sometimes we can just use direct injection approaches to analyze these compounds.

For the NPS, we typically use extraction methodologies, for example, with solid phase extraction, to concentrate the samples. Of course, the method now that we use around a 1000-fold concentration factor, which allows us to see these really low levels of compounds, we have detection limits in the sub-nanogram per liter range. And so this really likes to see these really low levels.

It allows us to quantify and understand what's happening within the community, even if these compounds aren't as prevalent as the more traditional drugs.

P.N.: Thanks, Richard, for that information. So you mentioned using sample pre-concentration methods to kind of concentrate your sample. Can you also tell us how critical it is for your lab to use sensitive instrumentation for this type of analysis?

R.B.: It's imperative for us to use various things that have instrumentation to allow us to see these really low levels, especially the more so the very potent compounds that potentially are, in sort of small quantities, even in the drugs that people are consuming. To allow us to see these really low levels. We really do need sensitive instrumentation.

So we've been using the SCIEX 7500 system for a lot of our work, both with direct injection analysis, and we've also got a direct injection method for NPS, as well as the SPE method. So that was a huge, dynamic range of NPS down for a very sub-nanogram per liter range up to tens or hundreds of ng/l, depending on the compound.

P.N.: Well, it's very impressive. Thanks for sharing that. I think one of the neat aspects about your fields is that these methods can be implemented locally, on a national level, or even

sometimes on an international level. Really, as you said, you provided insight on drug consumption and drug prevalence, drug trends, etc. I know you've been very involved in this community.

Can you talk about some of the international, national, or even regional wastewater surveillance programs that you've been involved with in the last couple of years?

R.B.: Yep. So, Australia, we're lucky enough to have a national wastewater drug monitoring program, and since 2016, we've been analyzing wastewater from around the country, covering around 50% of the population every 2 to 4 months. Looking for mainly the traditional illicit drugs, as well as some pharmaceuticals of abuse potential. And I've been sort of using some of these samples to also look for and to give us an idea of what's happening within Australia as well.

And we've been able to see some quite distinct spatial and temporal trends with some cathinone that are sort of coming and going within a year or two, and you see some other compounds that, some of the NPS, it sort of maintain their popularity throughout the years. And so, we've expanded this to look at other countries as well.

Over the past 4 or 5 years, I've started a sort of a New Year surveillance, project where samples are collected for around seven days over the New Year period, and the New year period was selected because this is the time of year where there's potentially risky behaviors taking place, people are potentially taking more drugs, sort of increased drug use, compared to other times of the year to give us a higher chance of detecting these NPS.

And so, this has been a fantastic collaboration with a lot of groups around the world. And now we've got samples from more than 20 countries, more than 60 sites around the world, over this New Year period to give us an understanding of a sort of a snapshot in time of what's happening in the NPS market around the world.

P.N.: Well that's great. It's such a cool collaborative effort, and really, getting a good sense of the trends for drug consumption around the world. It's very neat. I know you recently published a new study, showing the detection of nitazenes in wastewater from different states in the US. For those who don't know, nitazenes are novel synthetic opioids that have been implicated in overdose mortality, and they've become a really big public health concern due to their high potency.

Right. I was wondering if you could share some of the findings from this study and some of the drugs that you've targeted in this study.

R.B.: Yeah. So we've got around eight nitazenes currently in our methods. We're always trying to expand our method depending on which compounds are around, depending on conversations we have with other forensic agencies, both in Australia and also internationally. So in this study, we had around eight nitazenes. We were looking in various states in the US to see what we could find.

And interestingly enough, we found protonitazene in two sites in the US. And this was the first detection of any nitazenes in wastewater that had been published. It was really eye-opening for us to even be able to see these compounds because we all know how very potent they are and how dangerous they are to be within the community.

And so, it allows us to sort of show that wastewater can act as a pseudo-early warning system, helping us understand that these compounds are within a community. And, potentially, interventions could be put in place to reduce the harm associated with the consumption.

P.N.: That's great. It's very impressive because, as you mentioned, those drugs are present in such small concentrations that you really need to get a robust and sensitive method to be able to detect them. So it's very impressive. Last question before I let you go. I mean, you're involved in so many different projects. And, you are collaborating with different regional and international organizations.

Can you tell us about some of the upcoming projects that you've got in your pipeline?

R.B.: Yeah. So we're just in the process of organizing another sort of New Year collection. We always try to keep this annual. So that's been a bit of a logistical challenge, but it's a fantastic challenge. And I really love being involved in this project. I've got a student who's looking at exploring additional sampling methodologies to allow, sites that may have less easy access to wastewater to also be involved with analysis.

So we're exploring different ways to detect NPS through different sampling devices. And we're looking at transformation products to ensure we're looking at the correct biomarkers in wastewater. So we've got a few little projects that are ongoing. But yeah. So some exciting times coming up in the next year or so.

P.N.: That’s very exciting. We are looking forward to seeing the outcomes of these projects. And that’s a wrap for this episode on the use of mass spectrometry for the monitoring of NPS in wastewater. A big thank you to my guest Richard. Thank you for sharing your knowledge on a very unique yet super interesting topic. As I mentioned, we are very looking forward to seeing the outcome of those research projects because you are working on a very important public health and safety issue.

R.B.: No worries. Thank you for inviting me here.

P.N.: As always, thanks to the viewers for tuning in and watching this episode. If you want to know more about Richard's work, I'm going to drop the link to a technical note that we did in collaboration with his lab, and it really showcases the use and the sensitivity of the SCIEX 7500 system for NPS detection in wastewater. I'm also going to provide the links to Richard's most recent peer review papers.

Showcasing again, the use of the SCIEX 7500 system for wastewater-based monitoring of NPS. So make sure to check out those additional resources. And that's a wrap. Thank you so much once again for tuning in. And we'll see you next time.