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

Sara Walton is a forensic toxicologist at the Center for Forensic Science Research and Education (CFSRE). Her work falls under the NPS Discovery program and primarily focuses on the identification and characterization of NPS metabolites in forensic toxicology settings and their application to authentic biological samples. She is currently pursuing her Ph.D in analytical chemistry from Temple University. Her thesis is focused on method development and validation for NPS metabolism studies, where she performs pharmacodynamics and pharmacokinetics experiments to understand NPS metabolism in the human body better.

Pierre Negri (P.N.): Welcome to the fourth episode of Tox Chat Box, a SCIEX vodcast series, where we discuss the latest trends and applications in forensic toxicology.

I’m Pierre Negri from SCIEX, and in today's episode, we're going to talk about drug metabolism studies, forensic toxicology settings and the application to authentic biological samples. Today, we're joined by Sara Walton. Sara is a forensic toxicologist at the Center for Forensic Science Research and Education. Her work primarily falls under their NPS discovery program, where she's currently developing novel methods using accurate mass spectrometry to examine NPS trends and their metabolism.

Hi, Sara, how are you?

Sara Walton (S.W.): Hi. I'm good. How are you?

(P.N.):  I'm great. Thank you. Thanks for coming on. This is exciting. Sara, you've been working at the CFSRE for over three years now, where you've been supporting the NPS discovery program, but you're also currently pursuing your PhD in analytical chemistry from Temple University. Can you briefly tell me about the connection between your work there and your PhD thesis?

(S.W.): Yeah, I started working at the CFSRE back in 2019. And then, just in 2022, I started my PhD while working full-time at the center. The research that I do for my PhD thesis is based on NPS. So, it helps both Temple University and the Center by doing this novel research on NPS metabolism, development and validation. A lot of pharmacokinetics and pharmacodynamic studies are needed to get a better understanding.

(P.N.): That's great. Very exciting. As you know, we've talked extensively with your colleague Alex Krotulski in episode two and discussed the use and benefits of accurate mass spectrometry for the characterization and identification of NPS, specifically in toxicology case work for the NPS Discovery program. So, I know you're contributing extensively to this program, but as you mentioned, your work primarily focuses on NPS metabolism studies. Can you briefly tell us a little bit more about the scope of work and highlight the utility of developing methods to study drug metabolism?

(S.W.): Yeah. So, developing methods for drug metabolism, specifically for NPS is quite important. The problem with NPS is that no one has a ton of information. There is a significant knowledge gap when it comes to NPS. Metabolism studies help us broaden our scope and determine the best biomarker for drug identification, which is important when looking at toxicology samples, whether you're looking at blood or urine.

They are quite important and less focused on because NPS move in quick waves, so people don't normally have time to do these kinds of studies.

(P.N.):  That's right. For the listeners that may not necessarily be familiar with drug metabolism studies, can you briefly describe the goal of these studies and some insights on sample preparation, the type of data acquisition methods it's using and the data processing for these types of workflows?

(S.W.): Yeah. So, for drug metabolism studies, the goal is to determine the most abundant metabolites that you would find in authentic human samples. And there are many ways to do this. You can use human liver hepatocytes. But, at the center, we use human liver microsomes. We are able to acquire human liver microsomes that are pooled from multiple donors, so it has genetic variety across 50 different people. And then, as for sample preparation, it's quite simple. You have the microsomes, you have buffer, you add your drug and then it's just incubation in a hot water bath. And that begins the metabolism process. You need a cofactor. We use NADPH at the CFSRE as our cofactor to catalyze that enzymatic process. After our sample preparation, we have two LC Q-TOF MS systems that we run here at the center. This provides us with the accurate mass data that we need. We run them in independent data acquisition so we are able to get the most abundant components of the sample and information on accurate mass fragment data and percent area. After analysis, we use Metabolite Pilot for our data analysis. Metabolite Pilot is great. It gives us the most prevalent information. So, you have a table of biotransformations that you put in. When we're using human liver microsomes, we're primarily looking at phase one metabolism, which is hydroxylation, oxidation and those kinds of processes. And what the program does is it looks at what it thinks the biotransformation is, what the exact mass is, what the retention time is that we acquired from our system and then the fragments that we get from that. So, we do get a lot of important information as well as that PPMR, which is great for structural elucidation as well.

(P.N.):  So, Sara, I know one important area of focus in your work is centered around studying and understanding the metabolism of synthetic cannabinoids, which are arguably one of the largest and most structurally diverse classes of NPS. I was wondering if you could tell me a little bit more about the challenges of identifying and also characterizing these classes of NPS, and why forensic toxicology is so reliant on their metabolites to confirm their presence in authentic biological fluids.

(S.W.): Yeah. So synthetic cannabinoids are tricky. And they are, in my opinion, the most difficult class of NPS to identify and keep up with, because of all the legislation and everything. Aside from that, the chemistry of these compounds is just so complex and diverse. Synthetic cannabinoids are very quickly and highly metabolized in the human body. So, what happens is, by the time the person gets to the hospital or gets the blood drawn, it might not be in the blood anymore. And synthetic cannabinoids are normally found at very low concentrations, so that's tricky to identify and quantitate these compounds. And, because they quickly metabolize, they also quickly break down. An interesting thing about synthetic cannabinoids is that a lot of synthetic cannabinoids actually break down into their dimethyl butanoic acid metabolite. So, the importance of having metabolites in scope is so you can see these breakdown products, which are also metabolites of synthetic cannabinoids in blood, and then in urine, you will only use synthetic cannabinoid metabolites. So, if you don't characterize the synthetic cannabinoids and have them in your scope, you’re not going to be able to identify them.

(P.N.):  That's great information. Thank you, Sara. All your work on NPS metabolism is being used as a stepping stone to look at the presence of drug metabolites and authentic biological fluids as you mentioned. I know one of the interesting aspects of your work is looking at previously acquired data sets and performing data mining to kind of look at the presence of those metabolites in the previously acquired samples. Can you provide an example, or maybe just go over the process of doing data mining or what they call retrospective data analysis?

(S.W.): Sure. Yeah. So, data mining is big at the CFSRE. We use it continuously. Both of our LC QTOF MS systems are able to do all ions acquisition, SWATH acquisition where we can identify and fragment everything in the sample even if it's not in our library at the time. We do non-targeted data analysis and targeted data processing. A good example of this is N,N-Dimethylpentylone which is a popular synthetic cathinone in the United States and in Europe that has recently been scheduled. However, in 2021, when it first became popular, we were seeing a lot of instances of Pentylone, which was an NPS in its own right back in the late 2010s. So, we were seeing all these identifications of Pentylone that didn't make any sense, and then, once we finally realized what the problem was, we were able to get the analytical reference standard for Dimethylpentylone, which metabolizes into Pentylone. Then we did retrospective data mining and we were able to update our library for LC QTOF MS systems, open the data files that were acquired six months a year ago and reprocess it with this new mass and fragments and do retrospective identification of NPS. Dimethylpentylone was a great example of that. It worked very well. We saw a huge increase in identification sessions where we missed it the first time.

(P.N.):  Oh, that's a great anecdote. Thank you for sharing it. And that's a wrap for this week's episode on drug metabolism studies and their application to authentic biological samples. A big thank you to my guest, Sara, thank you for discussing your work on drug metabolism and sharing your knowledge on this topic with us today.(S.W.): Thank you so much, Pierre.

(P.N.):  And as always, thank you to the viewers for tuning in and watching this episode. Please make sure to check out the NPS Discovery website. As always, you can get the latest trend reports and public alerts about recently emerged NPS. Also, make sure to check out our NPS analysis page on the Science Forensic Toxicology webpage. We have a lot of good resources and some workflows on the latest screening and identification for NPS and metabolites. I'm also going to provide some links to some of those technical notes on NPS and metabolite screening using accurate mass spectrometry that we've developed over the years in collaboration with the Center for Forensic Science Research and Education. And again, a big thank you Sara, and the rest of your team for all the great collaboration over the years.

(S.W.): Thank you so much.

(P.N.):  Great. Thank you for joining us today. In the next episode, we'll be joined by Dr Tina Binz, who is the deputy head of the Center for Forensic Hair Analysis at the Zurich Institute of Forensic Medicine. We are going to discuss the advantages of hair and nail testing to determine long term drug exposure. Tina is going to be telling us a little bit more about the challenges associated with the detection of low-level drugs and metabolites and those keratinized matrices. Make sure to tune in for that episode. Thank you so much, and we will see you next time!