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

Dr. Alex Krotulski is the associate director at the Center for Forensic Science Research and Education (CFSRE) and program manager of their flagship program, NPS Discovery, the CFSRE’s drug early warning system and flagship program whose mission is to gather intelligence and general public information on the identification and distribution of NPS. Dr. Krotulski holds faculty appointments and serves as the Assistance Program Director for the Thomas Jefferson University Master of Science in Forensic Toxicology (MSFT) program and is an Associate Editor for the Journal of Analytical Toxicology.

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

I am Pierre Negri from SCIEX. And in today's episode, we're going to talk about detecting novel psychoactive substances in toxicology casework. More specifically, how accurate mass spectrometry has become the ultimate tool for their characterization and identification. And today we're joined by Alex Krotulski, who, in my honest opinion, is the best person to discuss this topic. Alex is the associate director at the Center for Forensic Science Research and Education and the program manager for their flagship program, NPS Discovery.

Alex Krotulski (A.K.): Hi, Pierre. Thanks for having me. It's great being here with you.

P.N.: Well, thanks for coming on. It's a pleasure to have you. So, Alex, I want to get us started and ask you about your scientific background and how that experience led to your current role at the CFSRE.

A.K.: Yes. My scientific background is heavily rooted in chemistry. I have a bachelor’s degree in chemistry. But I had a strong interest in learning about forensic science and applying chemistry to forensic science. I have a master’s degree in forensic science, and I ultimately got my PhD in chemistry with a focus, during my dissertation research, in forensic toxicology. So, really heavily rooted in chemistry, but with a lot of training and education in toxicology, pharmacology and other related subjects.

P.N.: That's great. So, I want to get right into it.  I am curious to learn more about the type of workflows that you and your team have developed over the years. I've been to your lab a few times, and I've seen all of the different types of applications and workflows that you've developed. I was wondering if you could tell us more about the instrumentation that you're using, and the types of analyses that you're currently using to identify and characterize novel psychoactive substances.

A.K.: Yeah. So, our laboratory tests everything from powders that are emerging within the recreational drug supply all the way through toxicology specimens from postmortem investigations. We use GC-MS to characterize powders, but in the field of forensic toxicology, we've really moved away from GC-MS and moved more toward liquid chromatography-mass spectrometry applications. Our first line of defense is generally screening by high-resolution mass spectrometry. Our laboratory has three LC-QTOF MSs that we use primarily for drug identification, and we have comprehensive screening methods on there that have library databases with more than 1200 targets in them. So, that's our first line of defense and then we do all our confirmations and quantitation by liquid chromatography triple quadrupole tandem mass spectrometry. So, LC triple quad mass spectrometry.

P.N.: So I wanted to have you come on the Vodcast to tell us a bit more about what you refer to as your first line of defense. The process of screening for NPS. I was wondering if you could tell us a little bit more about some of the steps that you're using and how you leverage accurate mass spectrometry for the characterization and identification of those drugs. Maybe share an example of an NPS your team recently identified for the first time and the processes behind it.

A.K.: Yes, so our NPS Discovery program is interested in identifying all types of drugs. They may be drugs that are more acidic or neutral, like your cannabinoids. They may be more basic, like your benzodiazepines or opioids. So, we have to have methods that are non-targeted in nature from the beginning. We use very non-targeted liquid-liquid extractions and sample preparation methods that will pull out drugs that we may not even know about yet. Our acquisition methods use SWATH acquisition and have focused specifically on data-independent acquisition modes to make sure we are gathering all the information from a sample, regardless of whether we know what the next new drug that is going to pop up may be. We have had a lot of success. And because we're gathering those precursor ions and those fragment ions, either going in and interrogating the data from scratch, it's a lot like finding a needle in a haystack or acquiring standard reference material and going back and comparing the reference material to the data. We've had a lot of success in that, and we've identified a number of new synthetic cannabinoids, actually, on papers from prisons through that process. Both on that sort of a drug material perspective in analyzing those by GCMS and by our LC-QTOF MS. And then we're continuing to identify new synthetic opioids in the recreational drug supply in forensic toxicology specimens.

P.N.: That's fascinating. Thank you for sharing that. I've always been a big fan of all the retrospective data analyses that you're doing and what you guys coin data mining. I think it's fascinating speaking about the power of those data-independent acquisition methods on your high-resolution mass spec. So, as you mentioned, and as I can imagine, software is very key in providing the ability to really dig deep into the data. So, I was wondering if you could tell us a bit more about the software and some of the bioinformatic tools that your using for this process.

A.K.: Yeah. So, we've used a number of SCIEX software tools over the years, whether that has been MasterView or PeakView, and are now all the way up to SCIEX OS. We've also used Metabolite Pilot as part of our program as well, which we know we have too much time to focus on now, but we have an interest in characterizing metabolites of NPS as they emerge in samples. But really, our goal has been to develop and cultivate huge library databases that have sophisticated information in them. We've got these large library databases that have data for precursor ions and product ions. We process our data in a manner similar to MRM, and really similar to MRMHR because we're using an HRMS system. And we're able to do overlaying because of the way that the data is acquired in that SWATH acquisition manner, we're able to do overlaying extracted ion chromatogram, we're able to track how product ions are migrating and it gives us a lot of confidence in the data that we're looking at. It gives us a lot of confidence in the new NPS we're identifying. It has been those processes that have been really successful, especially when you can't go back and identify and reanalyze the sample. Having as much certainty in the data that you've already collected is really important. And that's what's been important to us, being able to go back retrospectively, but also doing that in a prospective manner. We're doing this not only in real-time today in the lab, but we're doing it on samples that we acquired six months ago,a year ago or more.

P.N.: That's amazing. Thank you, Alex. What's great about the work that you're doing is it's all open access. You have worked over the past couple of years in developing your NPS Discovery program. And for those who are not familiar with it, all the information that Alex talked about is disseminated and shared on his NPS Discovery website. So, I wanted you to give us a quick summary of the work that you're doing and the type of information that people can look for on your website.

A.K.: Sure. Our website is easy to find. It's www.npsdiscovery.org. And as you mentioned, all of our reports are open-access. We have a whole bunch of different reports. Our NPS Discovery program really has exponentially grown over time. We have new drug monographs that have a lot of analytical data in them and are the signaling of our NPS emerging and being here in the United States for the first time. We have scope recommendations if laboratories are looking to understand what NPS they should include in their toxicology screening scopes. We've got data on what NPS are appearing in clinical populations. We have public alerts on what NPS are appearing in death investigations across the country. We have our drug-checking reports, which are, what NPS are appearing in our drug-checking populations in collaboration with public health. So, all of these reports are on our website. They're free to access and download. We're constantly adding to those reports each month, each quarter. So, please feel free to download those reports and use them to whatever level of use you think their utility has for you in your field.

P.N.: Alright. And that's a wrap for today's episode on the use of accurate mass spectrometry for NPS screening and more specifically, characterization and identification. So, a big thank you to my guest, Alex. Thank you so much for sharing your knowledge and all the valuable information on NPS screening. And thank you for sharing all the insights with the NPS Discovery program.

A.K.: Absolutely. Thanks, Pierre

P.N.: Great. And again, thanks to the viewers and listeners for tuning in and watching this. Please make sure to check out NPS Discovery. As Alex mentioned, you'll get a depth of information, the latest trend reports and public alerts about NPS. Also, make sure to check out the NPS analysis subpage on the SCIEX Forensic Toxicology webpage, where you can find the latest screening and identification workflows for NPS analysis.

I'm also going to include some links to the technical notes that we published in collaboration with Alex, so make sure to check below for the links. A big thank you to Alex for all the great collaboration over the years and for joining us today. In the next episode, we'll be joined by Dr Luke Rodda, who's the Chief Forensic Toxicologist and Director of Forensic Services at the San Francisco Office of the Chief Medical Examiner.

In the next episode, we'll be discussing how his laboratory is leveraging SCIEX OS software for data processing and reporting. And we're going to dig deep into some of the latest improvements in data flagging, custom calculations and reporting functions that are critical for his lab to provide the most comprehensive toxicology report. So, make sure to tune in for that.