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

Peng is a doctorate student at the Vrije Amsterdam University in Dr. Isabelle Kohler’s research laboratory. His doctorate research primarily focuses on in vitro metabolism studies of NPS and their metabolites, specifically on the characterization and identification of synthetic cathinones, a class of novel psychoactive substances (NPS) that are central nervous system (CNS) stimulants.

Pierre Negri (P.N.): Welcome to the sixth 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 the benefits of an alternative fragmentation technique such as electron-activated dissociation on the ZenoTOF 7600 system for the characterization and structural elucidation of novel psychoactive substances and their metabolites. And today we're joined by Peng Che, Peng is a doctorate student at the Vrije Amsterdam University in the Netherlands. His research primarily focuses on in vitro metabolism studies of NPS and their metabolites, and more specifically on the characterization and structural elucidation of synthetic cathinone metabolites.

Hi Peng, how are you doing today?

Peng Che (P.C.): Hi Pierre, I'm doing well. How are you?

P.N.: Great, thank you. I'm very excited to have you on the vodcast.

P.C.: Thank you for your inviting me. I'm really excited for this interview.

P.N.: So, Peng. I know you're in the last year of your PhD at the Vrije Amsterdam University. And as I understand, you're currently in the middle of writing your doctoral dissertation. Can you tell us what the focus of your thesis was and the different projects that you've been involved in during your time as a graduate student?

P.C.: Of course! Hello, everyone. My name is Peng. I'm a final year PhD student from Vrije Amsterdam University and as Pierre said my thesis is about identification of new psychoactive substances and their metabolites using innovative analytical strategies.

P.N.: Can you tell us about the importance of conducting metabolism studies on NPS, and maybe mention some of the benefits of using accurate mass spectrometry for conducting in vitro metabolism studies?

P.C.: Of course. We know that after people consume NPS, NPS will be transformed into metabolites. So, in many cases, if we only focus on parent compound, we will probably not see it in biological samples, for example, plasma or urine, we cannot find them anymore. But we can find their metabolites. So, they are important for us to select them as targets. Accurate mass spectrometry is an important technique for metabolite identification. For example, it can provide us with accurate mass of metabolites. Then we know what metabolites are generated. Also, from an MS/MS perspective, it can give us accurate mass value, and we can use these values to derive the elemental compositions of product ions. And finally, we can use this information for structural elucidation of metabolites.

P.N.: As you said, accurate mass spectrometry is a great tool for the characterization and identification of new drugs in their metabolites. Can you provide some of the challenges that remain when it comes to structural elucidation and more specifically for NPS metabolites formed using in vitro models?

P.C.: Of course. For identification and the characterization of NPS metabolites, in the last decade, CID has been widely implemented in this field. However, in some cases, the CID MS/MS spectra are not enough or not sufficient for confident and the complete identification and characterization. So, we surely need more other fragmentation techniques to get more confidence in our results.

P.N.: This is a great segway into my next question, because I know your lab recently acquired a ZenoTOF 7600 system, which is our last generation QTOF instrument. This system supports electron-activated dissociation, or EAD for short. So, EAD is an alternative fragmentation technique to the commonly used collision-induced dissociation or CID. And I was wondering if you could share how you are leveraging EAD on the ZenoTOF 7600 system for your NPS metabolism work, and more specifically, how it relates to your work using synthetic cathinones.

P.C.: Of course! The ZenoTOF 7600 system enables us to perform both collision-induced dissociation (CID) and electron-activated dissociation (EAD). And we can see that they have different names. So behind them, they have different fragmentation mechanisms. So, if we perform, both EAD and CID, we can see potential different product ions. And we can then use these product ions for structural characterization of all metabolites. At the end, we gain more confidence in our results.

P.N.: Great Peng. It's very exciting, and it's really opening new doors for the characterization of drugs and metabolites. You recently authored two peer review papers and really interesting that specifically looked at comparing CID and EAD for the characterization and structural elucidation of synthetic cathinone metabolites. Can you maybe tell us a little bit more about these publications and discuss some of the findings?

P.C.: Yes. Last year we published a paper, and in that paper, we combined both CID and EAD into a metabolite identification workflow. In this study, we compared both CID MS/MS spectra and EAD MS/MS spectra of synthetic cathinones and their metabolites. Interestingly, we found some exclusive product ions only discovered in EAD MSMS spectra. And those product ions are very important for structural characterization and identification. However, we didn't find those product ions in CID MS/MS spectra, which means EAD can play an important role for metabolite identification in some cases.

P.N.: Excellent. Thank you so much for sharing some of the findings of your research. Now, I'm kind of curious: can you briefly tell us about some of the future studies in the pipeline that your lab has using the ZenoTOF 7600 system and more specifically, how you are planning on leveraging EAD for this type of work?

P.C.: Of course! Now we are also developing some quantitative methods to determine the presence of NPS and their metabolites. And in the future, we are planning to do more and more quantification using the Zeno TOF 7600 system.

P.N.: That's excellent! We're really looking forward to seeing more exciting work coming out of your laboratory in years to come.

P.N.: And that's a wrap for this episode on the benefits of electron-activated dissociation for the characterization and identification of NPS and their metabolites. A big thank you to my guest Peng. Thank you so much for taking the time to discuss your NPS work using the Zeno TOF 7600 system, and really sharing your knowledge on the benefits of EAD for this application.

P.C.: Thank you so much!

P.N.: And good luck with the writing of your dissertation and defending your thesis. And as always, thanks to the viewers for tuning in and watching this episode. Please make sure to check out our NPS analysis sub-page on the SCIEX Forensic Toxicology web page. There you can find the latest screening and identification workflows for NPS analysis. I'm also going to include two links to two technical notes that highlight the use of the ZenoTOF 7600 system and more specifically, the use of EAD for NPS characterization and identification.

I'm also going to provide the links to Peng's most recent peer review publications on the use of EAD on the ZenoTOF 7600 system for NPS in vitro metabolism studies. Peng’s also recorded an on-demand webinar on the same topic. I'm also going to provide the link for that, so please make sure to check out all those additional resources.

Thanks for joining us today. In the next episode, we'll be joined by Dr. Alberto Salamone, who is the laboratory supervisor at the Regional Center of Anti-Doping and Toxicology in Turin, Italy. Alberto is going to talk about different strategies that his laboratory uses to monitor the prevalence of novel psychoactive substances. So, make sure to tune in for that. Thank you so much, and we'll see you next time.