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

Dr. Alberto Salomone is the Laboratory Supervisor of Forensic Toxicology at the Regional Center of Anti-Doping and Toxicology in Turin, where he oversees the forensic laboratory operations of Piemonte, Italy. He also holds an Associate Professor position in the chemistry department at the University of Turin, Italy.

Pierre Negri (P.N.): Welcome to the seventh episode of Tox Chat Box, a SCIEX vodcast series, where we discuss the latest trends in applications in forensic toxicology. I’m Pierre from SCIEX. In today's episode, we're talking about different strategies used to monitor the prevalence of novel psychoactive substances using LC-MS. Today, we're joined by Dr. Alberto Salamone, who is the laboratory supervisor at the Regional Center of Anti-Doping and Toxicology in Turin, Italy. He's also an associate professor in the Department of Chemistry at the University of Turin.

Hi, Alberto. How are you doing?

Alberto Salamone (A.S.): Hi, Pierre. I'm doing great. Thank you.

P.N.: So, Alberto, you're currently the laboratory supervisor at the Regional Center of Anti-Doping and Toxicology. You started working there about 18 years ago when the lab was just getting up and running. And an interesting fact is that the lab was originally built to serve as the anti-doping center for the 2006 Winter Olympics in Turin. I know you were just getting started back then, but can you tell us what it was like to be part of this unique experience running athletes' samples for the Olympic Games?

A.S.: Sure. You just said the perfect word for that experience. Unique. It was a unique combination of science and fun. Yeah, because we were a team made of world top-level scientists in the field of anti-doping, pharmacology, mass spectrometry, and more. We were working 24/7 for two weeks in a crazy atmosphere, not only from the city, but also inside the laboratory. It was like a school trip for adults. We were having a lot of fun. And the testing samples were special because those were samples coming straight from the field, from the slopes, from the athletes who were competing for medals during the Olympic Games. So, we really felt the responsibility of giving solid and sound results in a very short time because there was a medal ceremony very shortly after. So, we really worked hard. But it was an unforgettable experience. I wish everyone could do it.

P.N.: It sounds like a fun experience indeed. Now, I know your lab operations and focus have evolved since the Olympics. You've been serving the region of Piedmont for many years now, testing for the presence of Ethyl glucuronide in the hair of people who have lost a driver's license after driving under the influence of alcohol. But in recent years, you've also started looking for the presence of fentanyl analogs and other novel psychoactive substances in the hair to determine long-term exposure. Can you tell us a little bit more about how these came about?

A.S.: Yeah. So, after the Olympic Games, the laboratory looked for new opportunities. Anti-doping analysis and forensic toxicology are very similar disciplines, I believe, because there are many things in common. The biological samples are similar: urine and blood mostly. The target analytes include some that are common. The technology of mass spectrometry, for example, is the technical reference technique for both applications. So, it was quite natural for us to move to forensic toxicology. And I have to say that I have no regrets because in forensic toxicology, we usually have more positive samples. In anti-doping, you work your eight hours in the lab as an analyst, and you are happy when there are positive samples. Now, when you look at the instrument, you see a nice chromatogram with a nice peak. In forensic toxicology, we began working with different samples, and hair became one of the most important, especially in the context of driving. It is quite common in Europe to use hair testing for driving relicensing because of the larger diagnostic windows with hair. We can identify long-term use of alcohol, for example, and for driving relicensing, this becomes very important because you want to avoid someone repeating the offense of driving under the influence. So, when someone is using alcohol regularly, they are probably not fit for a driving license. This is the principle that stands at the base of hair analysis in the context of driving relicensing.

P.N.: I think one thing that strikes me about your lab and the evolution over the years is the extent to which you're changing matrices. Right? You mentioned running anti-doping samples with hair, but also in recent years we've collaborated extensively and you've, focused on the development of LC-MS/MS-based workflows looking at a variety of drugs fentanyl analogs, synthetic opioids, and other classes of NPS in other biological matrices like urine and meconium, using both accurate mass and nominal mass system. So, I wanted to ask you how those projects came about and maybe discuss the advantages of looking for NPS in those matrices using LC-MS/MS instrumentation.

A.S.: Yes, we indeed did publish a couple of papers together, and it was a really nice collaboration. Yes, you mentioned two important opportunities that we have today in forensic toxicology. One is the alternative matrix. You cannot choose every time what matrix you have to analyze. So, you need to be ready to analyze what you receive in the laboratory from maybe a dead body, maybe a newborn, or maybe drug seizures. So, you need to have different analytical methods to test for whatever you receive. So, meconium was a nice option for newborns, hair we discussed it before, and now we are looking at dried blood spots because it's less invasive sampling then venous blood. Each matrix has pros and cons, but when you combine the results of as many as samples you are able to analyze, you increase the probability of reaching a correct or defensible conclusion. Then you mentioned the mass spectrometry in high-resolution mass spectrometry. Today, the drug black market is probably unpredictable because we know that every month there are new drugs outside. We have observed in the last ten to fifteen years the introduction of hundreds of new compounds. So it's quite impossible for the laboratory to be always up to date with target methods and have a full list of what is outside in terms of illicit or licit drugs. And this is happening with nitazene, this new class of threatening opioids, and it is happening with fentanyl. So, we have to be ready to identify xenobiotics in our alternative or standard samples. So, with accurate mass spectrometry, you can screen your sample and possibly identify other compounds. You can also do retrospective analysis. So, you can go reprocess your raw data and look for a sample for a compound that you didn't screen for at the very beginning, but maybe six months after you get to know that the compound was already out in the market so you reprocess your sample to look for also for that compound that you missed.

P.N.: So now we discussed analysis of hair, urine, blood, and dried blood spots. And now I want to bring up one of the most recent projects that you've been working on over the past year or so, which is the detection and quantitation of drugs and metabolites, and more specifically, novel psychoactive substances in wastewater. Can you tell us why it's important to monitor the substances in wastewater and some of the benefits of using sensitive instrumentation for this type of analysis?

A.S.: Thank. Sensitive instrumentation is not a benefit. It is the key. The levels are too low in wastewater so if you don't reach a ppt-level sensitivity, you can forget wastewater analysis. Today, we know that the analytical instrumentation available can usually reach those levels so we have not concerned. And, we are trying to use wastewater samples, not for epidemiological investigation because this is a quite well-established the science, often coordinated in Europe by the European Union drugs agencies, which collects data from all over across Europe to investigate patterns and trends of drug use, but for us, it's different. We are trying to use wastewater to identify the presence of something new. We mentioned NPS before. We mentioned opioids. And, you know, if there is one, sporadic use of a new compound in a small population, which maybe is not even tested, you will never know it. But with wastewater analysis, you can get an aggregated sample that represents a much larger population, and maybe someone is using that new drug.

P.N.: Last question before I let you go. As I said earlier, one of the most exciting facts about your lab is the way you're constantly evolving and looking for the next big thing, changing matrices, and looking for different compounds. Can you briefly tell us some of the future projects that you have in the pipeline and what we can expect from your laboratory in the next couple of years?

A.S.: It is not forensic toxicology, but more environmental toxicology. We are trying to collaborate with different zoos across Europe to analyze the hair of penguins, which in this case, is the feathers of penguins or other animals to explore the presence of contaminants of other pollutants or pharmaceutical that are coming from the environment, and who knows, maybe there is a correlation between different habits, or different behavior, or pathologies, or diseases of the animals, and the presence of the emerging contaminants. For sure, there is climate change, which is a big problem for the environment, but the presence of these contaminants can also play a role. And, this is what we are trying to investigate.

P.N.: And that's a wrap for this episode on the different strategies to monitor the prevalence of novel psychoactive substances using LC-MS/MS. A big thank you to my guest, Alberto. Thank you so much for sharing all your knowledge and valuable insights. And thank you for the great collaboration over the years.

A.S.: Thank you.

P.N.: Thanks to the viewers for tuning in and watching this episode. Please make sure to check out our NPS analysis page on the SCIEX forensic toxicology page, where you can find the latest screening and identification workflows for NPS analysis from SCIEX. I'm also going to include some direct links to some of the technical notes on NPS screening in various biological matrices that we've done in collaboration with Alberto's lab over the years. So, if you're interested in learning more about his work, please make sure to check out the links below. In the next episode, we'll be joined by Victor Van Dam, who is the science director of the Pain Management Laboratory at Quest Diagnostics. In the next episode, we will be discussing the relocation of a fleet of over 80 LC-MS instruments to a new laboratory space. Victor will be sharing his recent experience and describing what went down behind the scenes to get those instruments relocated, serviced, and up and running in a full production environment in record time, so make sure to tune in for that. Thank you very much, and we'll see you next time.