The Toxpod
Forensic Toxicology: It's a matter of half-life and death
A deep dive into the fascinating world of forensic toxicology, supported by The International Association of Forensic Toxicologists (TIAFT)
The Toxpod
On the Spot at TIAFT2019
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During his recent trip to the TIAFT2019 conference in Birmingham, England, Peter caught up with a few toxicologists to discuss some of their recent work.
Barry Logan's group has published their findings on kratom
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The Toxpod is a production of The International Association of Forensic Toxicologists. The opinions expressed by the hosts are their own and do not necessarily reflect the views of TIAFT.
Hello and welcome to The Toxpod. I'm Tim Scott.
PeterAnd I'm Peter Stockham.
TimAnd Pete, you're fresh off the plane from the TIAFT conference in Birmingham.
PeterYes, I am pretty tired , but it was a good show.
TimAnd you managed to catch up with a few different people while you were there.
PeterYeah, while I was there, I tried to catch up with a few people. It was actually pretty hard to find people, it was such a large meeting but um, but I managed to catch up with Simon Elliott, the chair of the committee, who organized the meeting and Barry Logan who gave a talk on Kratom and how it's really going crazy in the US at the moment.
TimSo today we're going to bring you a couple of those interviews that Pete did while he was at TIAFT, similar to the on the spot we did at FACTA a few episodes ago.
PeterYeah and in addition to that, I also visited the University of Copenhagen for a meeting with Petur Dalsgaard who runs the high res NPS database and has also got some very interesting automation in the laboratory. So I wanted to go and see them and I had a chat to him about high res NPS, which you will hear mentioned in Barry's interview.
TimHope you enjoyed the interviews.
Simon Elliott
PeterOkay. I've just pulled aside Simon Elliot here at the penultimate day of the conference, how's it going Simon ?
SimonYeah, pretty good Peter. You're um the chair of the organizing committee and you've been trying to have this conference here for four years you were saying in the opening address. What's so important about Birmingham for you ? Well, Birmingham is Britain's second largest city really after London. So there's a lot of opportunities for people to come to Birmingham. There's good transport links, but importantly, the venue here, the ICC has been really good to allow people flow because we've had over 800 people throughout this week. So it's been uh quite a mammoth task to organize everybody and, herding cats, you know the phrase, yeah, it's been difficult.
PeterAnd that's , uh , one of the largest turnouts for a TIAFT meeting outside of the US. Is that what you were saying on, the other night?
SimonThat's right, yeah. The, the, the president informed me that would be the most well attended TIAFT ever aside from the US meetings.
PeterSo the scientific program has been pretty strong so far.
SimonYeah. So we've had lots of different sessions. So what we've tried to do with the scientific program here is create a variety, not just the talks but the different nations as well. So people can give a view of what's happening in their country. So we as an international organization can see what's happening in Asia, in the Middle East and also, not just Europe and Australasia and the usual places.
PeterThat's the whole thing about coming to these international conferences cause they're, you don't realize what's prescribed in other countries. I didn't realize that morphine's prescribed as cough medicine in some places. And midazolam is prescribed as pills in the US, in the UK, is that correct?
SimonYeah. Yeah. Well it's a , it's an eyeopener for all of us when you started hearing from someone in another country who, yeah, over the counter medicines that in your country is prescribed and controlled, it's, yeah, it's an interesting one.
PeterAnd you've got a bit of a passion for TIAFT haven't you, you've probably come to most of the TIAFT meetings during your career. Is that, would that be correct?
SimonYeah, I think that's , that's right. I was trying to think which ones I've not been to and it's only a couple, but yeah, I've been coming to TIAFT since 2000. I've been a member for over 20 years now. And yeah, it's a family. You know, it , you've, you've been here and been involved before Peter and all the listeners as well. TIAFT is a family and it's a, it's a network. It's a connection. It allows us to talk to each other about toxicology and that's what your podcast does, which is great.
PeterThank you very much. We're really enjoying it actually. I've got to say thank you very much for allowing The Toxpod to come to the meeting and allowing us to do a live recording on Friday. It's going to be very exciting. But , uh, as you were saying, TIAFT's got a , a real friendly atmosphere and you can approach anyone from the president , uh , any of the , the previous presidents. And they'll just have a chat to you and ask you what you're doing, and be interested. That's what I like about it.
SimonAbsolutely. And also for the youngsters as well. Cause we're obviously trying to encourage the next generation of toxicologists, you know, cause we've been around a little bit, but it's good that we can talk to the youngsters, they can talk to us and we can mix together. You know, there's no divides . That's the most important thing is that everyone is connected together in our big network and family of TIAFT.
PeterOkay Simon, thank you very much for those words and uh we'll see you for the rest of the meeting, I think we have to go to a talk.
SimonWe do have to go to a talk. Okay. Thanks Peter.
PeterDr
Barry Logan
PeterBarry Logan , thanks for joining the Toxpod.
BarryYou're welcome. Good morning.
PeterBarry yesterday you gave quite an interesting talk on mitragynine or Kratom, um can you give us a bit of background about this drug?
BarryUh, sure. So , uh, Kratom is a plant that grows in Southeast Asia , uh, in , uh , Malaysia, the Philippines and Thailand. Uh, it's been around for a long time. It's been a folk medicine there . Traditionally it was used as uh , a mild stimulant drug. The leaves are chewed , uh , more recently, um , people have discovered that if you use it in much higher doses then contrary to the stimulant effect that it was historically associated with, you can get more of an opioid like effect. And as word about that has gotten out in the United States certainly, it's become a popular recreational drug. And it's also used by people who are trying to manage their opioid use , um, to supplement maybe what prescription opioids they're getting from their physicians. Uh, or in some cases they're using it to try to manage their withdrawal.
PeterSo certainly there's been a lot of publications recently. I notice there's probably almost double the publications this year as there was last year, just looking on pubmed. And even just the last few days there's been a paper out from the UK on it, but it definitely does seem to be the US is the focus of the reemergence of mitragynine.
BarryThat's been our experience, as I said, traditionally it was a folk medicine in, in , uh , kind of the Pacific and in Asia, but , uh, it's use now has certainly been commercialized in the United States. Uh, specifically, it's completely unregulated currently. About two years ago, the Drug Enforcement Administration made an attempt to uh , place it on the schedule. In fact, a schedule one, which is our highest schedule , uh , no medical use and potential for abuse, but due to some political pressure, it was taken off or the , the notice was withdrawn and the DEA's still trying to collect , uh , data that will help us evaluate what its true harms might be.
PeterSo they're still doing a bit of consultation type thing or is it more of a reassessment of the hard data ?
BarryWell, I think what the DEA's very interested in are the publications that are coming out as the growth and interest in use of this drug has increased. Uh , there are more adverse event reports. There are now more , uh, reports of fatalities associated with its use. Um, we also see it in , uh, impaired driving cases from time to time.
PeterAnd so it's , um, active on the opioid receptors. So would people be using that as an alternative to their opioids or were they using it to come off opioids or is it just genuine recreation?
BarrySo I think part of its attraction is that it's an unregulated , uh , uncontrolled psychoactive substance that does produce, as I said, in lower doses , uh , more of a stimulant kind of buzz at higher doses, more of an opioid uh, effect. Uh, and then at very high doses causes significant sedation. So, so I think people are, people are always on the lookout for some mind-altering experience. And this is one that appears to be unregulated or, or, or in that legal gray area.
PeterIt's quite interesting, isn't it ? It does have these effects and it's still completely legal. And you showed um diagrams or pictures of some packaging yesterday and they look just like a synthetic cannabinoid. So that's an example of someone pretending that this is a natural substance, a leafy substance, but now it's , and putting in nice packaging, and now we're having a natural thing being put in the same sort of packaging. But it is a natural thing, it's sort of gone around in a circle.
BarryRight. So yeah, in a lot of the head shops and um, drug paraphernalia shops, smoke shops, you walk in there and they have a whole wall of displays of uh , different , uh, kratom products that are packaged, the packaging looks very much like synthetic cannabinoids, colorful characters , uh, um, mylar kind of packaging with different flavors or names for the, for the materials, hard to know really though what's in any of those packages.
PeterOf course, as with all of these things and it's uh , often the crushed very finely, from the diagram I've got, very finely crushed powder into capsules is generally what it's sold as?
BarryIt is, it's the leaves are finely ground. Some of the products are advertised as 20 x, 30 x, whatever that means. So some kind of concentrate perhaps of the leaves, which may have a lot more drug in them. We've also been, not so much currently, but when the drug first appeared , um, there were a number of reports of it being doped or adulterated with , uh , O-desmethyl tramadol. Uh, so there was a combined uh , effect and some of the early deaths actually were attributed more to the, the tramadol then to mitragynine itself.
PeterThat's interesting. I wonder what the pharmacological reason for that would be?
BarryUm yeah , somebody I think gave it a lot of thought because tramadol at, has actually some similar properties, at lower dose it does produce a , a mild stimulant effect, but you have to get to the um , uh , therapeutic threshold for pain control and then it looks more like an opioid.
PeterSo, analytically it's a bit of a problem. Quite a complex molecule.
BarryYeah, so in the plant there are over 20 related alkaloids, mitragynine being the most prevalent or most abundant. Um, and the rest are structurally related to that. Mitragynine itself has three chiral centers. So you kind of have a total of eight different conformers of the molecule. Uh , there are four that are , uh, the predominant ones. Uh, two pairs of Stereo, uh diastereomers. And because they are, they only differ in their conformation around , uh, whether the substituents are above or below the plane of the ring on the piperidine portion of the molecule. Um, when you put them into a mass spectrometer, they're going to fragment exactly the same way. So you'll get the same daughter ions and you'll get the same ion ratios. So this is one of these examples where if you're doing msms , um , or even uh high resolution mass spec, you're not going to be able to differentiate these based on their , uh, on their fragmentation. You really have to come back to , uh , making sure you have good , uh, chromatography.
PeterSo you showed a diagram of, you had at least four peaks, quite well separated, but another diagram, if they were all overlapping , then you'd never actually get an accurate concentration. Unless you had a really good high resolution method, you wouldn't be able to separate them.
BarryThat's right. That's right. So when we first brought this test up , uh, we bought a standard reference material for one of the unique stereoisomers from , uh, from one of the suppliers. And we developed our methods , uh, went through our optimization and then our, our , uh, method validation and when we got into method validation, one of the things we do is test authentic samples as part of that. And with the first iteration of the method, we not only saw a couple of other peaks there that had the same transitions in the same ion ratios, but we found that it was another substance with the same , uh , properties that was eluting under the, or just on the shoulder of the , the standard reference material. So that clued us into the fact that we were going to have to do some more chromatography to separate this out. And in talking to some other people who have methods and looking at some of the published methods, a lot of people , um, when they see that they'll just push the peaks in together , uh , cause they're , they look to be , uh , the same. The difficulty with that is you've got a number of species now under that peak that are going to have different pharmacological effect. So if you're quantifying it and it's a mixture of diastereomers with different levels of potency and everybody's testing it differently, you're not going to get good consistent reference data.
PeterSo has there been any work on whether any of these diastereomers are active anyway, or might it be better to combine them all in one peak or sum all of those peaks together?
BarryWell, so the, the mitragynine , uh, of the diastereomeric pairs is the most potent. Uh , there's a second one called speciogynine , uh , for which there's no uh standard reference material available, which has less potency. The other two, there's really been no assessment of what their potency is. Uh , there's another active substance in the plant, which is seven hydroxymitragynine, which is more important, maybe 10, as much as 10 times as potent as mitragynine, but is present only at a fraction of its abundance in the plant, probably 1 or 2%. So the focus has really been on this one, a diastereomer mitragynine.
PeterAnd the seven hydroxy's not a metabolite, I was sort of under the impression awhile ago that it was, but...
BarryWell there's, there's emerging evidence in that, in addition to being a natural product in the plant that it is in fact also , um , a metabolite.
PeterNow , um , what about dependency, uh people are getting dependent on this drug as well as, it's obviously got opioid effects so it's probably going to have the same sort of issues in that regard.
BarryYeah, I think the emerging evidence says that in , um, significant overuse or mega dosing of the drug, because of its opioid like effects, it does produce reinforcement and habituation and there are people who are seeking treatment for , uh , habituation or having developed some dependency on it. In our experience, it's most frequently used alongside other drugs, particularly opioids and , uh, frequently benzodiazepines. So I think it's hard oftentimes to disentangle how much of the adverse effect is due to the drug itself, the other drugs it's being taken along with or the incremental or perhaps , um, uh , enhanced effect that you get from taking a drug in combination.
PeterWell, I've had a couple of conversations here and whether it's anecdotal or whether there's true evidence, but is it, is polypharmacy on the rise? Everyone just seems to be having never ever have just the one drug. It's always uh opioids with benzos, with GHB, with everything else. Is it , do you think that's increasing?
BarryYeah, that's very much our experience across the board. So if people are , are taking mitragynine as an adjunct to their therapy, if it's for pain management or they're self-medicating to withdraw or to manage their illicit opioid use , uh , you're obviously going to get it in combination. But just across the board, we, the patterns we're seeing now in our regular legacy recreational drugs, we see combinations of fentanyl and cocaine, heroin, cocaine , uh, we've also had a number of episodes now of combined opioid and synthetic cannabinoid products. So powders that are being sold ostensibly as fentanyl or as an opiate , but they contain a mixture of the opioid plus synthetic cannabinoids.
PeterSo that sounds like the particular dealer trying to making his product more attractive. Is that what it is? Do they know what they doing?
BarryUh, it's hard to tell just how deliberate that is or somebody who's just trying to differentiate their product on the market, but it produces a very different kind of , uh , high. Uh, in one of the incidents we investigated there were about 200 intoxications in an area of Philadelphia over one weekend , um, which was very high. Uh , and what was unusual about them was when the , these patients went to the hospital, they were, they were obtunded, they were sedated, they were given narcan. And typically when somebody is reversed with Narcan, they'll come out of it. They will, within a short period of time, return to apparent normalcy. But these folks, when they got the narcan emerged into a very agitated, delirious state. It was initially diagnosed by some of the physicians as um, an anticholinergic kind of reaction. But , uh , we obtained some of the powder and then obtained some of the clinical specimens and we were able to verify that it was a combination of , uh , fentanyl, heroin and uh 5-fluoro-ADB. So giving the Narcan to these patients basically unmasked all the synthetic cannabinoid effects.
PeterNow I'm probably the first in line when it comes to getting a new source of NPS data on the Internet. And you also head up the NPS discovery centre. Is that right? Do you what you want to tell us a little bit about that and how you're getting involved in that?
BarrySure. So a lot of this activity is done through our uh nonprofit organization that , uh, our founder created about, actually, this is our 25th anniversary of the Fredric Rieders family foundation. About 10 years ago, we created a program within that called the Center for forensic science research and education where we really stepped up a lot of our research activities. We , uh, moved into high resolution mass spectrometry techniques , uh, as a way to explore , uh , new, new drugs, find out more about drugs and their metabolism. Uh , and over the years we , uh, created a number of different workflows for the identification of novel psychoactive substances. Some of that is in forensic case work. A lot of it is post-mortem , uh , through our partnership with NMS labs. Uh , we have access to discarded de-identified blood samples for testing.
PeterAre they blood samples or blood extracts?
BarrySo we started with blood samples for a variety of projects, particularly during the time of the rise of the opioid, of the fentanyl analog , uh, crisis or epidemic. But what we , uh, subsequently did was we had, we a number of ways of looking at that data. Uh, one was to get samples and test them for things that were outside of the scope of what , uh , the routine NMS test had been. As we became aware of novel opioid or fentanyl analogs, we would rescreen the samples for those. We eventually figured out that it was easier just to get the discarded extracts , uh, saved a lot of sample preparation time and NMS tests probably two or 300 of the, of these cases a day. So we had a very large pool of, of samples to, of extracts to retest. So that's been very productive in terms of our ability to rapidly update our scope of testing, to recognize things that look alike in terms of something that's already in the database but not exactly alike, and will trigger a series of workflows to, to dig down into the identification of those. Uh, we also took advantage of the fact that the NMS labs screening had been done by , uh, LC-TOF. Uh, so we've been archiving all of the, and it's a non-targeted acquisition, so we've been archiving all of that data and now we data mine that data. When we find , uh, could, get confirmation of the identity of a new substance, we'll retrospectively go back and reprocess months worth of data to see if perhaps it had been present in cases , uh , earlier that we just didn't recognize.
PeterYeah. When it comes to free information about NPS, the illicit drug, drug,seizure sort of industry that, the groups that do that sort of work, if they find a new drug, they send it to SWGDRUG and put it in their GC/MS library. But as yet there's no central repository for LCMS high resolution information. It'd be good if we could get something going like that, but I think it's going to be difficult to get a standardized approach across different vendors. So it was really pleasing to see that you had started to introduce um high res NPS MS/MS data into some of the later entries into database . So that's great.
BarrySo we, in addition to the postmortem , uh , biological,extracts, we also , uh , have a couple of other projects that give us some insight to what the trends are in the, in the market. One is , uh , NMS labs also , uh , perform , seized drug testing and has about six laboratories around the United States. So , um, the, our center receives , uh , samples from them if it's things that are outside of the scope of their library. Uh, and then we have access to high resolution platforms that we use for identification. We also run uh NMR, so that gives us the opportunity to identify things de novo even if we don't have a reference material on hand and then we'll either work with the vendors to get them to expedite synthesis of that or in some cases we've commissioned those synthesis , uh , ourselves.
PeterSounds like a good approach, joining up different arms of your business to, and hopefully disseminate information where you can.
BarrySure. And a complementary aspect of that is we also have a partnership with the United States Customs and Border Protection Service and they're monitoring illicit drugs coming into the United States through international express mail. So they have some screening that they do on site at the ports of entry. But again, anything that is unidentified there is forwarded to , uh , our NPS discovery program for these enhanced identification techniques. So as soon as we get something, typically we'll be able to make an identification within two to three weeks. And then we put that information out through a , an email list. We put it on our website. It's, we have secondary distribution through UNODC's website and EMCDDA and at various internal early warning systems in the United States. So the goal really is to get the information about the identity of these substances out, including their analytical characteristics. You're right that there is no repository or a comprehensive place to go for that high res mass spec data. Um , that's one of the things that we've been asked to do with these new identifications is to provide electronic, searchable , uh , Spectra. Uh, so in the next phase of the project, we'll be looking for places to host that data.
PeterThere's a website called um HighResNPS.com, which I'm involved with a little bit. And , um, unlike uh GCMS data which can be put into a certain format that all vendors have managed to get their software to read . This is in excel database form. So, makes it sort of universal because most vendors can sort of import that.
BarryUh , yes, we've certainly looked at HighResNPS as one of the sources that we go to for help with identification of new substances, you know , so we'd be happy to contribute uh data.
PeterAnd you already have contributed quite a few spectra, especially the ones that weren't even on the database before . So that's much appreciated by the people who run it. Okay, thank you very much Barry.
BarryYou're welcome.
PeterI appreciate your time.
BarryYou're welcome. Thank you.
PeterThank you.
Petur Dalsgaard
PeterI'm here at the University of Copenhagen speaking to Petur Dalsgaard about his database HighResNPS. How are you going Peter?
PeturOh good. How are you?
PeterSo you started this project up quite a long time ago and you've been working on it very hard ever since.
PeturYes. This project actually started back at the TIAFT conference , uh , in Madeira back in 2013 and I was working here at the section of Forensic Chemistry and I had the same problem as everybody else. I was trying to buy standards for NPS to put in the screening library. And I know that my colleagues from the Scandinavian countries, they also used uh high resolution mass spectrometry and they were struggling to do the same. So I asked them if we should just make a database together , uh, with NPS where we could share information.
PeterThat's a great idea because there's very little information out there in terms of the fragmentation information isn't there? There's a lot of stuff about uh which new drugs are coming out and maybe its formula. And so this is um , not necessarily as highly validated as maybe a commercially available gcms library or something like that. But nonetheless, for the purposes of suspect screening, which is where, or maybe you can explain suspect screening for us?
PeturSo suspect screening in, in my point of view is that you, you have a list of things that you would like to see. So you can have the names and the molecular formula. Um, but at this point you maybe don't have any fragment data. So we can just use that with a high resolution mass spectrometer and um , and screen for compounds. Now if you find something that you think is a NPS, then you of course you need to buy the standard and confirm the retention time and the fragmentation. But as a starting point its good to just use a suspect screening library, especially when there are so many new compounds coming out every week.
PeterThat's right. Every week there's new ones. And this of course saves you buying standards, the only another way to do this would be to buy standards or get standards from another laboratory. And that can be very expensive. So this suspect screening's just a place to start, can narrow down the list of compounds that you buy to verify the detection.
PeturYeah, that's correct. And it also seems that even if you are using different instrumentation, they tend to generate the same fragments. So it's quite straightforward . Why not make a database where you can share the exact mass of these fragments?
PeterAnd you've demonstrated that recently in a paper you published this year and which I also contributed to by analyzing some samples for you. Um , so tell us a little bit about that paper.
PeturSo we want this paper to be a proof of concept , uh, about this. So we , um , we took the whole database, exported into an excel spreadsheet and then generated libraries for the Agilent system, for the Bruker system, for the Waters system and for the Thermo system. And then we spiked a sample with some NPS and then we analyzed this sample with the different instrumentation and, just to see if this library could be a universal library that worked with all manufacturers.
PeterAnd so you found that basically you could detect all those compounds, except there were a couple of exceptions, but in general, the fragment data was the same regardless of what instrument you used , which has always been a suspicion, but no one's really ever proven that I guess, so it was a great proof of concept paper to start with.
PeturYeah, it seemed to work. So we continue this work now , um, we encourage people to add data into the database if they have the exact mass of some fragments , um, and they get some NPS, just add it to the database and you can share it with the rest of the world basically.
PeterAnd so, to contribute they just have to get a user name from you, is that correct?
PeturYeah, they can just uh, go into the website, HighResNPS.com. And um , from there they can request a login to the database.
PeterSo the database has actually got , uh , how many compounds? A couple of thousand?
PeturI think there are 2000 entries in the database now. Um, but there are duplicates in the database so there are probably a little bit more than 1000 NPS there . But , um, the reason for there , there are duplicates in the database is that we encourage people to post what they have on , uh, of , uh , data from their NPS. And this makes the database kind of self validating. If we could see that people get the same fragments, even if they recorded the data on a Bruker, Agilent, Waters, or Sciex system, it makes them more confident in the data.
PeterThat's a great way to , um, sort of, self validate the database. And so these are entered by the , the source of the data that, that people might enter, that may be from a seizure or it may be from an authentic standard or it may even be some data that someone's found in a paper and entered it in there . Is that correct?
PeturYeah , we also monitor some websites , uh , to see if there's any new NPS out there. And if we find something, we just , uh, enter the, the name, the molecular formula, the exact mass and uh , some structure information as well.
PeterSo that's a big job. You're doing largely most of this yourself I think. And with a little bit of help here and there, uh people who want to help, how are they, can get in touch with you ?
PeturWell they can first, they can sign up to be a member of the database and if they would like to contribute maybe they can add their own uh, parts of their own library to HighResNPS or they can monitor some, some website if they would like to, or they could maybe add the data from some scientific publication.
PeterOkay. Peter, thanks very much for joining us. I hope we can get some more contributors to your database.
PeturThank you very much.
PeterA well worthwhile project. And if you're interested in Petur's paper, it's in the Journal of analytical toxicology. HighResNPS.com an online crowdsourced HRMS database for suspect and non-targeted screening of NPS. And that's published , um , in May this year.
TimOkay, I hope you enjoyed those interviews. It's always good to catch up with what different toxicologists are doing around the world.
PeterI'm sure there are lots of very interesting talks, lots of interesting people I could have had a chat to, but didn't really have that much opportunity. But hopefully we can do more of this sort of thing in the future Tim.
TimSounds great. Well, thanks for listening, and if you want to contact us, remember, you can email us at thetoxpod@sa .gov .au. See you next time.