Identifying Novel Opioid Agonists From Metabolites

Novel drugs of abuse have been a growing problem for law enforcement and the medical profession alike in the last few years. Consequently, they have also become a challenge for analysts who must find ways to identify and characterize these substances, whilst keeping up with the constant emergence of new or variant chemicals. One way in which scientists can extend the window of detection is by looking for the degradation products of these substances too as they are broken down by the body.

We spoke to Alex Krotulski, Research Scientist at the Center for Forensic Science Research & Education at the Fredric Rieders Family Foundation, about the work he is doing to identify new and emerging illicit substances.

Karen Steward (KS): Can you tell us a bit about the emergence of novel opioid agonists and why you are looking to identify their metabolites?

Alex Krotulski (AK): Novel opioids began appearing in the US on illicit drug markets between 2013 and 2015 with fentanyl and its analogues, respectively. In the years that followed, several fentanyl analogues emerged and dominated certain drug markets, notably butyryl fentanyl and furanyl fentanyl. Then around 2016 marked the emergence of non-fentanyl derived synthetic opioids, most notably U-47700. The turnover of novel opioids during this time was relatively quick, ranging from about 6-9 months to a year or so, with new novel opioids popping up on a monthly basis. More recently in 2017 and 2018, several scheduling actions nationally (e.g. core structure scheduling of fentanyl analogues) and internationally (e.g. China scheduling some fentanyl analogues and U-47700) have made seemingly direct impacts on the emergence of novel opioids, as we have seen fewer new fentanyl analogues in recent months/the past year. Now, in 2019, we are seeing the emergence of new novel opioids not related to fentanyl or the “U-series” (following U-47700), including substances like 2-methyl AP-237.

It is important for our laboratory to study the metabolism of fentanyl analogues in order to identify metabolites or biotransformation products that can extend detection windows, specifically in urine. We typically conduct these studies to identify the primary metabolite of the novel opioids so we can then add that analyte to our screening and testing protocols. In addition, the metabolites of new psychoactive substances (NPS) have been found to be active, so studying the metabolites and then subsequently studying the potency of the metabolites provides a between understanding of the toxicity profile of a new substance.

KS: What were the biggest challenges you faced in looking to identify metabolites of the opioid agonists U-47700 and U-49900?

AK: The biggest challenge was accurately elucidating the structures of each metabolite without the ability to conduct additional testing (e.g. nuclear magnetic resonance (NMR)). This required a very good knowledge base and understanding of mass spectrometry (MS) fragmentation. For each metabolite, we acquired high resolution MS data of the fragment ions, and there were often 5-10+ fragment ions for each metabolite. Structural elucidation can be difficult but using the likes of the SCIEX MetabolitePilot™ identification software, this process was streamlined and straightforward. MetabolitePilot™ was extremely useful in pulling this data out for review, but in the end, it was necessary to have an analyst with MS expertise to determine where the sites of metabolites were occurring.

KS: What were the most surprising outcomes from your study?

AK: Going into this study, we knew that there would be the opportunity, or challenges, for/with isomeric or isobaric species, both among the metabolites identified and with other drugs or NPS. We found several isomeric hydroxylated metabolites throughout the study, as well as an isobaric metabolite of U-49900 and U-47700. The surprising aspect came when we realized that these two analytes (U-47700 and N-desethyl-U-49900) could be misidentified as each other. We highlighted that in the paper, but it was unknown to what extent that could have caused analytical problems.

KS: The number of “real” urine samples used to detect metabolites of the opioid agonists U-47700 and U-49900 were quite low (n = 1 and n = 5 respectively), is there a reason for this and do you plan to expand the study to ensure robust results?

AK: When this study was conducted, we were right in the middle of U-47700’s emergence and proliferation, and right on the front lines of U-49900’s emergence. We felt that, at the time, five urine samples were fine for the work that we were conducting. We certainly could have obtained more, but the goal was to conduct these studies as timely as we could, to get the data and information out to other forensic toxicologists and analytical chemists. For U-49900, this was the first case we had seen. We did not have any more urine samples to include. Looking back, we now have hundreds of U-47700 positive samples (and many for the metabolites since adding to the library) and a handful of U-49900 positive samples. Given the time and resources, it would be a great opportunity to re-examine these samples, especially if either of these two substances proliferated the illicit drug markets again.

KS: How do you foresee the findings of your work being used in a practical way to aid forensics or how might they lead to further work that could be of practical benefit?

AK: The direct results of this study are already being felt within forensic laboratories as the metabolites of U-47700 are added to library databases. In addition, laboratory scientists will be able to point to this study for metabolism comparison when new U-series novel opioids are found in laboratories, and specifically if unknowns are found that are suspected to be U-series metabolites.

Our laboratory has developed a rapid approach that allows for timely reporting of new NPS metabolites, using liquid chromatography high resolution mass spectrometry (LC-HRMS). Other laboratories will be able to adopt this method to further forensic toxicology and forensic science.

Alex Krotulski was speaking to Dr Karen Steward, Science Writer for Technology Networks.