By Stuart A. K. Kurtz, M.S., D-ABFT-FT
Forensic science has always been the field I wanted to work in. I made my parents buy me a book at the Scholastic Book Fair in 3rd grade on forensic science. From there, I started looking at what areas interested me the most. I also loved chemistry so decided I would work as a drug chemist working with seized materials. While at IUPUI for grad school, I took a class with Kevin Shanks on designer drugs and knew that’s what I wanted to do.
Forensic toxicology combines many different aspects but the one that sticks out to me the most is science communication. I think that is the most impactful part of what I do. I have to be able to explain how we got the results including all steps from receiving a case, the review and release of the case, and, most importantly, what the results mean and don’t mean. Lawyers, juries, families, investigators, coroners, medical examiners, and pathologists all have very different levels of understanding when it comes to forensic toxicology. I have to be able to cater my explanations to each person and make sure that I am meeting them where they are in terms of their understanding.
Being able to talk to families and help them understand is my favorite part even if it is difficult at times to talk to someone experiencing tragedy. It can be as important to explain what something doesn’t mean as it is to explain what it does mean. Sometimes a family wants to pursue certain testing because they think it will give them the answers they need. I explain as well as I can the reasons for and against pursuing the testing. We never want to practice toxicology in a vacuum so I make sure to explain whether I can or cannot interpret the results and why. Ultimately, I want the results to be able to provide information that leads to the best closure possible for families.
Chief Deputy Coroner Alfarena McGinty of the Marion County IN Coroner’s Office came and gave a very moving presentation on her experiences and how that affects her daily work. She says, “We speak for the dead but we serve the survivors.” I say, “Behind every case there is a person. Behind every person there is a family.” Both of these mantras get at the fact that the families are central to how she and I go about our days in our respective jobs. While we arrived at those mantras separately, we have similar experiences that lead us there.
An old colleague of our Lab Director and Chief Toxicologist Dr. Behonick said to him “Someone has to make sense out of all this mess.” My goal is to help gather information that can be used to help the survivors make sense of the mess. No one person can clean up the mess but I can do my best to help others understand it.
By Kevin G. Shanks, M.S., D-ABFT-FT
I always had an interest in science and math at a young age. And for some reason, I was enamored with drugs and poisons too. Some of my favorite television shows as a teenager were Murder She Wrote and Quincy, M.E. – yes, I know I’m showing my age there. I also watched the now dreaded CSI television shows, including ever sunglasses-wearing Horatio in Miami. I also was a big fan of Agatha Christie or any work that involved poisons. Yes, I was (and still am) a big nerd.
My first role in the laboratory years ago was doing the analytical testing on a pharmaceutical drug: a urinary tract medication that had three active ingredients (methenamine, phenylsalicylate, and hyoscyamine). The pharmaceutical company had outsourced the regulated testing to my former lab and we had to test the tablets for regulatory compliance, which included content uniformity and testing for excipients and degradation in simulated gastric and intestinal fluids. It was very boring, monotonous work, but I learned a lot during that first year including how to make simulated gastric and intestinal fluid, which was quite interesting. I operated HPLC, flame photometers, and UV-VIS instruments. I also learned I didn’t like GMP testing very much. Just think of all the documentation you have to do in your job every day (it’s a lot, I know) and then multiply that by 1,000,000. I’m not joking. It was actually pretty stressful work too as it was all FDA-regulated work so we had multiple instances of FDA inspectors in the lab to watch us do the testing. And those FDA inspectors (at least the ones I’ve met) aren’t the nicest of people. After a year, an opportunity arose in method development and validation and I jumped at it. A few years went by and I got my hands on a LC-ToF in 2006 and I was hooked at that point. We became the first production forensic toxicology lab in the country to utilize an LC-ToF in screening. Around that time I also acquired the duty of handling the non-routine casework in the lab – things like syringes, tablets, liquids, drug paraphernalia, seized drug evidence, and foodstuffs. Also, another duty was developing methods for drugs that neither we nor a reference lab had a method for. A few more years later and a forensic toxicologist position opened up and I happily took it and as they say, that’s all she wrote. I’ve been in an official forensic toxicologist position for the last 12 years. If you ever want to know more, I’d gladly tell you more, but that’s enough about me in particular.
I’ve been lucky to see a lot of change in this field over the last 20 years. Firstly, instrumental analysis is quite different than it was a couple of decades ago. When I first was on the job we were using Thin Layer Chromatography, GC-MS, and immunoassay for screening. We had HPLC with UV and fluorescence detectors, GC-MS, and LC-single quadrupole MS instruments for confirmation testing – LC-triple quadrupole MS wasn’t really a thing yet in forensic toxicology. And no one inside of toxicology had even dreamt of using high resolution accurate mass instruments such as single stage time of flights or quadrupole time of flight mass spectrometers for anything at that point. But you look around the lab today and all you see is LC-QToFs and LC-MS/MS. It’s wild how much change has occurred in a relatively short amount of time.
Secondly, the sheer breadth of available drugs has substantially increased as well. In the early 2000s, novel psychoactive substance (NPS) wasn’t a term that was familiar. No one spoke about them. But sometime around 2008, NPS such as substituted cathinones, designer benzodiazepines, fentanyl analogs, synthetic opioids, and synthetic cannabinoids changed the toxicology landscape. We can no longer just worry about the classical drugs of abuse (such as methamphetamine or heroin or cocaine) or prescription medications (such as oxycodone, hydrocodone, or alprazolam). A challenge we face in forensic toxicology is what substances do we need to include in our scope of testing? In the early 2000s, most labs weren’t even testing for fentanyl. Can you imagine that? Illicit fentanyl is by far and away the most important drug that is driving overdoses in the United States these days and has been for the last several years. The next question after scope is how do we analyze them in the effective and efficient way as possible? And finally, after all that, the most pressing issue is the arduous challenge of results interpretation and expressing a scientific opinion in courts of law – how does the substance play a role in a medical-legal death investigation or human impairment? Does it play a role? Is it an incidental finding? Someone much smarter than me used to say, “Never practice toxicology in a vacuum”. And it’s truer today than it has ever been.
There’s that old adage that the only constant in life is change. It’s a saying for a reason. If this was social media, I’d end the sentence with #truth. That saying exactly describes the last 20 years of forensic toxicology.
By George S. Behonick, Ph.D. F-ABFT
In recognition of National Forensic Science Week (NFSW), September 18-22, 2023, I am putting pen to paper to capture some thoughts on what this means to Axis Forensic Toxicology. The euphemism, “Dead men tell no tales”, is oft used as a metaphor. To be certain, it is an exaggerated phrase, but it does get a point across. Similarly, medical examiners (ME), forensic pathologists and coroners hold as a credo, “We speak for the dead”. It is a creed extending to other members of the medico-legal death investigation team, chiefly the men and women who comprise the ranks of the various disciplines within the field of forensic science. We at Axis Forensic Toxicology are part of that team and we are charged with the responsibility of trying to provide answers and context to what often are the final moments, or acts, of a person before departing this planet. Forensic postmortem toxicology is uniquely set apart from the other forensic science specialties. Think about this for a moment, it is the only branch within forensic science that provides the ME, coroner or forensic pathologist with explanation for, or reason for a decedent’s demise; that is, a cause of death (COD). All of the other forensic science disciplines may provide supporting evidence integral to unraveling the circumstances and details of a death. For example, DNA and latent fingerprint scientists provide definitive proof in establishing a decedent’s identity, or likewise may be able to establish the identity of a subject who may have had close contact with the decedent before or at the time of death. Criminalist analysts may categorize and document trace evidence such as hairs and fibers, for eventual comparison to known materials from a death scene or decedent. Firearms examiners provide weapon function tests in cases of suicide by suspected self-inflicted gunshot wound (that is, was it an accident or was it self-intentional?). Projectile fragments and bullets recovered at autopsy can be matched to a specific weapon. Note however, that none of these examples provide potential for COD. Postmortem forensic toxicology can offer plausible reason and evidence for the pathophysiological mechanisms to cause one’s death (e.g. the respiratory depression and accompanying apneic and anoxic pathology associated with an opioid poisoning or intoxication).
The past quarter century has borne witness to rapid change and advancement in the field of forensic toxicology. Toxicologists and analysts were at the forefront of the nationwide epidemic of prescription drug abuse and misuse which ignited in the mid to late 1990s with OxyContin® (dubiously dubbed “Hillbilly Heroin” because of its scourge inflicted to middle Appalachia) and then morphed to other opioids such as methadone and prescription derived fentanyl. Within the first decade of the new millennium, the United States experienced a re-emergence in heroin. Heroin-related deaths surged for a brief period 2010-15, to be followed by the nationwide infiltration of illicitly manufactured fentanyl (IMF) into the street drug supply chain. The synthetic modification and manipulation of IMF then resulted in the proliferation of potent fentanyl analogs such as carfentanil and furanylfentanyl. More recently, other designer opioids, novel psychoactive substances, and clandestinely manufactured psychotropic substances such as synthetic cannabinoids (‘K2 Spice’), cathinone compounds (‘bath salts’), nitazene compounds and designer benzodiazepines such as bromazolam and flualprazolam have come to make their mark in the United States. Not to mention, mitragynine (‘kratom’) and the adulterant drug xylazine (‘Tranq’). Fortuitously, forensic toxicology has enjoyed a golden age in the last twenty-five years with respect to technology. This encompasses not only new and improved methods for the extraction and recovery of drugs and drug metabolites from postmortem blood and other fluids and tissues, but also a robust cavalcade of sophisticated instrumentation and automation. Utility and versatility of high-resolution mass spectrometry (HRMS) which empowers laboratories with the ability to detect and identify, in real time fashion, literally hundreds of drug compounds and drug metabolites of interest is astounding. Liquid Chromatography-Quadrupole Time of Flight HRMS imbues laboratories with a tool to meet the challenges of an ever-changing illicit drug landscape. Indeed, working as a forensic scientist, technician, or analyst in a modern forensic toxicology laboratory is both exciting and rewarding; moreover, it is imperative we also acknowledge all of the actors in this play. It is not an exaggeration in stating that it takes a village to do what we do day to day, so it is we recognize during this NFSW 2023 the executive and administrative clerical staff, the logistics staff, the IT support, managers and supervisors, the accessioning staff, and everyone associated with Axis Forensic Toxicology.
In closing, I leave you with the sage words of a grizzled ME whom I had the pleasure of working with in Virginia. His name is Dr. William Massello III, recently retired in the last several years as the Chief Medical Examiner for the state of North Dakota. He once posited to me, “Someone has to make sense out of all of this mess”. Words I have never forgotten, but still echo today. Despite some of the most horrific, tragic circumstances that can befall a human being, we are called upon to do our jobs. Be proud of what you do, realize the essential contributions you make to the public at large, the criminal justice system and to the decedent families and next of kin we indirectly serve. Be proud to work in forensic science!
By Kevin Shanks, M.S., D-ABFT-FT
As mentioned in previous blog posts, novel psychoactive substances (NPS) come in different varieties and the Novel Emerging Compounds (NEC) Panel offered by Axis Forensic Toxicology helps to detect the most newly emerged NPS on the drug market and is meant to evolve over time as new drugs emerge on the street. In the first post in the series, we took a look at two of the more recently emerged NPS benzodiazepines, bromazolam and flubromazepam, and in the second post, we looked at two of the most recently detected stimulants, alpha-PiHP and N,N-dimethylpentylone. In this third post in the series, we will take a brief look at two more recently emerged compounds: phenibut and tianeptine.
Phenibut is a substance that was originally developed in the Soviet Union in the 1960s and is currently marketed as a medication in Belarus, Kazakhstan, Latvia, Russia, and Ukraine and used in the treatment of anxiety and insomnia, as well as many other disease states and disorders. Phenibut is not approved for use in the United States, but it has been sold on the internet as a dietary supplement and nootropic and has been used recreationally. The name phenibut is derived from the chemical name (B-phenyl-y-aminobutyric acid). It is an analog of the neurotransmitter gamma-aminobutyric acid (GABA) and is thought to act as a GABA receptor agonist (similar to baclofen), but it also has dopaminergic effects. Reported effects of the substance include sedation, tiredness, drowsiness, nausea, headache, irritability, agitation, and euphoria. Phenibut is currently considered an uncontrolled substance in the United States.
Tianeptine is a drug that was developed by the French Society of Medical Research in the 1960s and is currently approved as a prescription medication in France and other European and Asian countries. It is used for the treatment of major depressive disorder, as well as anxiety, irritable bowel syndrome, and asthma. Tianeptine is not approved for use in the United States as a medication. It has been found as a drug of abuse throughout the years in Russia and has become an emerging public health risk in the United States as a recreational drug. Tianeptine is a mu opioid receptor agonist and also has glutamate receptor activity via the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Reported effects of tianeptine use include headache, drowsiness, nausea, agitation, anxiety, and euphoria. Tianeptine is not a controlled substance in the United States, but has been controlled at the state level in Alabama, Michigan, Ohio, and Tennessee as either a Schedule I or II substance.
As both substances are not controlled substances at the Federal level, the Drug Enforcement Administration (DEA) has not released any detection or prevalence data via the National Forensic Laboratory Information System (NFLIS), but both phenibut and tianeptine have been implicated in human toxicity which have led to hospitalizations and have also been associated with fatalities in the United States.
Axis qualitatively monitors both of these compounds in our NEC panel (order code 13710) and Comprehensive Panel, Blood with Analyte Assurance (order code 70510) using liquid chromatography with quadrupole time of flight mass spectrometry (LC-QToF-MS). Over the time range 01/30/2023 – 06/30/2023, Axis has detected phenibut in 5 blood specimens in 5 states (Arizona, Florida, Indiana, Ohio, and Tennessee) and tianeptine in 6 cases in 4 states (Florida, Indiana, Kentucky, and Tennessee). In Axis Forensic Toxicology casework, phenibut was typically found alongside THC/THC-COOH (n=2), tianeptine (n=2), caffeine (n=2), dextromethorphan (n=1), and fentanyl/norfentanyl (n=1). Tianeptine was simultaneously detected with bromazolam (n=2), phenibut (n=2), cotinine (n=2), fentanyl/norfentanyl (n=1), and methamphetamine (n=1).
Axis also monitors other NPS in our Novel Psychoactive Substances panel (order code 13610). These additional compounds include 25B-NBOMe, 25C-NBOMe, 25I-NBOMe, 2C-B, 2C-E, 2C-I, 5-MeO-DALT, adinazolam, alpha-PVP, butylone, clonazolam, dibutylone, dimethylone, ethylone, etizolam, eutylone, flualprazolam, flubromazolam, MDPV, mephedrone, methcathinone, methedrone, methoxetamine, methylone, N-ethylpentylone, pentylone, and TFMPP.
As always, if you have questions about these substances and how they may play a role in your medical-legal investigation, please reach out to our subject matter experts by email ([email protected]) or phone (317-759-4869, Option 3).
Be on the lookout for the fourth and final post in the NEC Panel series! To round out the panel, we will take a brief look at three more recently emerged novel compounds: AP-237, brorphine, and fluorofentanyl.
By George S. Behonick, Ph.D., F-ABFT, Laboratory Director, Chief Toxicologist
Synthetic compounds designed to mimic the pharmacology of various illegal drug classes infiltrated the illicit drug market during the last decade plus; this includes synthetic cannabinoids (“K2 Spice”), cathinones (“Bath Salts”), hallucinogens, and designer fentanyl analogs and opioids (examples such as fluorofentanyl, brophine and the nitazene compounds). In particular, the fentanyl analogs, along with illicitly manufactured fentanyl (IMF), imposed significant impact to the morbidity and mortality of drug-related exposures. Heroin cases experienced a dramatic resurgence around 2010; in large part, its unprecedented purity (users could ‘snort’ or insufflate the drug) and its relative low cost compared to prescription opioids (e.g. OxyContin®) fueled its unbridled demand as an alternative among opioid addicts and neophyte users. Gradually with time, solid dose materials (paraphernalia) and autopsy specimens began to demonstrate the concomitant presence of heroin and IMF. Ostensibly, the addition of IMF was designed to increase the potency of the product. Eventually, beginning in 2014 the proportion of IMF became greater than heroin in these mixtures. By 2015 the greater number of drug-involved cases included fentanyl, and not heroin. Simultaneous to this trend, numerous synthetic fentanyl analogs were being manufactured outside of the United States, but made available to illicit drug traffickers over the Internet. In 2016-17, carfentanil began appearing in seized solid dose drug products and medical examiner death investigation cases .
Carfentanil is a mu (µ) opioid receptor agonist; it is about 10,000 times more potent than morphine and demonstrates 30-100 times the potency of fentanyl . Between September 1, 2016 and January 1, 2017, Axis Forensic Toxicology detected carfentanil in 262 postmortem blood specimens . We described the specific details of 13 fatalities, from Indiana, Michigan, Kentucky and Ohio in this data set. Other investigators reported the detection of carfentanil in blood specimens obtained from impaired drivers and postmortem toxicology submissions [7, 4]. In response, state and federal agencies issued health alerts in 2018 which detailed the rising numbers of deaths involving fentanyl and fentanyl analogs to include carfentanil [2, 1].
2016 and 2017 demonstrated a surge increase in the number of drug-related deaths in the United Sates; that is, ↑11,228 (+21.4%) in 2016 and ↑6605 (+10.4%) in 2017; however, notably in 2018, drug-related deaths declined by 2,870 or -4.1%. The rise and fall coincided with increases, and then, decreases of carfentanil detections in seizure exhibits from 2015-2018. The overall availability of carfentanil was attributed to be a major factor in the accelerated, then diminished rate of drug-related deaths in the period 2016-2018 . China banned carfentanil on March 1, 2017.
In CY 2022 Axis Forensic Toxicology detected carfentanil in two cases. Carfentanil was detected in one case in February 2023 however, in late spring and summer (May 26 – Aug 18), Axis Forensic Toxicology detected carfentanil in blood specimens from 9 postmortem cases. The time span being four months with the cases originating from four different states: Indiana (4), Wisconsin (2), Kentucky (2), and Ohio (1). A cursory review of a subset of these cases (n = 7) indicated 6 of 7 decedents to be male, with an age range of 29-61 years and median age of 41 years. Carfentanil postmortem blood concentrations exhibited a range of 47.2 to 409 picograms per milliliter (pg/mL); carfentanil was identified qualitatively in two of the cases. Multiple drugs were detected in all but one case. Fentanyl was detected in all cases with multiple drug detections. Methamphetamine and amphetamine was detected in 5 of the 7 cases. Other detections included: Acetylfentanyl (4 cases), 4-ANPP (4 cases), fluorofentanyl ( 1case) and morphine (1 case).
The aforementioned case accounts underscore the continued awareness and vigilance medico-legal death investigators and forensic toxicology laboratories must demonstrate to surveilling the drug carfentanil. In one case reported herein, the decedent, a 61 year old male, was found deceased in his vehicle at a truck stop that he was known to frequent on a near daily basis. The sole toxicological finding in this case was a carfentanil postmortem blood concentration of 403 pg/mL. In the two Wisconsin cases, the deaths occurred in two males who apparently knew each other and were suspected to have secured drugs from the same supplier within a close approximate time to each other. And in one Indiana case, investigators believed there was an association or a link to two other cases with respect to the source of the carfentanil. All three deaths occurred proximate to each other in time with a belief that the carfentanil supplier was one of the decedents. Detecting carfentanil in blood is a challenge because of the low (sub nanogram/mL or picogram/mL) presenting concentrations of the drug. Axis Forensic Toxicology presumptively screens for carfentanil in the Comprehensive panel (order code 70510) and the Indiana State Department of Health panel (test code 70575); this is accomplished by high resolution mass spectrometry ( Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry) via the Analyte Assurance™ feature of the Comprehensive panel. Confirmation and quantitation of carfentanil is accomplished by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) in the Designer Opioids panel (order code 13810) with a carfentanil Lower Limit of Quantitation (LLOQ) of 10 pg/mL , or 0.010 ng/mL. Of note, carfentanil is not presumptively screened in the Drugs of Abuse panel (order code 70530), or in the customized Drugs of Abuse panel (Kentucky, order code 70555).
In summary, carfentanil exhibited a significant nationwide prevalence in postmortem cases in 2016-17; subsequent scheduling of the drug in the United States and a ban in China in 2017 resulted, in part, to a precipitous decline in positive postmortem cases. Axis Forensic Toxicology recognized a re-emergence of carfentanil in postmortem case work notably beginning the latter part of May 2023 and extending into August 2023 with a total of 9 detections from four states (IN, WI, KY and OH). Medico-legal death investigators, medical examiners, coroners and law enforcement agencies are urged to be alert to this observation. Death scene investigation, decedent social/drug history, autopsy and an appropriate scope of toxicology testing are of paramount importance in suspected drug-related cases. Drug intelligence trends in a given jurisdiction or region, as provided by local sources such as the Drug Enforcement Agency (DEA), law enforcement drug task forces together with information from state and federal drug testing laboratories are essential tools in identifying real time drug patterns in a specific location. Thus far in 2023, with the exception of 1 case from the total of 10, all of the carfentanil detections noted in the laboratory were punctuated by the parallel detections of IMF; likewise, methamphetamine and amphetamine was detected in 5 of the 7 cases reviewed as a subset to the ten case total observed thus far in 2023. Axis Forensic Toxicology will continue to monitor carfentanil case trends in the remainder of 2023.
The following individuals and jurisdictions are recognized for their contributions to this article by the provision of case investigative details, circumstances and decedent histories: Amy Lay, Medicolegal Death Investigator/Pathology Assistant, Lake County Coroner’s Office, IN; Steve Lockyear, Vanderburgh County, IN Coroner; Joe Hudson, Grayson County, KY Coroner; Josh Garvey, Iowa County, WI Deputy Coroner, and Dr. Robert F. Corliss, Professor and Autopsy Director-Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison.
 Centers for Disease Control Health Update, CDCHAN-00413, Rising numbers of deaths involving fentanyl and fentanyl analogs, including carfentanil, and increased usage and mixing with non-opioids. July 11, 2018
 Delaware General Health District, Delaware County, Ohio Health Alert, Rising numbers of deaths involving fentanyl and fentanyl analogs, including carfentanil, and increased usage and mixing with non-opioids. July 12, 2018
 Jalal, H, Burke, DS. Carfentanil and the rise and fall of overdose deaths in the United States. Addiction. 2021 Jun; 116(6):1593-1599
 Papsun, D, Isenschmid, D, Logan, BK. Observed carfentail concentrations in 355 blood specimens from forensic investigations. J Analytical Tox. 2017; 41:777-778
 Schueler, HE. Emerging synthetic fentanyl analogs. Acad Forensic Path. 2017 7(1):36-40
 Shanks, KG, Behonick, GS. Detection of carfentanil by LC-MS-MS and reports of associated fatalities in the USA. J Analytical Tox. 2017; 41:466-472
 Tiscione, NB, Alford, I. Carfentanil in impaired driving cases and the importance of drug seizure data. J Analytical Tox. 2018; 42:476-484
 Wilde, M, Pichini, S, Pacifici, R, Tagliabracci, A, Paolo Busardo, F, Auwarter, V, Solimini, R. Metabolic pathways and potencies of new fentanyl analogs. Frontiers in Pharmacology. 2019 10:1-16
By Kevin Shanks, M.S., D-ABFT-FT
Novel psychoactive substances (NPS) are compounds designed or consumed to mimic the effects of typical recreational substances such as diacetylmorphine (heroin), cocaine, methamphetamine, cannabis, or even prescription medications. As these NPS emerge and become prevalent, the United States Federal government can use its scheduling powers to effectively ban the substances as Schedule I controlled substances.
Schedule I controlled substances are defined as a substance that has a high potential for abuse and no currently accepted medical use in the United States.
Effective July 26, 2023, the United States Federal government controlled the following NPS as Schedule I controlled substances: etizolam, flualprazolam, clonazolam, flubromazolam, and diclazepam. Over the past few years, each of these drugs have been associated with or implicated in numerous cases of driving under the influence of drugs as well as toxicity and fatality.
Axis tests for clonazolam, etizolam, flualprazolam, and flubromazolam in the Novel Psychoactive Substance panel (order code 13610) as well as the Comprehensive Panel with Analyte AssuranceTM (order code 70510). Diclazepam (as metabolites delorazepam and lorazepam) is also included in order code 70510. If you have questions about these substances and how they may apply to your casework, please reach out to our subject matter experts by email ([email protected]) or phone (317-759-4869, Option 3).
Department of Justice, Drug Enforcement Administration, 21 CFR Part 1309, Docket No. DEA-989. Federal Register. Volume 88, No. 142. Schedules of Controlled Substances: Temporary Placement of Etizolam, Flualprazola, Clonazolam, Flubromazola, and Diclazepam in Schedule I. July 26, 2023.
By Kevin Shanks, M.S., D-ABFT-FT
As mentioned in the previous post, novel psychoactive substances (NPS) come in different flavors – and opioids, cannabinoids, stimulants, hallucinogens, and benzodiazepines are just a few of them. The Novel Emerging Compounds (NEC) Panel offered by Axis Forensic Toxicology helps to detect the most newly emerged NPS on the drug market and is meant to continually evolve over time as new substances emerge on the street.
In the first post in this series, we took a look at two of the more recently emerged NPS benzodiazepines, and in this second post, we are looking at two of the most recently detected stimulants: alpha-PiHP and N,N-dimethylpentylone.
Alpha-PiHP, also known as 4-methyl-1-phenyl-2-(pyrrolidin-1-yl)pentan-1-one or alpha-pyrrolidinoisohexanophenone, is a substituted cathinone that is a positional isomer of the prescription medication pyrovalerone, a norepinephrine-dopamine reuptake inhibiting drug that is used for the treatment of chronic fatigue and as an appetite suppressant. Alpha-PiHP was first reported as being a drug sold on the illicit drug market in 2016 by the National Forensic Laboratory of Slovenia when it was detected in an off-white colored powder. In the United States in 2018, the Center for Forensic Science Research and Education (CFSRE) first reported the detection of the substance in a white solid material via the Department of Homeland Security.
N,N-dimethylpentylone is also known as 1-(1,3-benzodioxol-5-yl)-2-(dimethylamino)pentan-1-one or dipentylone and is a substituted cathinone that is a positional isomer of a previously reported cathinone, N-ethylpentylone, a norepinephrine-dopamine-serotonin reuptake inhibitor. Pentylone is a common metabolite produced by the body via de-ethylation after consumption of N,N-dimethylpentylone. The drug was first identified in Sweden in 2014, but didn’t emerge in the United States until 2021-2022.
Similar to other classical stimulants such as methamphetamine and amphetamine, these two compounds act pharmacologically as central nervous system stimulants with activity involving the neurotransmitters serotonin, norepinephrine, and dopamine. While the human pharmacology of these substances hasn’t been readily tested, it is surmised that they act as either direct releasers of the neurotransmitters or as transporter inhibitors. Reported effects after use of substances such as these include increased alertness, increased energy, euphoria, feelings of well-being, restlessness, and hallucination. Other physiological effects are hyperthermia, tachycardia, hypertension, mydriasis, diaphoresis, dehydration, and hyponatremia.
While not explicitly listed as controlled substances in the United States, both of the substances may be considered positional isomers of already controlled drugs and therefore be considered “analogues”. According to the Drug Enforcement Administration’s (DEA) National Forensic Laboratory Laboratory Information System (NFLIS) 2022 Midyear Drug Report, N,N-dimethylpentylone was the third most common phenethylamine detected by the DEA – behind only methamphetamine and amphetamine. Alpha-PiHP was the sixth most commonly detected phenethylamine in 2022. Alpha-PiHP and N,N-dimethylpentylone have been implicated in human toxicity which have led to numerous hospitalizations and fatalities in the USA.
Axis qualitatively monitors both of these compounds in our NEC panel (order code 13710) and Comprehensive Panel, Blood with Analyte Assurance (order code 70510) using liquid chromatography with quadrupole time of flight mass spectrometry (LC-QToF-MS). Over the time range 01/30/2023 – 06/30/2023, Axis has detected alpha-PiHP in 11 blood specimens with all detections located in Florida and N,N-dimethylpentylone in 42 cases with 41 cases in Florida and 1 case in Indiana. The reason why these compounds have been predominately found in Florida is not known, but NPS and classical drug trends tend to be regional phenomena.
In Axis Forensic Toxicology casework, Alpha-PiHP was typically found alongside fentanyl/norfentanyl (n=6), 4-ANPP (n=5), N,N-dimethylpentylone (n=5), cocaine/benzoylecgonine (n=3), and naloxone (n=3). N,N-dimethylpentylone was simultaneously detected with fentanyl/norfentanyl (n=30), 4-ANPP (n=29), pentylone (n=21), cocaine/benzoylecgonine (n=18), and naloxone (n=14).
Axis also monitors other NPS stimulants in our Novel Psychoactive Substances panel (order code 13610). These additional compounds include alpha-PVP, butylone, dibutylone, dimethylone, eutylone, MDPV, mephedrone, methcathinone, methedrone, methylone, N-ethylpentylone, pentylone, and TFMPP. As always, if you have questions about these substances and how they may play a role in your medical-legal investigation, please reach out to our subject matter experts by email ([email protected]) or phone (317-759-4869, Option 3).
Stay tuned for the third post in the NEC Panel series! We will take a look at two more recently emerged novel compounds: phenibut and tianeptine.
By Kevin Shanks, M.S., D-ABFT-FT
Novel psychoactive substances (NPS) come in different varieties – opioids, cannabinoids, stimulants, hallucinogens, and benzodiazepines are just a few of the families on the illicit drug market. Axis developed the Novel Emerging Compounds (NEC) Panel to help detect the most newly emerged NPS on the drug market. The panel is meant to continually evolve in scope as new substances emerge on the street and then either continue to be prevalent or possibly disappear from the market.
This is intended to be a series of multiple posts briefly describing the compounds in the current iteration of the NEC Panel.
In the recent past, we have seen the emergence of NPS benzodiazepines such as etizolam, clonazolam, and flualprazolam. In this first summary of the NEC Panel, we are looking at two of the more recently emerged compounds are bromazolam and flubromazepam.
Bromazolam is a drug that has a history in pharmaceutical drug development as it was first synthesized in the 1970s as XLI-268, but it was never approved for medicinal use. The first emergence of bromazolam on the illicit drug market in the United States was in 2019, but it never became prevalent until more recently. The substance is the brominated analog of alprazolam, meaning it has a bromine atom in the place of the typical chlorine atom.
Flubromazepam is a drug that also has a history in pharmaceutical drug development. It was first synthesized in 1960, but it did not receive any further study as a medicine. It appeared on the illicit drug market in 2012, but didn’t gain any traction for several years. Flubromazepam is a fluorinated analog of phenazepam, a benzodiazepine used as a medicine in Russia, meaning it has a fluorine atom in the place of the typical chlorine atom.
Similar to other classical benzodiazepines such as alprazolam (Xanax), diazepam (Valium), or clonazepam (Klonopin), these compounds act pharmacologically as GABA (gamma-amino butyric acid) receptor agonists. They bind to the GABA receptor and initiate a change in chloride ion channels which leads to inhibition in the central nervous system. Common adverse effects reported by users of NPS benzodiazepines are consistent with those reported after using the classical benzodiazepines and include drowsiness, tiredness, sedation, loss of motor coordination, slurred speech, amnesia, and respiratory depression. Both bromazolam and flubromazepam are not considered controlled substances by the United States Federal government, but may be controlled at a state level as Schedule I controlled substances. Data from the Drug Enforcement Administration’s (DEA) National Forensic Laboratory Information System in 2022 showed that bromazolam was now in the top 15 reported tranquilizers and depressants in the United States. Both bromazolam and flubromazepam have been previously implicated in human intoxication cases involving driving motor vehicles as well as being involved in toxicity leading to fatality.
Axis qualitatively monitors both of these compounds in our NEC panel (order code 13710) and Comprehensive Panel, Blood with Analyte Assurance (order code 70510) using liquid chromatography with quadrupole time of flight mass spectrometry (LC-QToF-MS). Over the time range 01/30/2023 – 06/30/2023, Axis has detected flubromazepam in 3 blood specimens across 3 states (Indiana, Ohio, and South Dakota) and bromazolam in 72 blood specimens across 15 states (California, Colorado, Florida, Indiana, Iowa, Kansas, Kentucky, Louisiana, Michigan, Missouri, Nebraska, Ohio, Tennessee, Texas, and Wisconsin). Bromazolam was commonly detected alongside fentanyl/norfentanyl (n=59), 4-ANPP (n=46), acetylfentanyl (n=27), THC/THC-COOH (n=21), methamphetamine (n=20), and cocaine/benzoylecgonine (n=19). Flubromazepam was typically detected alongside gabapentin/pregabalin (n=3) and fentanyl (n=2).
Axis also monitors other NPS benzodiazepines in our Novel Psychoactive Substances panel (order code 13610). These additional compounds include adinazolam, clonazolam, etizolam, flualprazolam, and flubromazolam. As always, if you have questions about these substances and how they may apply to your toxicology casework or investigation, please reach out to our subject matter experts by email ([email protected]) or phone (317-759-4869, Option 3).
Stay tuned for the second post in the NEC Panel series – where we will take a look at two of the more recently emerged stimulant compounds: alpha-PiHP and N,N-dimethylpentylone.
Dear Valued Client,
In the spirit of continuous improvement, to provide the most relevant panels and tests in the industry, our products are periodically updated to provide new and relevant offerings. It is with that goal in mind that we announce an update to our 44050: Cannabinoids, Blood Order Code. This panel, and likewise the 70510: Comprehensive Panel with Analyte Assurance and 70530: Drugs of Abuse Panel, will include Delta-8 THC and 11-Hydroxy Delta-9 THC beginning with orders placed on or after July 17th, 2023. You can always find the most recent publication for our panel offerings on our Test Catalog, found at www.axisfortox.com.
The cannabis industry continues to grow and with it, new challenges arise as laws and regulations change surrounding it. One such challenge is the rise of different cannabinoid isomers being sold to try and circumvent these laws. Delta-8-THC, pictured below beside delta-9-THC, is one such isomer that has gained attention.
The claim of manufacturers and sellers is that the isomer exists in a legal gray area due to the 2018 Farm Bill. We are toxicologists, not lawyers or lawmakers, so we cannot give our opinion on whether this is correct or not. No matter what is decided, the compound is being widely sold and may be relevant to your casework and we can help with the toxicology of that.
Delta-8-THC is very similar to delta-9-THC which is considered the primary component in cannabis products. Its potency is less than that of delta-9-THC but has a similar psychoactive effect. The metabolism of each produces an 11-hydroxy-THC metabolite which is an active metabolite. This is further metabolized into a carboxylic acid metabolite, 11-nor-9-carboxy-THC.
The cannabinoids that occur naturally are not going to typically be contributing directly to cause of death. The presence of such substances may indicate that the individual was under the influence but toxicology testing alone cannot be used to determine if an individual was under the influence. Toxicology results must be paired with the scene investigation and witness statements to make this determination.
If you have any questions regarding these new analytes and application to your casework, please reach out to us via email at [email protected] or via phone at 317-759-4869 option 3.
By Stuart Kurtz, D-ABFT-FT
In April, I spoke at the Midwest Association of Toxicology and Therapeutic Drug Monitoring (MATT) annual conference in Columbus, OH. The title of the talk was “Detection of Xylazine in Postmortem Specimens With Fentanyl, Morphine, Methamphetamine, and Cocaine From 2021-2022.” The conference was a great opportunity to talk shop with peers from the Midwest region and hear what they are doing. In this post, I will summarize what was presented at the MATT conference.
Xylazine was first synthesized as an antihypertensive by Bayer in 1962. During clinical trials, it was found to have additional central nervous system (CNS) depression effects. Despite being investigated for use as an antihypertensive agent, some patients would initially experience hypertension before the desired effect of a reduction of blood pressure. Some patients also experienced much more severe hypotension than intended. Due to these findings, it was rejected for human medical use and only approved for veterinary purposes.
A survey done by Texas Poison Control looked at the reported effects of xylazine toxicity. The major reported adverse effects were drowsiness and lethargy (47%), bradycardia (20%), hypotension (11%), hypertension (9%), and slurred speech (8%). Analgesic effects have been reported but the mechanism is not well understood, and the effect is minimal compared to its other adverse effects. There are two theories behind the mechanism. One immunoassay study found that xylazine could weakly displace opioids at binding sites. In this study, naloxone was not seen to reverse the effects. The other theory is that xylazine stimulates the release of endogenous opioids which could be reversed by naloxone. These are theories that have been reported but further study is needed.
Published case reports involving xylazine typically describe the presence of at least one other additional substance in combination with xylazine which may lead to an additive effect. When xylazine is the primary substance of toxicological interest detected in casework, there is a competing cause of death or an intentional overdose as determined by the scene investigation. The blood concentration range in these cases was 2,300-11,000 ng/mL. In cases of intentional overdose, the decedents worked in the veterinary field and had access to xylazine. The case involving a xylazine blood concentration equal to 2,300 ng/mL was ruled suicide by hanging after injection of xylazine. One attempted suicide case had a serum concentration of 4,600 ng/mL with the individual making a recovery with medical intervention. Two other fatal cases involving xylazine had blood concentrations of xylazine (200 ng/mL)/nordiazepam (2,500 ng/mL)/ and xylazine (300 ng/mL)/flualprazolam (1,100 ng/mL). In driving under the influence of drugs (DUID) casework, a range of 5.1-450 ng/mL of xylazine in blood was found. Full case histories were not available so it is unknown if any of those individuals died from effects of xylazine with other drugs that may have been present. A case of 570 ng/mL in blood was reported from a driver who passed out in their car but could be awakened.
For the years 2021-2022, we had 442 total detections of xylazine. For reference, we receive about 22,000-24,000 cases per year. The following drugs were commonly detected alongside xylazine: fentanyl (92%), benzoylecgonine (29%), methamphetamine (26%), morphine (12%). In 26% of the cases, the combination of xylazine, fentanyl, and methamphetamine was observed, while in 4% of the cases, the combination of xylazine, fentanyl, methamphetamine, and benzoylecgonine was detected.
Because of the lack of data surrounding human clinical use of xylazine and the lack of reports of sole xylazine use, it is hard to determine what postmortem blood concentrations could be considered fatal. Xylazine is almost always found in combination with another drug due to its popular use as an adulterant. Its CNS depression effects can mimic other compounds such opioids and benzodiazepines and it is not scheduled as a controlled substance by the Drug Enforcement Administration (DEA) in the United States, so access is not very restricted. Because of its additive effects with other CNS depressants, it can still be relevant in postmortem toxicology, and it is included in our comprehensive panel of testing.
Wounds associated with xylazine use have been reported in Philadelphia, PA. We have not received any reports of wounds potentially associated with xylazine use. Also, in talking with other forensic toxicologists around the country, we have not heard from them any other reports of these wounds being present in their locations.
As always, please reach out to us if you have any questions about this post. We can be emailed at [email protected] or you can call us at 317-759-4869 option 3.
Sherri L Kacinko, Amanda L A Mohr, Barry K Logan, Edward J Barbieri, Xylazine: Pharmacology Review and Prevalence and Drug Combinations in Forensic Toxicology Casework, Journal of Analytical Toxicology, Volume 46, Issue 8, October 2022, Pages 911–917, https://doi.org/10.1093/jat/bkac049
Karla A. Moore, Mary G. Ripple, Saffia Sakinedzad, Barry Levine, David R. Fowler, Tissue Distribution of Xylazine in a Suicide by Hanging, Journal of Analytical Toxicology, Volume 27, Issue 2, March 2003, Pages 110–112, https://doi.org/10.1093/jat/27.2.110
Malayala SV, Papudesi BN, Bobb R, Wimbush A. Xylazine-Induced Skin Ulcers in a Person Who Injects Drugs in Philadelphia, Pennsylvania, USA. Cureus. 2022 Aug 19;14(8):e28160. doi: 10.7759/cureus.28160. PMID: 36148197; PMCID: PMC9482722.
Way Koon, Teoh & Muslim, Noor & Chang, Kah Haw & Abdullah, Ahmad Fahmi Lim. (2022). Abuse of Xylazine by Human and its Emerging Problems: A Review from Forensic Perspective. Malaysian Journal of Medicine and Health Sciences. 18. 190-201. 10.47836//mjmhs18.4.26.