In October, toxicologist Stuart Kurtz presented a poster at the annual National Association of Medical Examiners (NAME) meeting in Dallas, TX. The abstract is below.

A Case Report Involving the Detection of Five New Psychoactive Substances in Postmortem Analysis.  

Stuart A. K. Kurtz, MS (1), Billy Scott (2), George S. Behonick, Ph.D., F-ABFT (1), and Kevin G. Shanks (1), MS, D-ABFT-FT 

(1) Axis Forensic Toxicology, Indianapolis, IN, USA; (2) Clark County Coroner, Jeffersonville, IN, USA 

Scheduling of fentanyl analogs in recent years has created a shift in new synthetic opioids (NSO) that are being detected by drug and toxicology laboratories. While the detection of fentanyl analogs has decreased, other NSOs have risen to fill the space. The intention of these NSOs is to mimic the effects on the body of prescription medications and previously available illicit drugs. They are often drugs that were synthesized by pharmaceutical companies in the mid-1900s, but studies were halted leaving a gap in information as to how the drugs behave pharmokinetically and pharmacodynamically. The constant emergence of these compounds creates detection challenges for laboratories, medical examiners, and coroners. Flualprazolam, a designer benzodiazepine, has also emerged in recent years in the illicit drug market. This case report involves the detection of four NSOs (brorphine, fluorofentanyl, flunitazene, metonitazene) with three different class types (benzimidizol-2-one, fentanyl analog, nitazene analog) and a designer benzodiazepine (flualprazolam). 

Jugular blood was submitted for toxicological analysis. The screen utilizes an extraction followed by high resolution mass spectrometry via liquid chromatography quadrupole time of flight mass spectrometry (LC-QToF-MS). Novel psychoactive substance subclasses screened for include NSOs, designer benzodiazepines, synthetic cathinones (bath salts), and synthetic cannabinoids. Toxicological findings include methamphetamine (245 ng/mL), fentanyl (40.1 ng/mL), norfentanyl (3.3 ng/mL), and the qualitative presence of cotinine, quinine, 4-ANPP, brorphine, fluorofentanyl, flunitazene, and metonitazene. 

There are a few things I would like to highlight here. The first is the instrumentation that we use to identify compounds of interest in a sample. We use liquid chromatography paired with a quadrupole and time-of-flight mass spectrometer (LC-QToF-MS). This allows us to collect data in such a way that we can go back and reprocess the data to see if something is present in the sample. 

The second thing is the whack-a-mole game that is ongoing when it comes to identifying these NPS compounds. The lifecycle of an NPS in the drug supply is often determined by government scheduling of either the NPS itself or the materials that are used to synthesize it. They can show up abruptly and gradually begin to replace one or more compounds. An example of this is the emergence of flualprazolam and isotonitazene mixtures in 2019. The scheduling of isotonitzene led to the emergence of brorphine in that mixture in 2020. 

Thirdly, detection of NPS in post-mortem casework can have a lag time of weeks to months depending on the intelligence data available. Information that can greatly improve our ability to upgrade our testing to including compounds of interest is scene data. Testing the unknown substances at the scene is the best way to determine what to look for. The data from the LC-QToF-MS can be processed to look for compounds that were previously not monitored in our methods. However, this is best done when there is the identification of something specific in seized drugs in a jurisdiction but is even more precise with scene identification of a compound. 

Lastly, it is important to utilize identification techniques that are specific. These include LC-QToF-MS, liquid chromatography paired with triple quadrupole mass spectrometry, and gas chromatography paired with mass spectrometry. These techniques are significantly less prone to false positives and false negatives. The methods that use these techniques often go through rigorous validation to show what the limits are to prevent false positives and false negatives. Less specific techniques such as immunoassays, color tests, and test strips are prone to false positives and false negatives. These types of tests tend to rely on core structures and/or functional groups. The core structure of morphine is different from fentanyl so one of these non-specific tests that work for morphine may not be able to detect fentanyl. 

There were 5 different portions of powder collected at the scene. A plastic baggy and folded up receipt were described to contain a blackish/grayish substance. 3 additional folded up receipts were collected and described to contain white powders. Brorphine and flualprazolam are often seen together in seized drug material and sometimes known as “benzo dope.” Metonitazene and flunitazene were also identified with flualprazolam in our casework. We do not have any information on whether they were mixed with the flualprazolam and consumed. A limitation of toxicology testing is it does not tell you if something was consumed in the same mixture as something else. The wider investigation would have to determine if that was a possibility. The MOD and COD were determined to be accidental due to methamphetamine and fentanyl toxicity. The methamphetamine level was 245 ng/mL and the fentanyl level was 40.1 ng/mL. 

As always, please reach out to us with questions. We are happy to help guide toxicology testing and interpretation however we can. 

Keary CJ, Wang Y, Moran JR, Zayas LV, Stern TA. Toxicologic testing for opiates: understanding false-positive and false-negative test results. Prim Care Companion CNS Disord. 2012;14(4):PCC.12f01371. doi: 10.4088/PCC.12f01371. Epub 2012 Jul 26. PMID: 23251863; PMCID: PMC3505132. 

Marthe M Vandeputte, Alex J Krotulski, Donna M Papsun, Barry K Logan, Christophe P Stove, The Rise and Fall of Isotonitazene and Brorphine: Two Recent Stars in the Synthetic Opioid Firmament, Journal of Analytical Toxicology, Volume 46, Issue 2, March 2022, Pages 115-121, https://doi.org/10.1093/jat/bkab082 

Truver MT, Chronister CW, Kinsey AM, Hoyer JL, Goldberger BA. Toxicological Analysis of Fluorofentanyl Isomers in Postmortem Blood. J Anal Toxicol. 2022 Mar 11:bkac014. doi: 10.1093/jat/bkac014. Epub ahead of print. PMID: 35277721. 

The Center for Forensic Science Research & Education. 2022 Q2 NPS Opioids Trend Report. https://www.npsdiscovery.org/wp-content/uploads/2022/07/2022-Q2_NPS-Opioids_Trend-Report.pdf 

Blanckaert, P, Balcaen, M, Vanhee, C, et al. Analytical characterization of “etonitazepyne,” a new pyrrolidinyl-containing 2-benzylbenzimidazole opioid sold online. Drug Test Anal. 2021; 13( 9): 1627– 1634. https://doi.org/10.1002/dta.3113 

Sara E Walton, Alex J Krotulski, Barry K Logan, A Forward-Thinking Approach to Addressing the New Synthetic Opioid 2-Benzylbenzimidazole Nitazene Analogs by Liquid Chromatography–Tandem Quadrupole Mass Spectrometry (LC–QQQ-MS), Journal of Analytical Toxicology, Volume 46, Issue 3, April 2022, Pages 221–231, https://doi.org/10.1093/jat/bkab117 

If you would like a copy of the poster, please email [email protected].

Dr. George Behonick presented the following poster at the annual NAME meeting in Dallas, TX.  This is the first of several articles to share recent presentations by our toxicologists.

Postmortem Redistribution of Fentanyl as Evidenced by Central and Peripheral Blood Concentrations

George S. Behonick, Ph.D., F-ABFT (1), Michael H. Heninger, MD (2), Stuart Kurtz, MS (1), and Kevin G. Shanks (1), MS, D-ABFT-FT 

(1) Axis Forensic Toxicology, Indianapolis, IN, USA; (2) Fulton County Medical Examiner, Atlanta, Georgia, USA 

The most recent, complete calendar year overdose death rates compiled by the National Center for Health Statistics (NCHS) at the Centers for Disease Control and Prevention reveal 91,799 persons in the US succumbed to fatal drug intoxications in 2020. In this same year, 56,516 deaths were attributed to synthetic opioids other than methadone (primarily fentanyl); this represents 61.5% of the total overdose deaths reported in 2020. Licit pharmaceutical fentanyl abuse during the 1990s was demonstrated in a variety of activities (e.g. sucking, chewing or ingesting transdermal patches, drinking fentanyl brewed tea, inserting a transdermal patch into the rectum, or onto the scrotum, and heating and inhaling the contents of a patch). In 2013-14 heroin laced with fentanyl was being distributed and determined to be responsible for at least 700 deaths nationwide. Drug traffickers were adding either pharmaceutical grade or illicit fentanyl to heroin to increase potency of the product. Today in the US illicitly manufactured fentanyl dominates the landscape of abused drugs; it can be delivered as itself in powder form, be ad-hoc mixed into other drugs such as cocaine and methamphetamine, or be incorporated into counterfeit pills and tablets. The interpretation of fentanyl postmortem blood concentrations is paramount to establishing cause of death. A confounding factor to interpreting postmortem fentanyl blood concentrations is postmortem redistribution (PMR) of the drug. 

Herein we describe a case which demonstrates the significant challenges which arise from PMR of fentanyl. Our case depicts a stark contrast in postmortem blood fentanyl concentrations between central (310 ng/mL) and peripheral (17.6 ng/mL) autopsy collected specimens. The C:P (central blood to peripheral blood) concentration ratio is calculated to be 17.6. Second to illustrating the wide differential observed between the central and peripheral blood specimens in this case, we intend to highlight and briefly discuss the various factors which influence PMR of fentanyl to thereby provide insight to the interpretation of fentanyl postmortem blood concentrations by medical examiners and forensic pathologists. 

Post-mortem interpretation of toxicology results is often tricky due to post-mortem redistribution (PMR). The case that Dr. Behonick looked at is a great example of this and it’ll be used it to highlight some important factors to consider. Central and peripheral blood sources in the same case are not routinely tested and compared in our lab. When they are, we have an opportunity to see how the results compare. 

When it comes to PMR, there aren’t a lot of hard and fast rules that can be used to interpret the results in a black and white manner. In general, the more fat-like, also called lipophilic, a drug is, the more prone to PMR it is. The volume of distribution (Vd) of a drug can be used to estimate its ability to undergo PMR. A drug with a Vd of 3 L/kg or more is said to be more prone to PMR. As a body decomposes, it gradually acidifies which results in basic drugs, such as fentanyl, to ionize. When they ionize, they become more readily distributed into the fluids in the body. 

The history of use also plays a part. Tricyclic antidepressants are typically used over a long period of time and tend to sequester in tissue such as the liver. On death, the drugs in the liver and other organs can start to redistribute into blood around the heart, lungs, or any other blood source near them. The longer a drug is used, the longer it builds up in tissue, and the more available to redistribute upon death. 

The next factor that plays a big part in PMR is the post-mortem interval (PMI). This is the length of time that elapses from death until samples are taken. A longer PMI is usually associated with PMR. In the case Dr. Behonick examined, there was a PMI of approximately 24 hours for toxicology sampling and 48 hours for a full autopsy. There is almost always some sort of delay in sampling so having a rough idea of the PMI is always helpful for interpretation later on. The family initially declined the autopsy being done but later approved it. 

On a similar note, the interval from exposure to death can also affect concentrations. Blood taken from around an injection site in a case where death was rapid can have significantly increased concentrations compared to a site that is further away. For example, injection into a leg can lead to femoral blood concentrations being higher than expected when compared to femoral blood from the other leg or a subclavian draw. 

In this case, the central blood had a fentanyl concentration of 310 ng/mL and the peripheral blood had 17.6 ng/mL. It is likely that all the factors above played a part in the stark contrast of the two values. Fentanyl is a lipophilic drug that is prone to PMR, there was a PMI of about 24 hours for toxicology specimens taken, and the body was moved several times due to the initial denial of an autopsy. Norfentanyl, sildenafil, and levamisole were all detected in the central blood but not the peripheral blood. The confirmation levels for norfentanyl and sildenafil were close to the lower limit of quantitation for each compound. This would account for why they weren’t detected in the peripheral blood. Levamisole is reported qualitatively off the screen so it is likely that it is just above our screening cutoff in the central blood and below it in the peripheral blood. Interpreting the toxicology results in the context of the overall investigation is vital to ensuring all appropriate factors are considered. 

Baselt, RC. In: Disposition of Toxic Drugs and Chemicals in Man, 12th ed., Biomedical Publications, Seal Beach, California, 2020, p. 846 

Cook, DS, Braithwaite, RA, Hale, KA. Estimating antemortem drug concentrations from postmortem blood samples: the influence of postmortem redistribution. J Clin Pathol. 2000;53:282-85 

Dolinak, D. Drug concentrations and postmortem changes. In: Forensic toxicology: a physiologic perspective, Academic Forensic Pathology Incorporated, Calgary, Canada, 2013, pp. 278-86 

Kennedy, M. Interpreting postmortem drug analysis and redistribution in determining cause of death: a review. Path Lab Med Int. 2015;7:55-62 

Langille, RM.  S33 Aggressive resuscitation as a cause of post-mortem redistribution. 2006;30:157 

Olson, KN, Luckenbill, K, Thompson, J, Middleton, O, Geiselhart, R, Mills, KM, Kloss, J, Apple, FS. Postmortem redistribution of fentanyl in blood. Am J Clin Pathol. 2010;133:447-53 

Pelissier-Alicot, A-L, Gaulier, J-M, Champsaur, P, Marquet, P. Mechanisms underlying postmortem redistribution of drugs: a review. J Anal Toxicol. 2003; 27:533-44 

Watson, WA, McKinney, PE. #7 Necrokinetics: the practical aspects of interpreting postmortem drug concentrations. Clin Toxicol. 2001;39(3):213-14 

Yarema, MC, Becker, CE. Key concepts in postmortem drug redistribution. Clin Toxicol. 2005;43:235-41

If you would be interested in obtaining a copy of the poster, please email [email protected].

By Kevin Shanks, D-ABFT-FT

Over the last several years, the Drug Enforcement Administration (DEA) has moved to ban various newly emerged illicit opioids as Schedule I controlled substances. From 2015-2017, they controlled 19 fentanyl analogs and other opioids. In 2018, the DEA banned any substitutions to the fentanyl core chemical structure and classified them as “fentanyl-related substances”. 

Waves of Federal Legislation for Opioids 
DEA, 2015 – 2017

After this legislation in 2018, compounds that were chemically dissimilar from fentanyl and analogs began to emerge. The major family of non-fentanyl related compounds to emerge is known as the nitazenes, which are based on a benzimidazole chemical structure. This family of opioids was first synthesized in the 1950s in the pharmaceutical industry as potential analgesic and anesthetic medications. The first compound, also the most potent, is etonitazene. Other compounds in this family include butonitazene, flunitazene, isotonitazene, metonitazene, and N-pyrrolidinoetonitazene. Pharmacologically, these compounds are mu opioid receptor agonists, much like morphine, heroin, and fentanyl. In vitro data suggests that these compounds have analgesic potentcies similar to or greater than fentanyl, and because of this potency and potential for respiratory depression, they have never been investigated further or approved for use in medicine. 

Chemical Structure of Isotonitazene. 
Kevin G. Shanks (2022)

We screen for nitazene compounds by liquid chromatography with quadrupole time of flight mass spectrometry (LC-QToF-MS) and confirm their identity by liquid chromatography with triple quadrupole mass spectrometry (LC-MS/MS) test. Test specifics can be found in the Axis online catalog. 

From June 1, 2021 to May 1, 2022, we detected a total of four nitazene compounds (metonitazene, isotonitazene, flunitazene, and N-pyrrolidinoetonitazene) in 128 postmortem toxicology blood samples across eight states (Florida, Illiniois, Indiana, Michigan, Nebraska, Ohio, Texas, and Wisconsin). Fentanyl was most commonly found alongside nitazene compounds, but other substances included 4-ANPP, acetylfentanyl, naloxone, methamphetamine, THC, cocaine/benzoylecgeonine, and morphine.  

If you have any questions about these newly emerged nitazene compounds, please reach out to subject matter experts at Axis by email at [email protected]. 

References 

Axis Forensic Toxicology internal data for nitazene analysis. 06/01/2021 – 05/01/2022. 

By Kevin Shanks, D-ABFT-FT

Mitragyna speciosa is a tree or shrub that grows in southeast Asia, particularly Thailand and Malaysia. The plant is locally known as kratom or biak-biak. It exists in the Rubiaceae family of plants, which includes the genera Coffea or caffeine-containing plants, with the most-widely known species being Coffea arabica and Coffea canephora (coffee plants). In regions of Asia, the plant has been used by either chewing the leaves or brewing them into a liquid beverage such as a tea. The leaves can also be pulverized and fashioned into a powder and then smoked or consumed orally in a capsule. 

Mitragyna speciosa 
Image by Ahmad Fuad Morad (CC BY-SA 2.0)

Mitragyna contains the alkaloids, mitragynine and 7-hydroxymitragynine. Approximately 60% of the plant’s alkaloid content is mitragynine and 7-hydroxymitragynine makes up about 2% of the overall alkaloid content. In lower dosages, the alkaloids produce stimulant-type effects, but at larger dosages, both compounds function as mu opioid receptor agonists. Mitragynine is considered to be approximately 13 times more potent than morphine as an analgesic, but 7-hydroxymitragynine is considered to be approximately 4 times more potent than mitragynine. 7-hydroxymitragynine is also a product of mitragynine biotransformation in the human body, thus mitragynine can be considered a prodrug for 7-hydroxymitragine. The alkaloids have also been shown to have other effects such as the blocking of serotonergic receptors and inhibition of CYP1A2, CYP2D6, and CYP3A4 enzymes.  

Chemical structures of Mitragynine and 7-hydroxymitragynine 
Structure drawn by Kevin G. Shanks (2022)

An interesting pharmacological characteristic of mitragynine and 7-hydroxymitragynine is that when binding to opioid receptors, they exhibit biased agonism. Normally, when an opioid binds to an opioid receptor, the β-arrestin pathway is initiated – the β-arrestin pathway is responsible for most of the respiratory depression and sedation observed in opioid use and overdose. There exists evidence that shows mitragynine and 7-hydroxymitragynine do not initiate this pathway. 

The United States Federal government moved to control mitragynine and 7-hydroxymitragynine as Schedule I controlled substances in 2016-2018, but backed off the legislation after public comment on the matter. They remain uncontrolled at the Federal level, but some states have passed legislation making them controlled substances in their locale.  

Most forensic toxicology laboratories include only mitragynine in the scope of their testing and do not include the 7-hydroxymitragynine alkaloid/metabolite. Typical detection limits for the compound are 5-20 ng/mL in blood. At Axis Forensic Toxicology, mitragynine is included in the Comprehensive Panel (order code 70510) and/or as a directed confirmation test for mitragynine (order code 42090). Specific information about our testing can be found in the online test catalog. 

Experts at Axis, alongside the Coconino County (Arizona) Medical Examiner’s Office, recently published a manuscript in the Journal of Analytical Toxicology titled “Two Single-Drug Fatal Intoxications by Mitragynine”. There have been many mitragynine-associated or related intoxications and fatalities reported over the last several years, but most have involved multiple drugs including other central nervous system depressants such as opioids, benzodiazepines, and ethanol. Sole intoxications with mitragynine leading to fatality are rare. In the published article, an analytical method for the detection of mitragynine by liquid chromatography with triple quadrupole mass spectrometry (LC-MS/MS) is detailed as well as presentation of two cases where mitragynine was certified as the single agent in the cause of death of an individual. To request a copy of this new manuscript, please contact  us at [email protected].   

References 

Opioids. Principles of Forensic Toxicology. Fourth Edition. Barry Levine. AACC, Inc. Pages 271-291. (2017). 

Mitragynine. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages 1414-1415. (2020). 

“Two Single-Drug Fatal Intoxications by Mitragynine” (2022) G.S. Behonick, C. Vu, L. Czarnecki, M. El-Ters, K. Shanks. J Anal Tox, DOI: https://doi.org/10/1093/jat/bkac016 

By Kevin Shanks, D-ABFT-FT 

The presence of fentanyl in the street drug supply has rapidly exploded throughout the United States since approximately 2014. Drug overdose deaths have increased as well over the last several years and topped 100,000 deaths in the USA in 2021, with the major driving factor being fentanyl. 

Fentanyl Trends. DEA Annual Report, 2020. NFLIS.

We discussed fentanyl in a previous blog post, but briefly, fentanyl is a mu opioid receptor agonist and is metabolized in the human body by the cytochrome P450 enzyme system, primarily CYP3A4, into various products. It can be dealkylated, hydroxylated, methylated, and hydrolyzed. 

In the modern forensic toxicology laboratory, we monitor for the presence of unchanged fentanyl, alongside its primary metabolite, norfentanyl, in blood and urine. But, over the last several years, laboratories have added a third substance to their scope of analysis for fentanyl – 4-ANPP. 

Chemical structure of 4-ANPP 
Drawn by Kevin G. Shanks (2022)

4-ANPP, also known as N-phenyl-1-(2-phenylethyl)-4-piperidinamine or despropionyl fentanyl, is formed via amide hydrolysis. It is a minor metabolite of fentanyl, but it is also a precursor or starting material used in the synthesis of illicitly manufactured fentanyl and various related fentanyl analogs. 4-ANPP is reacted with propionyl anhydride to form fentanyl or some other reagent to form a related fentanyl analog such as acetylfentanyl (acetic anhydride) or cyclopropylfentanyl (cyclopropane carbonyl chloride). 

Pharmacologically, 4-ANPP is inactive – it does not produce any specific effect on the body. Ultimately, its presence is merely a marker for fentanyl use or exposure. Detection of this substance in the body is highly dependent on the dose and purity of the product consumed by the individual. The toxicology alone cannot determine if 4-ANPP is present due to metabolism or if it was ingested by using an impure illicit product. 

Axis Forensic Toxicology tests for the presence of 4-ANPP in our Designer Opioids panel (order code 13810), which is completed by liquid chromatography with triple quadrupole mass spectrometry (LC-MS/MS). The reporting limit is 50 pg/mL and the substance is reported as qualitatively positive or negative. 

If you have any questions regarding the presence or absence of 4-ANPP or its role in your toxicology casework, please reach out to Axis’ subject matter experts at [email protected]. 

References 

Fentanyl. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages 844-847. (2020).  

Opioids. Principles of Forensic Toxicology. Fourth Edition. Barry Levine. AACC, Inc. Pages 271-291 (2017). 

2020 Annual Drug Report. National Forensic Laboratory Information System (NFLIS). Drug Enforcement Administration. Springfield, VA. NFLIS-Drug 2020 Annual Report (usdoj.gov). (Accessed March 20, 2022). 

Labroo, R.B., Paine, M.F., Thummel, K.E., Kharasch, E.D. (1997) Fentanyl Metabolism by Human Hepatic and Intestinal Cytochrome P450 3A4: Implications for Interindividual Variability in Disposition, Efficacy, and Drug Interactions. Drug Metabolism and Disposition, 25: 9. 1072-1080. 

Drug Primer: Fentanyl (2021). Axis Forensic Toxicology Blog. Drug Primer: Fentanyl – Axis Forensic Toxicology (axisfortox.com). 

by Kevin Shanks, M.S., D-ABFT-FT

Marijuana, also known as cannabis, is a genus of annual flowering plants in the family Cannabaceae, and consists of the species, Cannabis sativa and Cannabis indica. The plants are native to Eastern Asia, but are cultivated all over the world. Cannabis is considered a Schedule I controlled substance by the United States Federal government, but it is legalized for medical use in 36 states and the District of Columbia (DC) and for recreational use in 18 states and DC. 

Marijuana plant. 
‘Legal Colorado Marijuana Grow” by Brett Levin Photography is licensed under CC BY 2.0.

The plant contains more than 500 different compounds. At least 113 of the compounds are classified as cannabinoids. The major cannabinoid is Delta-9-tetrahydrocannibinol (THC), but others include tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), and tetrahydrocannabinolic acid (THCA). Other compounds in the plant include terpenes, such as alpha-pinene, limonene, linalool, and myrcene. 

Chemical Structure of Delta-9-THC 
Structure drawn by Kevin G. Shanks (2021)

THC binds to the endocannabinoid system in the body. Cannabinoid receptor 1 (CB1) is primarily located in the brain and spinal cord, while cannabinoid receptor 2 (CB2) is found in the gastrointestinal system, the immune system, and the peripheral nervous system. THC binds to these receptors and acts as a partial agonist, which means it activates them, but only has partial ability to produce a maximal response. 

THC’s effects will vary according to the dose consumed, the potency of the substance, the route of administration, and the individual’s use history with the substance. When inhaled via smoking the plant or vaping THC oil, effects typically occur within minutes and last for a few hours. If taken orally via an edible such as THC-infused gummy candy and cookies, effects normally take 30-90 minutes to occur and last for 4-6 hours. Effects on the body include drowsiness, relaxation, relaxed inhibitions, altered time, altered perception, impaired learning and memory, difficulty in concentration and thought formation, and euphoria. Physiological effects include dry mouth, increased appetite, red eyes, and tachycardia. 

Cannabis Edibles. 
“THC-Infused Gummies” by THCProductPhotos is licensed under CC BY-ND 2.0

THC is metabolized in the liver primarily by the CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes to hundreds of detectable metabolites, with the main metabolites being the pharmacologically active 11-hydroxy-THC (11-OH-THC) and the pharmacologically inactive 11-nor-9-carboxy-THC (THC-COOH). THC-COOH is further conjugated with glucuronic acid and then excreted out of the body via the feces and urine.  

Detection windows for the metabolite can be quite extensive and will vary according to the dose used, duration of use, and the individual using the substance. Frequent users of THC could have detectable levels of THC-COOH in their urine for up to 30 days or longer after cessation of use. Most infrequent users eliminate the metabolite within a few days up to a week after use. 

 A forensic toxicology lab can test for THC using many different types of instrument platforms. The initial screening test can be an immunoassay test, but can also be completed via gas chromatography with mass spectrometry (GC-MS), liquid chromatography with high resolution mass spectrometry (LC-QToF-MS) or liquid chromatography with triple quadrupole mass spectrometry (LC-MS/MS). Confirmatory testing is usually completed by either GC-MS or LC-MS/MS. In blood, both parent THC and, at minimum, the THC-COOH metabolite is monitored. Normal reporting limits for blood testing are 0.5-1 ng/mL for THC and 1-10 ng/mL for THC-COOH. In urine, labs typically only monitor THC-COOH with reporting limits for positive determination varying widely (5-300 ng/mL). The current scope of testing and screening and confirmation reporting limits offered by Axis Forensic Toxicology can be found in the online test catalog.  

References 

• Tetrahydrocannabinol. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages 2041-2045. (2020).  
• Cannabis. Principles of Forensic Toxicology. Fifth Edition. Marilyn A. Huestis, Barry S. Levine, Sarah Kerrigan. Springer Nature Switzerland AG. Pages 389-448 (2020). 

by Kevin Shanks, M.S., D-ABFT-FT

Note: This is an excerpt of a presentation given to the LTG (London Toxicology Group) in June 2021. 

Packages of SCRA products 
Photograph by Kevin G. Shanks (2015)

Synthetic cannabinoid receptor agonists (SCRA) are laboratory synthesized chemicals which bind to cannabinoid receptors in the human body. There are thousands of these compounds in existence and there is no way to discuss all of them, but since 2009, many of these compounds have been sold as ingredients in smoking blends and herbal incense/potpourri in the United States. Over the last several years, the government has enacted various pieces of legislation controlling these compounds as Schedule I controlled substances and, currently, there are 43 SCRA considered to be controlled substances. The prevalence of specific synthetic cannabinoids vary over time and new compounds routinely emerge and disappear. 

Chemical structures of 5 Common SCRA
Structures drawn by Kevin G. Shanks (2021)

According to data from the Center for Substance Abuse Research (CESAR) via quarterly Emerging Threats Reports (ETR), the prevalence of 5F-ADB and FUB-AMB have waned over the last couple of years, and the most prevalent compounds reported in 2020 were 5F-MDMB-PICA, followed by the MDMB-4en-PINACA and 4F-MDMB-BINACA. If you look at data from the National Forensic Laboratory Information System (NFLIS), compiled by the Drug Enforcement Administration (DEA) via non-biological evidence seizure, for 2020, the most common SCRAs were 5F-MDMB-PICA, MDMB-4en-PINACA, and 4F-MDMB-BINACA. 

Detection of SCRA in Blood at Axis, 2018
Data compiled by Kevin G. Shanks (2021)

Detection of SCRA in Blood at Axis, 2019
Data compiled by Kevin G. Shanks (2021)

Detection of SCRA in Blood at Axis, 2020
Data compiled by Kevin G. Shanks (2021)

If we look at Axis Forensic Toxicology from the last few years, we can see that our data for postmortem toxicology mirrors what is reported by ETR and NFLIS. In 2018, 5F-ADB was the most prevalent SCRA (80.4% positivity in all sample tested for SCRA), while in 2019, 4F-MDMB-BINACA was the most prevalent compound (42.3% positivity). During 2020, the most prevalent compound reported in postmortem toxicology casework for synthetic cannabinoids testing was 5F-MDMB-PICA (60.0% positivity). During 2020, three compounds (5F-MDMB-PICA, 4F-MDMB-BINACA, and 5-ADB) accounted for 90+% of positive synthetic cannabinoid detections in blood samples tested.  

Chemical structures of 3 Emerging SCRA
Structures drawn by Kevin G. Shanks (2021)

Recognizing this ever changing scope of testing, we must always look towards the future and we need to be aware of newly reported SCRA compounds. Some of these compounds include 4F-MDMB-BICA, ADB-BINACA, and ADB-HEXINACA. What more should we expect in the future? We should expect more of the same. The beat goes on and new compounds emerge. They become prevalent. They disappear. And then new compounds take the place of the old ones. 

The Puzzle Pieces of an SCRA
Image drawn by Kevin G. Shanks (2021)

Axis Forensic Toxicology continues to adapt SCRA testing to provide a relevant scope of analysis and aid in the medical-legal investigation of death and poisoning. For more information, the current scope of testing offered by Axis Forensic Toxicology can be found in the online test catalog (Order Code 42130, Synthetic Cannabinoid Panel, Blood).  

If you would like a full copy of this presentation, please contact [email protected]. 

References 

• Synthetic Cannabinoid Receptor Agonists (2020 – 2021). K. Shanks. London Toxicology Group (LTG) Virtual Meeting. (2021). 
• Synthetic Cannabinoids. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages 1979-1986. (2020).  
• Synthetic Cannabinoid Receptor Agonists. Novel Psychoactive Substances: Classification, Pharmacology, and Toxicology. Paul Dargan and David Wood. Academic Press (Elsevier). 317-338. (2013). 
• Axis Forensic Toxicology. Laboratory Data. Indianapolis, IN. (accessed July 2021). 

by Kevin Shanks, M.S., D-ABFT-FT

Synthetic cannabinoids are laboratory synthesized chemicals which bind to cannabinoid receptors in the human body. The JWH series of cannabinoids was developed by Dr. John Huffman at Clemson University. UR-144 is an Abbott Labs compound which was developed during investigational drug research. ADB-FUBINACA is a synthetic cannabinoid which was synthesized by Pfizer. Numerous synthetic cannabinoids, such as 4F-MDMB-BINACA and 5F-MDMB-PICA, exist, which have no formal history in academic or pharmaceutical industry research.

In the USA, since 2008-2009, synthetic cannabinoids have been sold as ingredients in herbal incense, potpourri, or smoking blends. Throughout the last decade, various waves of legislation have been passed by the Federal government classifying several synthetic cannabinoids as Schedule I controlled substances. There are currently 43 synthetic cannabinoid substances that are controlled at the Federal level in the USA. These substances have no recognized medicinal application. Each state has its own controlled substance laws and may vary according to their regional drug trends.

Chemical structures of 3 Synthetic Cannabinoids
Structure drawn by Kevin G. Shanks (2021)

Synthetic cannabinoids are cannabinoid receptor agonists. The cannabinoid receptor 1 (CB1) is found primarily in the central nervous system and is responsible for mediating the psychoactive effects of cannabis and related-substances. Cannabinoid receptor 2 (CB2) is located in the peripheral nervous system, the spleen, and the immune system, and is thought to be involved in pain perception mediation and immunosuppression. Unlike delta-9-tetahydrocannabinol (THC), which is a partial agonist of CB1 and CB2 receptors, the majority of synthetic cannabinoids are considered to be full agonists of the receptors.

Pharmacokinetics such as blood elimination half-life and volumes of distribution are not known for these compounds. Most synthetic cannabinoids are biotransformed to hydroxylated or carboxylic acid metabolites. Reported effects of synthetic cannabinoid use or exposure include poor coordination, sedation, slurred speech, nausea, vomiting, tachycardia, hypertension, hyperthermia, agitation, delusions, paranoia, hallucinations, psychosis, and acute kidney injury. Fatalities have occurred after the use of these substances.

Detection of Synthetic Cannabinoids in Blood at Axis Forensic Toxicology, 2020.
Data Compiled by Kevin G. Shanks (2020).

As the prevalence of specific synthetic cannabinoids vary over time, the modern forensic toxicology laboratory should have a relevant scope of analysis. Typically only parent drugs are monitored in blood specimens, but some compounds require a metabolite be monitored in lieu of the parent drug. Detection limits for both parent drug and metabolite vary in biological matrices, but are generally 0.1 – 2 ng/mL. During 2020, three compounds (5F-MDMB-PICA, 4F-MDMB-BINACA, and 5-ADB) accounted for 90+% of positive synthetic cannabinoid detections in blood samples tested.

There current scope of testing offered by Axis Forensic Toxicology can be found in the online test catalog (Order Code 42130, Synthetic Cannabinoid Panel, Blood), and these compounds are detected by the Comprehensive Panel with Analyte Assurance™ (70510).

Axis Forensic Toxicology prides itself on its expertise in the field of novel psychoactive substances. Here are citations for papers published by Axis on the topic of synthetic cannabinoids:

• “Three Cases of Fatal Acrylfentanyl Toxicity in the United States and a Review of the Literature”, D.C. Butler, K. Shanks, G. Behonick, D. Smith, S.E. Presnell, L.M. Tormos. J Anal Tox. 42, e6-e11 (2018).
• “Synthetic Cannabinoid Product Surveillance by LC/ToF in 2013-2015”. K. Shanks, G. Behonick. J Forensic Toxicol Pharmacol, 4:3 (2016)
• “Death After Use of the Synthetic Cannabinoid 5F-AMB”. K. Shanks, G. Behonick. For Sci Int, 262, e21-e24 (2016)
• “Death Associated with the Use of the Synthetic Cannabinoid ADB-FUBINACA”. K. Shanks, W. Clark, G. Behonick. J Anal Tox, 40:3, 24-242 (2016)
• “Case Reports of Synthetic Cannabinoid XLR-11 Associated Fatalities”. K. Shanks, D. Winston, J. Heidingsfelder, G. Behonick. For Sci Int, 252, e6-e9 (2015)
• “Four Postmortem Case Reports with Quantitative Detection of the Synthetic Cannabinoid, 5F-PB-22”. G. Behonick, K. Shanks, D. Firchau, G. Mathur, C. Lynch, M. Nashelsky, D. Jaskierny, C. Meroueh. J Anal Tox, 38, 559-562 (2014)
• “Identification of Novel Third Generation Synthetic Cannabinoids in Products by Ultra Performance Liquid Chromatography and Time of Flight Mass Spectrometry”. K. Shanks, T. Dahn, G. Behonick, A. Terrell. J Anal Tox, 37: 517-525 (2013)
• “Detection of Synthetic Cannabinoids and Synthetic Stimulants in First and Second Generation Legal Highs by Ultra Performance Liquid Chromatography with Time of Flight Mass Spectrometry (UPLC/ToF)”. K. Shanks, T. Dahn, G. Behonick, A. Terrell. J Anal Tox, 36: 360-371 (2012)
• “Detection of JWH-018 and JWH-073 by UPLC/MS/MS in postmortem whole blood casework”. K. Shanks, T. Dahn, A. Terrell. J Anal Tox, 36: 145-152 (2012)

If you would like a copy of these papers, please contact [email protected].

References

• Synthetic Cannabinoids. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages 1979-1986. (2020).
• Tetrahydrocannabinol. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages 2041-2045. (2020).
• Synthetic Cannabinoid Receptor Agonists. Novel Psychoactive Substances: Classification, Pharmacology, and Toxicology. Paul Dargan and David Wood. Academic Press (Elsevier). 317-338. 2013).
• Axis Forensic Toxicology. Laboratory Data. Indianapolis, IN. (accessed April 23, 2021).