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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.