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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 

This post has been updated since its original posting Apr 7, 2022.

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. There is consensus among pathologists and medical examiners to not include 4-ANPP in cause of death because of its pharmacological inactivity.

As of January 22, 2024, Axis Forensic Toxicology screens for the presence of 4-ANPP in our Comprehensive Panel with Analyte Assurance™ and reflexively confirms it with our Fentanyl & Metabolites Panel (order code 40410), which is completed by liquid chromatography with triple quadrupole mass spectrometry (LC-MS/MS). The reporting limit is 0.1 ng/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).