By Stuart Kurtz, M.S. D-ABFT-FT

As discussed in the previous blog post, vitreous humor has many caveats when interpreting its results in the context of postmortem toxicology. For those considerations and some background on vitreous humor, check out that post first (https://axisfortox.com/vitreous-chemistries-pt1). 

Vitreous humor can be used in instances where a drug or dug metabolite is quickly eliminated from the blood and it is known to cross into the vitreous and be detectable. One such metabolite is 6-acetylmorphine (6-AM) which is a marker for heroin exposure. Heroin is not typically looked for in blood samples because of its extremely short half-life at around 5-10 minutes. It metabolizes into 6-AM, which has a longer half-life at around 30 minutes, but it still may not be detectable in blood. Once 6-AM metabolizes into morphine, it is impossible to determine pharmaceutical morphine vs. heroin as the source. Urine would be the best matrix to look for the presence of 6-AM but vitreous humor is also a good option if urine isn’t available. 

Electrolytes testing is one of the most common uses for vitreous humor. This usually includes sodium, potassium, chloride, glucose, urea nitrogen, and creatinine. Sodium and chloride concentrations in vitreous will approximate serum concentrations. Potassium concentrations will rapidly increase postmortem. Urea nitrogen and creatinine are similar to sodium and chloride in terms of correlation with serum concentrations and can be used as indicators for dehydration. Glucose concentrations in vitreous are more reliable than blood glucose concentrations prior to death. However, glucose vitreous concentrations dissipate rapidly in the postmortem interval. Therefore, although a diagnosis of hyperglycemia may be made from elevated vitreous glucose concentrations, a diagnosis of hypoglycemia cannot be made because of the time rate disappearance of glucose from the matrix. Postmortem blood glucose concentrations are considered unreliable and vitreous should be used when possible. 

The significance of vitreous glucose is in helping to determine if diabetes played a part in cause of death. Elevated vitreous glucose is a good indicator that someone was experiencing a diabetic event. This can often be corroborated with acetone presence in the blood and/or vitreous humor. Additional testing for beta-hydroxybutyrate (BHB) can be done in blood if a diabetic event is suspected. Diabetes tends to not have any signs at autopsy so blood and vitreous testing can be helpful. BHB is the primary ketone that is produced during ketoacidosis. Diabetic ketoacidosis is typically associated with elevated glucose in vitreous humor. Medical history can also be very important if vitreous volume is limited or unobtainable. 

The presence of acetone and normal glucose can be an indicator of some other form of ketoacidosis. Alcoholic ketoacidosis may occur in cases of binge drinking alcoholic beverages. As is typical with chronic alcoholism, the person will have evidence of liver disease. The underlying cause is malnourishment which can also occur independently of alcoholism. Malnourishment may also be associated with dehydration which would be indicated by elevated urea nitrogen, sodium, and chloride concentrations. Ultimately, diabetic vs. alcoholic vs. malnourishment ketoacidosis requires additional information beyond just the toxicology testing. 

Medical history and an autopsy are very important for contextualizing the results we can provide for you in these cases. If your case potentially involves dehydration, it may be prudent to check the vitreous for sodium, potassium, or chloride, whereas if your case involves potential issues with diabetes or diabetic ketoacidosis, it would be necessary to order testing for vitreous chemistries to include glucose.  If kidney function is a potential area of concern, evaluation of urea nitrogen and creatinine would be important. Again, as always in forensic toxicology, case circumstances and underlying pathology will aid in determining the best course of action when considering vitreous humor chemistry and electrolytes testing. 

Please reach out to us if you have any questions regarding this topic or want help with deciding what testing may be necessary. Our email is [email protected] and our phone number is 317-759-4869 option 3. 

References 

Guidelines for the Interpretation of Analytical Toxicology Results. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages xxx-xlii. (2020).  

Introduction to Forensic Toxicology. Clarke’s Analytical Forensic Toxicology. Sue Jickells and Adam Negrusz. Pharmaceutical Press. Pages 1-12. (2008).  

Postmortem Toxicology. Clarke’s Analytical Forensic Toxicology. Sue Jickells and Adam Negrusz. Pharmaceutical Press. Pages 191-218. (2008).  

Postmortem Forensic Toxicology. Principles of Forensic Toxicology. Fourth Edition. Barry Levine. AACC, Inc. Pages 3-14. (2017). 

By Stuart Kurtz, M.S. D-ABFT-FT

The purpose of these next two blog posts is to discuss the utility that vitreous humor provides in the context of a case. This first post will focus on what vitreous humor is and the caveats associated with its interpretation. The second post will discuss some scenarios in which it can be very useful. 

Vitreous humor is the fluid found in the eyes between the lens and retina. There is approximately 2 mL of fluid in each eye meaning the volume is limited for testing. Because of this, we don’t recommend the use of vitreous humor as the primary specimen, and we do recommend careful consideration before attempting testing on it. The context of the case is always important and sometimes that is the most useful sample to test. Therefore, from a whole case investigation approach, we recommend always collecting vitreous humor, alongside blood and/or urine, at autopsy – if possible. Collection should be done in a tube without sodium fluoride or potassium oxalate as a preservative. The clear top tubes that are provided in our toxicology kits are well suited for vitreous collection, having no preservatives.

The primary components of vitreous humor are a connective tissue gel and transparent liquid that is primarily water. Its composition and location mean that it is quite stable and resistant to putrefactive changes postmortem. Movement of substances in and out of the vitreous happens by diffusion, osmotic pressure, convection, or active transport. Low molecular weight substances, such as ethanol, acetone, methanol and isopropanol, will travel in and out via diffusion. Most drugs that are important in postmortem testing are a much higher molecular weight and will travel in and out via convection. 

Ultimately, the mechanisms for most substances entering the vitreous are mostly unknown and/or not well studied. Drugs such as fentanyl, methamphetamine, and cocaine might be found in vitreous humor but the ability to correlate to a blood concentration is extremely limited and not recommended. Most drugs are very fat soluble and the vitreous humor is mostly water. So just like with oil and water, drugs will not be easily dissolved in the small volume present. 

Whereas with urine we expect that the majority of drugs will be found there in one form at some point post-exposure, that may not be the case with vitreous humor. There may be some drugs that are so poor in their ability to enter the vitreous humor that they would be undetectable. It can have some ability to show recent exposure, similar to urine, of a drug, but the absence of a drug should be interpreted with caution. 

Our next post will discuss vitreous humor’s important use in forensic toxicology. As always, we are here to assist you. Please reach out to us if you have any questions regarding this topic or want help with deciding what testing may be necessary. Our email is [email protected] and our phone number is 317-759-4869 option 3. 

References 

Guidelines for the Interpretation of Analytical Toxicology Results. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages xxx-xlii. (2020).  

Introduction to Forensic Toxicology. Clarke’s Analytical Forensic Toxicology. Sue Jickells and Adam Negrusz. Pharmaceutical Press. Pages 1-12. (2008).  

Postmortem Toxicology. Clarke’s Analytical Forensic Toxicology. Sue Jickells and Adam Negrusz. Pharmaceutical Press. Pages 191-218. (2008).  

Postmortem Forensic Toxicology. Principles of Forensic Toxicology. Fourth Edition. Barry Levine. AACC, Inc. Pages 3-14. (2017). 

The purpose of this blog post is to take a look at some of our data regarding nitazene detections. For some background on nitazene compounds, take a look at our blog post of June 9, 2022 titled Drug Primer: Nitazenes. As a reminder, nitazenes are a structurally distinct class of opioids so non-specific tests such as color strips and immunoassays will not detect them. 

For the SOFT 2022 meeting in Cleveland, Stuart Kurtz presented a poster titled Emergence of the Nitazene Class of Novel Synthetic Opioids in Postmortem Toxicology and Detection by LC-QToF-MS that looked at areas of detection as well as compounds found with nitazenes. Since that presentation, we examined the data through Q4 in 2022 to see what trends, if any, we could see in our casework. A big caveat is that the number of detections is a product of the testing being ordered. Nitazene screening is done as part of the comprehensive panel and not every case has comprehensive testing ordered. 

One of the main takeaways from the poster was that only 11 out of the 128 cases examined had a nitazene present with no detection of fentanyl, morphine, methamphetamine, or cocaine. 83% of the cases had fentanyl detected with a nitazene. This is consistent with the trend of polysubstance overdose deaths rising and fentanyl being the dominate opioid detected. 

Metonitazene was most commonly detected (n=102) with a big drop off to isotonitazene (n=22). Flualprazolam was the most common non-opioid NPS detected. Detection of opioids and NPS benzodiazepines has been an increasing trend in seized materials. Non-NPS detected included diphenhydramine (n=23) and gabapentin (n=16) which are suspected as cutting agents. It’s impossible to tell from postmortem toxicology work if a substance was cut with something but seized material data suggests that these are common cutting agents. Xylazine (n=7) has emerged as a common cutting agent and is gaining popularity in media. We will have more on xylazine in a future post. It is important to note that the presence of diphenhydramine or gabapentin does not necessarily mean they are a cutting agent since they are available over-the-counter and with a prescription respectively. Medical and prescription history is important in helping to determine where they may have come from. 

The data from the SOFT poster was from June 2021 through May 2022. We went back and looked at the data through the end of 2022 to see if there were any trends regarding detection. We didn’t look at other drugs detected as we feel the sampling for the SOFT poster represents general trends well. We did see that the detections of nitazenes peaked in Q1 of 2022 and has dropped off through the end of 2022. 

We updated the heat map regarding number of detections in each state. Keep in mind that Axis does not necessarily service the entirety of a state so actual numbers may be higher than what is shown. Indiana (n=130) is the leader in detections from our data. While the compounds that we test for may not be detected as often, that doesn’t mean that nitazenes as a class have completely disappeared. 

The NPS landscape is always changing and requires us to adapt our testing scope. The best information we can have to help inform testing is testing unknown powders at the scene. A powder at a scene doesn’t mean that the person was exposed to it but it can be a big help in knowing what to look for and guiding you on future testing. The next best piece of information is knowing what is being found in seized materials in the region. Again, this doesn’t guarantee that a person was exposed to the NPS but helps make us aware of what we may need to test for. 

As always, please feel free to reach out to us with any questions either by phone at 317-759-4869 option 3 or by email at [email protected]. 

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].