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

To quantify or not? That is the question. If you regularly read forensic toxicology reports, then you know laboratory tests can be qualitative or quantitative. Qualitative tests only provide a positive/present or negative result.  Quantitative tests are tests that have a numerical result. So, why do we report some tests as qualitative and some as quantitative? There are a couple of different answers that resolve the question. 

Firstly, in forensic toxicology, blood is the gold standard when it comes to analytical testing as blood gives a snapshot of drug exposure and potential toxicity of circulating drugs in the window of a few minutes to several hours. Studies compile reference ranges or sets of blood drug or metabolite concentrations used as a guideline for interpreting results. These can normally be classified as therapeutic, toxic, and fatal ranges. Therapeutic blood concentration is the concentration of a drug or its active metabolite which is present in the blood following a therapeutic dosage of the drug. Most therapeutic ranges originate from data acquired during pharmaceutical medication human clinical trials or controlled dosing drug studies. Toxic blood concentration is the concentration of a drug, or its active metabolite, present in the blood that is associated with serious adverse symptoms. Fatal blood concentration is the concentration of a drug, or its active metabolite, present in the blood that has been reported to cause fatality or is so far above reported therapeutic or toxic concentrations, that one may judge it might cause fatality. Alternative matrices such as liver and brain tissue have very limited reference data available, and the availability is typically relegated to the classical drugs of abuse. Urine, on the other hand, gives a much wider window of drug detection – usually on the order of 1-5 days – but any drug or metabolite that is detected in urine is not imparting a pharmacological effect on the body. As urine is an excretory product, quantitative results do not and cannot correlate to or be associated with impairment, toxicity, or fatality. 

For those drugs that have clinical human trial data, for those drugs that have controlled dosing studies, and for those drugs that have established toxic and lethal reference ranges, quantitative testing is applicable. A forensic toxicologist can use the data and the established blood concentration ranges as a guide to aid in interpreting the analytical result and potentially come to an opinion on the role of a substance in an incident such as driving while impaired or the cause of death of an individual. On the other hand, alternative matrices such as tissue or urine may be reported as qualitative only due to the limited amount of reference data available or the nature of the specimen being a waste product. 

What about those substances that are new to the drug scene? What about those new street drugs or novel psychoactive substances (NPS)? We do not have human clinical trial data for these substances. We do not have controlled dosing studies for these substances. For some newly established compounds on the street, we may not even know their true pharmacological action in the body. What receptors do they bind to? How tightly do they bind to the receptor? What metabolites does the body produce? Are those metabolites active or inactive? What effects does the drug elicit? In a controlled setting, what blood concentrations are typically observed? Are those blood concentrations correlated to or with a specific effect or behavior or toxicity?  

There is a paucity of information when it comes to new drugs as well as alternative matrices. And for this reason, many laboratories will choose to report qualitative results only. When there is a lack of this quantitative reference data, the mere presence of the drug is the important part in the interpretation of the toxicology results. 

A second reason why a laboratory may choose to report qualitative results over quantitative results is two-fold – the extensive work and costs that goes in to developing and validating the analytical method to determine the result in the lab in combination with the rapidly changing illicit drug market. 

NPS such as fentanyl analogs, nitazene opioids, synthetic cannabinoids, and designer benzodiazepines ebb and flow as time goes on. They can appear and disappear rapidly over weeks to months. By the time the analytical method is validated and on-boarded to the laboratory, there is a good chance that the drugs that were prevalent on the market are no longer out there and have been replaced by other new drugs not in the newly updated method. 

As an example, for a while around 2015-2020, synthetic cannabinoids were prevalent in the USA and the street drug market was very rapidly changing. Compounds were emerging on the street, becoming prevalent, and then disappearing from the market within approximately a 3–6-month time span. In the span of a year or two, there were 10-20 new synthetic cannabinoids on the street. To either create a new quantitative test for these newly emerged substances or update a current test to include these new compounds, the development and validation process would take approximately 3-6 months. By the time the quantitative method was validated and approved for use in the laboratory, the entire drug scene on the street had changed and the new method was outdated. By validation protocols, qualitative tests are much quicker to validate than quantitative tests. So, as a forensic toxicology laboratory aiming to produce relevant (in time and scope) toxicology results in the aid of medical-legal investigations, it makes sense to develop qualitative tests for those newly emerged compounds. 

Ultimately, whether it is a qualitative test or a quantitative analysis – the interpretation of results hinges on the context and circumstances of the case. Axis Forensic Toxicology understands that one should never practice toxicology in a vacuum, and we are here to help with interpretation of the relevant toxicology in your casework. If you have any questions or concerns regarding a substance’s reference range or its role in your medical-legal investigation, please reach out to our subject matter experts at [email protected]. 

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).  

Pharmacokinetics and Pharmacodynamics. Principles of Forensic Toxicology. Fourth Edition. Barry Levine. American Association for Clinical Chemistry (AACC). 2017. 77-93. 

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). 

Reference Ranges. Axis Forensic Toxicology Blog. https://axisfortox.com/reference-ranges/ (2022).  

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

As mentioned in previous blog posts, novel psychoactive substances (NPS) come in different varieties and the Novel Emerging Compounds (NEC) Panel offered by Axis Forensic Toxicology helps to detect the most newly emerged NPS on the drug market and is meant to evolve over time as new drugs emerge on the street.  In this series, we have taken a look at the recently emerged substances bromazolam, flubromazepam, alpha-PiHP, N,N-dimethylpentylone, phenibut, and tianeptine. In this fourth and final post in the series, we will take a brief look at two more recently emerged opioids: AP-237 and brorphine. 

AP-237, also known as 1-butyryl-4-cinnamylpiperazine or Bucinnazine, is an opioid analgesic substance that is used as a prescription medication in China to treat pain. AP-237 is considered to be equipotent to morphine as an analgesic. AP-237 and a methylated derivative, 2-methyl-AP-237, recently emerged on the illicit drug market in the United States. AP-237 is currently unscheduled in the United States and not considered a controlled substance. 

Chemical Structures of AP-237 
Structures drawn by Kevin G. Shanks (2023)

Brorphine is an opioid substance that was originally synthesized in 2018 by researchers who were investigating various opioids with the intention of finding safer analgesics that produce less respiratory depression than the typical prescription opioids used as medications. Brorphine was first detected on the illicit drug market in the United States in 2019 – 2020, but now has also been found in Europe. It is currently controlled as a Schedule I controlled substance in the United States. 

Chemical Structures of Brorphine 
Structures drawn by Kevin G. Shanks (2023)

Both of these substances function as opioid receptor agonists in the human body. Similar to other opioids, such as morphine and fentanyl, they bind to opioid receptors in the central nervous system (brain and spinal cord) and produce an analgesic effect. In overdose, they may cause severe central nervous system depression to include respiratory depression. When the breathing slows down, apnea can occur. Apnea leads to hypoxia – or lack of oxygen distribution to the surrounding tissues including the brain. Hypoxia can lead to cardiac arrest and death. 

Axis qualitatively monitors both of these compounds in our NEC panel (order code 13710) and Comprehensive Panel, Blood with Analyte Assurance (order code 70510) using liquid chromatography with quadrupole time of flight mass spectrometry (LC-QToF-MS). Over the time range 01/30/2023 – 06/30/2023, Axis did not detect AP-237, but did detect brorphine in 1 blood specimen in Indiana. In Axis Forensic Toxicology casework, brorphine was detected alongside diphenhydramine, clonazolam, acetylfentanyl, fentanyl, morphine, 6-acetylmorphine, butonitazene, isotonitazene, metodesnitazene, metonitazene, and protonitazene. 

As you can see from the prevalence data, both AP-237 and brorphine are rarely detected in blood by the toxicology laboratory, but it is still vital that we monitor them for the immediate future as NPS are a geographical and temporal phenomenon. 

 Axis also monitors other NPS in the following available panels of testing.  

• The Novel Psychoactive Substances panel (order code 13610) include 25B-NBOMe, 25C-NBOMe, 25I-NBOMe, 2C-B, 2C-E, 2C-I, 5-MeO-DALT, adinazolam, alpha-PVP, butylone, clonazolam,  dibutylone, dimethylone, ethylone, etizolam, eutylone, flualprazolam, flubromazolam, MDPV, mephedrone, methcathinone, methedrone, methoxetamine, methylone, N-ethylpentylone, pentylone, and TFMPP.   
• The Designer Opioids panel (order code 13810) includes 4-ANPP, acetylfentanyl, acrylfentanyl, betahydroxythiofentanyl, butyrylfentanyl, carfentanil, cis-3-methylfentanyl, cyclopropylfentanyl, furanylfentanyl, isobutyrylfentanyl, methoxyacetylfentanyl, ocfentanil, parafluorobutyrylfentanyl, parafluoroisobutyrylfentanyl, tetrahydrofuranfentanyl, and U-47700. 
• The Nitazenes Analog panel (order code 13910) includes butonitazene, etodesnitazene, etonitazene, flunitazene, isotodesnitazene, isotonitazene, metodesnitazene, metonitazene, N-pyrrolidinoetonitazene, and protonitazene. 

As always, if you have questions about these substances and how they may play a role in your medical-legal investigation, please reach out to our subject matter experts by email ([email protected]) or phone (317-759-4869, Option 3).