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By Kevin Shanks, D-ABFT-FT

It has been a little over 4 years since Axis mentioned synthetic cannabinoids on this blog. So, briefly, synthetic cannabinoids are man-made compounds that bind to cannabinoid receptors 1 (CB1) and 2 (CB2) in the body and mediate effects of the endocannabinoid receptor system.  While delta-9-tetrahydrocannabinol (THC), the main psychoactive cannabinoid found in cannabis, is a partial agonist of the cannabinoid receptors, most synthetic cannabinoids are full agonists at the same receptors. Synthetic cannabinoids are comprised of many structural variants, which change over time as compounds are controlled by Federal and state governments. Synthetic cannabinoids have been implicated in several outbreaks of illnesses and hospitalizations in the USA as well as associated with cause of death in postmortem toxicology. 

Chemical Structure of 5F-ADB  Drawn by Kevin G. Shanks (2025)

Methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate, also known as 5F-ADB or 5F-MDMB-PINACA, is a potent synthetic cannabinoid compound that first emerged in  Japan in late 2014 when it was identified on plant material products. Synthetic cannabinoids are commonly sprayed on plant material or soaked into small pieces of paper. 5F-ADB’s molecular formula is C20H28FN3O3 and its molecular weight is 377.4 g/mol. 5F-ADB is of the indazole-3-carboxamide family of compounds and consists of an indazole core structure, a fluorinated pentyl chain, and a carboxamide linking group. Common adverse effects after consumption of the substance include psychomotor agitation, tachycardia, confusion, anxiety, and loss of consciousness, psychosis, and fatality. 

5F-ADB Detections by US DEA Reported in NFLIS Annual Drug Reports (2015-2022)

The drug was first reported by the United States Drug Enforcement Administration (DEA) in 2014 and emerged in toxicology testing in 2015. It was made a Schedule I controlled substance in the USA in 2017. After emergence, 5F-ADB hit peak prevalence in 2018 and then rapidly disappeared from the illicit drug market with just a handful of reports by the DEA by 2022. During 2021-2022, Axis Forensic Toxicology had zero detections of 5F-ADB in postmortem toxicology testing. Because of the combined DEA and Axis data, the lab decided to remove it from the scope of synthetic cannabinoid testing in 2022. 

Until recently, the compound was considered to be non-existent on the street, but new reports suggest 5F-ADB has made a comeback as one of the top synthetic cannabinoids of choice in the USA. Published reports from NPS Discovery and the Center for Forensic Science Research and Education (CFSRE) in 2025 detail 5F-ADB’s comeback as either the first or second most identified synthetic cannabinoid in cases sent to them. Axis Forensic Toxicology has also spoken to pathologists who have had positive casework for 5F-ADB, over the last few months, especially in the prison population. Because of these recent events, the lab has added 5F-ADB back into the screening scope of analysis in the comprehensive panel (order code 70510). 5F-ADB is rapidly metabolized/degraded to its 3,3-dimethylbutanoic acid metabolite by enzymes in the blood and therefore must be analyzed by monitoring the metabolic product. The reporting limit for 5F-ADB 3,3-dimethylbutanoic acid on the screening test is 2 ng/mL. The result is reported as qualitative. Instrumentation used for analysis is liquid chromatography with quadrupole time of flight mass spectrometry (LC-qToF-MS). Upon publication of this blog post, Axis has had recent detections of 5F-ADB via its metabolite in 5 different states – Florida, Indiana, Missouri, Ohio, and South Dakota. 

It is important to remember that while most novel psychoactive substances emerge and then disappear rapidly from the illicit market, some persist for months and years. And in the case of 5F-ADB, it emerged, peaked, rapidly disappeared, and now has made a resurgence years later. As a toxicology laboratory, it is prudent to monitor the ever-changing scope of the drug market and adapt testing panels accordingly. The current scope of testing offered by Axis Forensic Toxicology can be found in the online test catalog at https://axisfortox.com. As always, if you have questions about 5F-ADB or any other synthetic cannabinoid 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).  

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). 
• NPS Discovery and CFSRE Trend Reports, Q1-Q3, 2025.  
• Axis Forensic Toxicology. Laboratory Data. Indianapolis, IN. (accessed October 2025). 

By Stuart Kurtz, D-ABFT-FT

Axis Forensic Toxicology has previously written on kratom and cases involving kratom fatalities. As a quick recap, Mitragyna speciosa, is a tree or shrub that grows in southeast Asia. It’s part of the Rubiaceae family which includes coffee plants. It can be consumed by chewing leaves, brewing a beverage, or pulverized and smoked or put in a capsule. Fatalities have been reported from kratom use with mitragynine as the primary alkaloid responsible for toxic effects. 

7-OH mitragynine is naturally occurring in the kratom plant at about 2% of the alkaloid content and is 10 times more potent than mitragynine. Reported side effects include agitation, nausea, vomiting, confusion, seizures, and fast heart rate. Due to activity at the mu opioid receptor, the receptor primarily associated with opioids such as fentanyl and morphine, severe cases can present similar to opioid overdoses. 7-OH mitragynine is further metabolized into mitragynine pseudoindoxyl which is 100 times more potent than mitragynine. Mitragynine pseudoindoxyl is also found in the kratom plant in low concentrations. Labs that test for mitragynine may not currently be testing for 7-OH mitragynine. Historically, the concern with kratom related toxicity has been centered on testing for mitragynine since that was the primary alkaloid in kratom products. However, new products may contain higher amounts of 7-OH mitragynine and/or mitragynine pseudoindoxyl and low or no amount of mitragynine which investigators and labs should be aware of moving forward with these cases. 

Image from United States Food and Drug Administration showing 7-OH mitragynine extract (left), powder (middle), and gummies (right). 

7-OH mitragynine products are manufactured by converting mitragynine in the kratom plant to 7-OH mitragynine. Available products containing primarily 7-OH mitragynine include concentrates, edibles, extracts, beverages, and tablets for chewing or swallowing. Testing of these products shows that they are primarily 7-OH mitragynine with a variable amount of unconverted mitragynine and other products that could be related to the synthesis process. These can often be found in stores that also sell kratom products and lead to confusion as to what the buyer is actually getting. Given the increase in potency of 7-OH mitragynine compared to mitragynine, the primary alkaloid in kratom, a naïve user may experience a toxic event if they are expecting the product to behave like kratom. 

Image from United Nations Office on Drugs and Crime Laboratory and Scientific Services Portal showing 7-OH mitragynine tablets and testing results of the contents. 

Interpretation can be tricky when it comes to mitragynine presence. It can be associated with kratom use or synthesis of mitragynine to produce the more potent 7-OH mitragynine instead of, or in addition to, mitragynine. A good indicator will be what products are found at the scene and if they are consistent in how they are labeled between products. A product labeled as kratom or mitragynine may actually be a 7-OH mitragynine product especially since the FDA is currently in the process of scheduling mitragynine only, which may lead to more products containing 7-OH mitragynine to avoid legal issues. Therefore, testing for both mitragynine and 7-OH mitragynine will be useful. Axis recommends that you check the testing scope with your lab to see if they test for one, both, or neither of these compounds. Axis currently screens with LC-QToF-MS for mitragynine and 7-OH mitragynine in our 70510 Comprehensive Panel with Analyte Assurance. Confirmation of mitragynine is quantitative using LC-MS/MS and 7-OH mitragynine is qualitative using LC-QToF-MS. 

If you have any questions regarding this or other topics, please reach out to us via email [email protected] or call at 317-759-4869 option 3. 

Krotulski, AJ; Denn, MT; Brower, JO; Papsun, DM; Logan, BK. (2025), Evaluation of Commercially Available Smoke Shop Products Marketed as “7-Hydroxy Mitragynine” & Related Alkaloids, Center for Forensic Science Research and Education, United States. 

Anderer S. What to Know About 7-OH, the New Vape Shop Hazard. JAMA. 2025;334(12):1045–1046. doi:10.1001/jama.2025.13592 

Smith, K.E., Boyer, E.W., Grundmann, O., McCurdy, C.R. and Sharma, A. (2025), The rise of novel, semi-synthetic 7-hydroxymitragynine products. Addiction, 120: 387-388. https://doi.org/10.1111/add.16728 

Pullman MK, Kanumuri SRR, Leon JF, Cutler SJ, McCurdy CR, Sharma A. Cardio-pulmonary arrest in a patient revived with naloxone following reported use of 7-hydroxymitragynine. Clin Toxicol (Phila). 2025 Sep 30:1-2. doi: 10.1080/15563650.2025.2565428. Epub ahead of print. PMID: 41025553. 

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

We have previously discussed both fentanyl and 4-ANPP in this blog (1-2), but briefly, fentanyl is a mu opioid receptor agonist that is used as both a pharmaceutical medication and as an illicit drug found on the street typically as a powder or tablet. Fentanyl itself is metabolized primarily by the CYP3A4 enzyme in the human body. It can be dealkylated, hydroxylated, methylated, and hydrolyzed (3-4). At Axis, we monitor for the presence of the unchanged drug, fentanyl, alongside its primary metabolite, norfentanyl, in blood and urine. Several years ago, we added a second fentanyl-related substance to the scope of testing – 4-ANPP. 

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

So, what is 4-ANPP? The substance is known chemically as N-phenyl-1-(2-phenylethyl)-4-piperidinamine, but is also referred to as despropionyl fentanyl. It is formed via amide hydrolysis of the parent substance. It is considered to be a minor inactive metabolite of fentanyl – meaning it does not produce a pharmacological effect on the body (5-6). But it is also a precursor or starting material used in the synthesis of illicitly manufactured fentanyl and various related fentanyl derivatives or analogs. As an example, 4-ANPP can be reacted with propionyl anhydride to form fentanyl or acetic anhydride to form acetylfentanyl. 4-ANPP is not used in the synthesis of pharmaceutical-grade fentanyl. 

Why is all of this important? The presence of illicitly manufactured fentanyl in the street drug supply in the USA has rapidly increased over the past 10-12 years (7). And with that increased presence comes a large increase in overdose deaths due to the substance. Recent CDC data shows that over 100,000 people died yearly from drug overdoses in the USA in 2022-2024, with the major driving factor being the prevalence of illicit fentanyl in the street drug supply (8). 

Ultimately, the presence of 4-ANPP in a biological specimen such as blood or urine is merely a marker for fentanyl use or exposure. The product used may have been either pharmaceutical fentanyl or illicitly manufactured fentanyl. The origin of the fentanyl – pharmaceutical vs. street – cannot be determined by the toxicology results alone. In order to ascertain if 4-ANPP is present in the body from metabolism or if it was ingested by using an impure illicit street product, testing must be conducted on evidence from the scene which may include unidentified powders or tablets. If 4-ANPP is detected in the non-biological evidence, then it may be determined that the fentanyl exposure is from a non-pharmaceutical origin.  

Axis Forensic Toxicology tests for the presence of 4-ANPP in our Comprehensive Drug Panel with Analyte Assurance™, Blood (order code 70510), which is completed by liquid chromatography with quadrupole time of flight mass spectrometry (LC-QToF-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The reporting limit is 100 pg/mL and the substance is reported as qualitatively positive or negative – it is not reported quantitatively. 

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

1. Drug Primer: Fentanyl (2021). Axis Forensic Toxicology Blog. Drug Primer: Fentanyl – Axis Forensic Toxicology (axisfortox.com). 
2. Drug Primer: 4-ANPP (2022). Axis Forensic Toxicology Blog. Drug Primer: 4-ANPP – Axis Forensic Toxicology (axisfortox.com). 
3. Fentanyl. Disposition of Toxic Drugs and Chemicals in Man. Twelfth Edition. Randall C. Baselt. Biomedical Publications. Pages 844-847. (2020).  
4. Opioids. Principles of Forensic Toxicology. Fourth Edition. Barry Levine. AACC, Inc. Pages 271-291 (2017). 
5. 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. 
6. Wilde, M., Pichini, S., Pacifici, R., Tagliabracci, A. et al. (2019) Metabolic Pathways and Potencies of New Fentanyl Analogs. Frontiers in Pharmacology, 10: 238. 1-16. 
7. 2022 Annual Drug Report. National Forensic Laboratory Information System (NFLIS). Drug Enforcement Administration. Springfield, VA. 
8. Provisional Drug Overdose Death Counts. National Vital Statistics System. Center for Disease Control. Vital Statistics Rapid Release – Provisional Drug Overdose Data. (Accessed 07/31/2025).

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

As mentioned in several previous posts on this blog, novel psychoactive substances (NPS) are arranged into different families. Opioids, cannabinoids, stimulants, hallucinogens, and benzodiazepines are the main ones we encounter in forensic toxicology. Axis makes an effort to be able to detect the most newly emerged of these NPS as the drug market evolves over time. In this post, we will take a brief look at two stimulants which have recently appeared in the United States.  

Chemical structure of N-isopropyl butylone Drawn by Kevin G. Shanks (2025)

N-isopropyl butylone and N-cyclohexyl methylone are considered substituted cathinones, which are a class of compounds related to cathinone, a naturally occurring stimulant alkaloid found in the plant Catha edulis, also known as khat. Substituted cathinones are chemically and pharmacologically similar to amphetamine and methamphetamine and act as central nervous system stimulants. These compounds primarily act on monoamine transporters in the body and inhibit the reuptake of dopamine, norepinephrine, and/or serotonin and may also act as releasers of the same neurotransmitters. The result of this pharmacological action is an increased concentration of neurotransmitters in the nerve cell synapse, which leads to stimulant and empathogenic physiological effects on the body. Desired effects of stimulants include euphoria, increased energy, sociability, and alertness. Adverse effects include anxiety, agitation, paranoia, hallucinations, and aggression. Acute toxicity is manifested via hyperthermia, hypertension, and tachycardia, severe agitation, hallucinations, and potential seizure, rhabdomyolysis, kidney failure, and death. Chronic toxicity includes psychological dependence, cognitive impairment, sleep disorders, depression, and neurotoxicity within the dopaminergic and serotonergic neutrotransmitter systems. 

Chemical structure of N-cyclohexyl methylone Drawn by Kevin G. Shanks (2025)

N-isopropyl butylone was first identified in the United States in 2024 in the state of Georgia, where it was detected in drug material alongside methamphetamine. It was also detected in drug material sold as MDMA in New Zealand in 2024 and in drug material purported to be MDMA/ecstasy sold in Europe in 2025. In 2025, it was found alongside ketamine and methamphetamine. N-isopropyl butylone is a positional isomer of other substituted cathinones including N-ethylpentylone, N, N-dimethylpentylone, and N-propylbutylone. 

N-cyclohexyl methylone was first identified in the United States in 2022 in drug material in the state of Florida when it was sold as an ecstasy tablet and in Indiana sold as a beige/off-white crystalline powder. The substance is a derivative of the older cathinone, methylone, which is itself a derivative of MDMA (3,4-methylenedioxymethamphetamine). 

While not explicitly listed as controlled substances in the United States, both of the substances may be considered positional isomers of already controlled drugs and therefore be considered “analogues”. According to the Drug Enforcement Administration’s (DEA) National Forensic Laboratory Information System (NFLIS) March 2023 Drug Snapshot, N-cyclohexyl methylone was the third most common detected substituted cathinone in the United States with 257 reports. And in the December 2024 Drug Snapshot, N-isopropyl butylone was the most prevalent cathinone with 178 detections while N-cyclohexyl methylone was number 5 with 12 detections. There have not yet been any published reports of these two substances being implicated in human toxicity, including hospitalizations and fatalities. 

Axis qualitatively monitors both compounds in our Novel Emerging Compounds (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).  Axis also monitors other NPS cathinones in the NEC panel including alpha-PHP, alpha-PiHP, and N, N-dimethylpentylone. Additional cathinone/stimulant compounds in our Novel Psychoactive Substances panel (order code 13610) include alpha-PVP, butylone, dibutylone, dimethylone, eutylone, MDPV, mephedrone, methcathinone, methedrone, methylone, N-ethylpentylone, pentylone, and TFMPP. 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). 

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

As we have discussed on this blog several times in the past, novel psychoactive substances (NPS) come in different varieties, which include cannabinoids, stimulants, opioids, benzodiazepines, and hallucinogens. One of Axis Forensic Toxicology’s objectives it to be able to detect the most recently emerged NPS on the street in a timely manner. With that said, a new fentanyl derivative has emerged! 

Chemical Structure of ortho-Methylfentanyl Drawn by Kevin G. Shanks (2025)

Ortho-Methylfentanyl is a fentanyl analog or derivative that has emerged in the United States as an NPS in both powders and counterfeit tablets. It has a chemical formula C23H30N2O and a molecular weight equal to 350.5 g/mol. It is structurally-related to fentanyl with the fundamental difference in chemical structure being in the position of a single methyl (-CH3) group, which is attached to the ortho- position of the phenyl ring. There are two other positional isomers: meta- and para-methylfentanyl. The ortho isomer of methylfentanyl was first detected in 2023 in British Columbia, Canada, but over the last two years, it was been detected across Canada and the United States. In the United States, the substance is considered a Schedule I controlled substance and is illegal to synthesize, possess, and distribute. 

Pharmacologically, it is a mu (µ) opioid receptor agonist, much like morphine, oxycodone, and fentanyl. It also shares a similar analgesic potency with fentanyl. As a mu opioid receptor agonist, the effects of the substance would include analgesia, tiredness, drowsiness, sedation, and in overdose, severe respiratory depression, hypoxia, apnea, coma, and potentially, death. 

In early 2025, we first detected ortho-methylfentanyl in our postmortem toxicology sample population and have now added it to our Designer Opioids panel (order code 13810) and the Comprehensive Panel, Blood with Analyte Assurance™ (order code 70510) using liquid chromatography with quadrupole time of flight mass spectrometry (LC-QToF-MS). Reporting limit is 50 pg/mL.  

Axis also has the ability to monitor other novel opioids to include acetylfentanyl, acrylfentanyl, AP-237, AP-238, betahydroxythiofentanil, brorphine, carfentanil, cis-3-methylfentanyl, cyclopropylfentanyl, furanylfentanyl, isobutrylfentanyl, methoxyacetylfentanyl, ocfentanil, para-fluorobutyrylfentanyl, para-fluoroisobutyrylfentanyl, tetrahydrofuranfentanyl, tianeptine, U-47700, and valerylfentanyl. 

As always, if you have questions about ortho-methylfentanyl and how it (or any other novel emerging compound) 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). 

Stay tuned to this blog for more quick drug primers on more novel compounds we have recently added to our testing scope!