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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!