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).