4-Anilino- <i>N</i> -phenylethylpiperidine (4-ANPP): the potential caution flag for illicit fentanyl

The ability to distinguish illicit fentanyl use is becoming increasingly critical in toxicological investigations. 4-Anilino-N-phenylethylpiperidine (4-ANPP), also known as despropionylfentanyl, is both a precursor in illicit fentanyl production and a minor metabolite frequently detected alongside fentanyl in forensic toxicology. Its presence may assist in distinguishing medical and illicit fentanyl sources. This study evaluated 4-ANPP concentrations and 4-ANPP: fentanyl (4-ANPP to fentanyl) ratios in clinical (presumed medicinal) and postmortem (forensic) submissions to ascertain trends that may aid source attribution and toxicological interpretation. Blood and serum/plasma (s/p) samples were analyzed via liquid-liquid extraction followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) throughout 2023. A total of 32,723 forensic and 1,015 clinical cases positive for fentanyl were included in the analysis. Most clinical 4-ANPP concentrations (69%) were below 0.50 ng/mL, compared to 20% of forensic cases. Forensic blood samples with reportable 4-ANPP concentrations (N = 29,701) had a median of 2.1 ng/mL (mean ± std dev: 6.5 ± 42 ng/mL, range: 0.20-4100 ng/mL). In clinical serum/plasma samples with reportable 4-ANPP (N = 451), the median was 0.96 ng/mL (mean ± std dev: 3.9 ± 17 ng/mL, range: 0.20-306 ng/mL). In cases with reportable 4-ANPP concentrations, the median 4-ANPP: fentanyl ratio was 0.141 (mean ± std dev: 0.22 ± 0.95; range: 0.000078-140) for forensic, while the clinical median was 0.105 (mean ± std dev: 0.44 ± 3.0; range: 0.005-60). Notably, 91% of forensic cases had reportable 4-ANPP concentrations (≥0.2 ng/mL) compared to 44% of clinical cases, excluding more than half of the clinical cases from ratio calculations. Although overlapping 4-ANPP: fentanyl ratios limit its utility as a clear indicator of illicit fentanyl use, elevated 4-ANPP concentrations are more strongly associated with non-pharmaceutical sources and may serve as valuable support in forensic interpretation.