Quantitative measurement of acetyl fentanyl and acetyl norfentanyl in human urine by LC-MS/MS

Anal Chem. 2014 Feb 4;86(3):1760-6. doi: 10.1021/ac4036197. Epub 2014 Jan 15.


Opioid abuse involving emerging opioid compounds is a growing public health problem, which was highlighted recently by cases of human morbidity and mortality linked to acetyl fentanyl abuse. Unfortunately, the lack of information available on the toxicology and metabolism of acetyl fentanyl precludes its detection in human samples. The following study was conducted to test a new analytical procedure for the simultaneous quantification of acetyl fentanyl and its predicted metabolite, acetyl norfentanyl, in human urine. Metabolic reference standards and deuterium-labeled internal standards were synthesized for use in an assay that coupled solid-phase extraction (SPE) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The accuracy (% Relative Error <5%) and inter- and intrarun precision (%CV <20%) of this new method resulted in low levels of quantification (∼1 ng/mL). Similar results were obtained using liquid chromatography columns manufactured with phenyl-hexyl and biphenyl stationary phases (r(2) > 0.98). Preliminary human liver microsomal and in vivo rodent studies demonstrated that acetyl fentanyl is metabolized by cytochrome P450s to acetyl norfentanyl. Urine samples from rats treated with a toxic dose of acetyl fentanyl contained high concentrations of acetyl fentanyl and acetyl norfentanyl. Further toxicokinetic studies are required to fully elucidate the metabolic pathways responsible for acetyl fentanyl detoxification and excretion.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Analgesics, Opioid / metabolism
  • Analgesics, Opioid / urine*
  • Animals
  • Chromatography, Liquid
  • Cytochrome P-450 Enzyme System / metabolism
  • Fentanyl / analogs & derivatives*
  • Fentanyl / metabolism
  • Fentanyl / urine
  • Humans
  • Male
  • Rats
  • Tandem Mass Spectrometry
  • Urinalysis / methods*


  • Analgesics, Opioid
  • norfentanyl
  • Cytochrome P-450 Enzyme System
  • Fentanyl