Qualitative studies on the metabolism and the toxicological detection of the fentanyl-derived designer drugs 3-methylfentanyl and isofentanyl in rats using liquid chromatography–linear ion trap–mass spectrometry (LC-MSn)

Abstract

The opioid 3-methylfentanyl, a designer drug of the fentanyl type, was scheduled by the Controlled Substance Act due to its high potency and abuse potential. To overcome this regulation, isofentanyl, another designer fentanyl, was synthesized in a clandestine laboratory and seized by the German police. The aims of the presented study were to identify the phase I and phase II metabolites of 3-methylfentanyl and isofentanyl in rat urine, to identify the cytochrome P450 (CYP) isoenzymes involved in their initial metabolic steps, and, finally, to test their detectability in urine. Using liquid chromatography (LC)-linear ion trap-mass spectrometry (MS(n)), nine phase I and five phase II metabolites of 3-methylfentanyl and 11 phase I and four phase II metabolites of isofentanyl could be identified. The following metabolic steps could be postulated for both drugs: N-dealkylation followed by hydroxylation of the alkyl and aryl moiety, hydroxylation of the propanamide side chain followed by oxidation to the corresponding carboxylic acid, and, finally, hydroxylation of the benzyl moiety followed by methylation. In addition, N-oxidation of isofentanyl could also be observed. All hydroxy metabolites were partly excreted as glucuronides. Using recombinant human isoenzymes, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were found to be involved in the initial metabolic steps. Our LC-MS(n) screening approach allowed the detection of 0.01 mg/L of 3-methylfentanyl and isofentanyl in spiked urine. However, in urine of rats after the administration of suspected recreational doses, the parent drugs could not be detected, but their common nor metabolite, which should therefore be the target for urine screening.