In vitro comparative assessment of metabolic stability and structure-metabolism relationships of five fentanyl analogs with varying N-acyl side chain lengths.

Fentanyl analogs, a major subclass of synthetic opioids, have emerged worldwide and pose serious public health threats owing to their widespread misuse. This study investigated the comparative in vitro metabolism of five fentanyl analogs with varying N-acyl side chain lengths (acetylfentanyl, fentanyl, butyrylfentanyl, valerylfentanyl, and crotonylfentanyl) to elucidate the relationship between drug structure and metabolism. Each analog (5 μmol/L) was incubated with human liver microsomes for 1 h, and after deproteinization, the samples were analyzed using liquid chromatography-high-resolution mass spectrometry. Elongation of the N-acyl side chain resulted in shorter half-lives and higher clearance values. Among the four analogs with N-acyl alkyl side chains, the formation of the nor-metabolites, the metabolites hydroxylated at the ethyl linker, and the metabolites hydroxylated at the piperidine ring increased with increasing side chain length, peaking with fentanyl or butyrylfentanyl, and then decreasing with valerylfentanyl, thereby exhibiting an overall inverted U-shaped trend. In contrast, the metabolites hydroxylated at the phenyl ring of the phenethyl group declined as the side chain lengthened, whereas the metabolites hydroxylated at the acyl side chain and the carboxylated metabolites increased. Crotonylfentanyl, featuring an N-acyl alkenyl side chain, deviated from the structure-metabolism relationship observed among the other analogs. These findings highlight distinct structure-dependent metabolic stabilities and biotransformation pathways among fentanyl analogs, providing valuable information for identifying diagnostic metabolites to confirm fentanyl analog use in humans.