In vitro metabolic profiling and structure-metabolism relationships of substituted acetyl fentanyl-type new psychoactive substances.

The abuse of fentanyl-type new psychoactive substances (F-NPS), which exhibit the four defining characteristics of new psychoactive substances (third-generation drugs), poses a severe threat to social stability and public health. The derivatization strategy investigated in this study, involving six substituted acetyl F-NPS across two substitution patterns, represents the primary approach for generating a new F-NPS. Using an in vitro human liver microsome metabolic model coupled with liquid chromatography-ion trap tandem time-of-flight mass spectrometry, we identified characteristic metabolism profiles of F-NPS corresponding to derivatization modifications while elucidating the structural effects on metabolism. This study revealed that, first, metabolism via amide hydrolysis was affected by concurrent hydrolysis at adjacent positions, rather than being solely determined by carbonyl carbon electrophilicity. Second, metabolism via N-oxidation and N-dealkylation shared a common initial intermediate, with the latter being triggered by α-hydroxylation of the phenethyl group. Third, metabolism via N-oxidation exhibited reduced susceptibility to structural changes owing to the contradictory bond orientations of the substituents on the piperidine ring between the parent drug and its N-oxide metabolite. Fourth, stable geminal diol metabolites were identified in the substituted acetyl F-NPS metabolites via mass spectrometric fragmentation. This research deepens the understanding of structure-metabolism relationships among F-NPS, providing critical foundational data for developing predictive metabolisms for emerging F-NPS and offering scientific support for drug abuse surveillance and prevention strategies.