A novel substrate for CYP3A4 phenotyping: in vitro biotransformation of hubamine into butanone

CYP3A4 is a major drug-metabolising enzyme responsible for the biotransformation of many commonly used pharmaceuticals. However, its interindividual variability strongly influences pharmacokinetics, and thereby efficacy and adverse drug reactions. This variability underscores the need for accessible, minimally invasive phenotyping. Exhaled volatile analysis using unlabelled substrates offers a promising approach, provided that metabolism generates specific volatile products. In this study, we report the potential use of N-(sec-butyl)-N-(3,3-diphenylpropyl)butan-2-amine (hubamine) as a novel substrate for a CYP3A4 phenotyping assay. In vitro studies using HepG2 cells demonstrated that hubamine is metabolised to N-dealkylated hubamine (a non-volatile) and butanone (a volatile). Metabolite formation was significantly higher in HepG2 cells overexpressing CYP3A4 compared with CYP2C9 and CYP2D6 overexpressing cells, indicating high CYP3A4 specificity. Hubamine demonstrated superior performance compared with the substrates tolterodine, diisopromine and gstachamine. Method optimisation identified 75 µM substrate concentration and 90 min incubation time as ideal conditions, while preserving high cell viability. Inhibition assays confirmed CYP3A4-dependent metabolism. We demonstrate that gas chromatography-ion mobility spectrometry can be used as a sensitive, selective and portable analytical technique for detecting butanone, highlighting its suitability as a compact analytical platform for volatile metabolite analysis in complex headspace samples and its potential for decentralised analytical applications.