Mass spectrometric detection of short-lived drug metabolites generated in an electrochemical microfluidic chip

The costs of drug development have been rising exponentially over the last six decades, making it essential to select drug candidates in the early drug discovery phases before proceeding to expensive clinical trials. Here, we present novel screening methods using an electrochemical chip coupled online to mass spectrometry (MS) or liquid chromatography (LC) and MS, to generate phase I and phase II drug metabolites and to demonstrate protein modification by reactive metabolites. The short transit time (∼4.5 s) between electrochemical oxidation and mass spectrometric detection, enabled by an integrated electrospray emitter, allows us to detect a short-lived radical metabolite of chlorpromazine which is too unstable to be detected using established test routines. In addition, a fast way to screen candidate drugs is established by recording real-time mass voltammograms, which allows one to identify the drug metabolites that are expected to be formed upon oxidation by applying a linear potential sweep and simultaneously detect oxidation products. Furthermore, detoxification of electrochemically generated reactive metabolites of paracetamol was mimicked by their adduct formation with the antioxidant glutathione. Finally, the potential toxicity of reactive metabolites can be investigated by the modification of proteins, which was demonstrated by modification of carbonic anhydrase I with electrochemically generated reactive metabolites of paracetamol. With this series of experiments, we demonstrate the potential of this electrochemical chip as a complementary tool for a variety of drug metabolism studies in the early stages of drug discovery.