We demonstrated the reference-free identification of the main metabolites of MDZ formed by incubation in human hepatocytes using a combination of LC-MS/MS, IRIS and quantum-chemical computations. The metabolic pathway elucidated in this study is shown in Figure . We found that MDZ metabolizes via hydroxylation to 1'-OH-MDZ, which is followed by glucuronidation on the newly introduced OH-group. Additionally, we observed direct glucuronidation of the drug resulting in an MDZ-N-glucuronide. This is in general agreement with the literature, which reports these compounds as the major metabolites as well . Some studies report minor amounts of 4-OH-MDZ and 1,4-dihydroxy-midazolam . Indeed, we observed minor amounts of a second OH-MDZ isomer (m/z 342, RT 4.04 min), which was however not selected for further characterisation because of its low abundance, but it likely corresponds to 4-OH-MDZ. A metabolite with the molecular mass of 1,4-dihydroxy-midazolam was not observed in this study.