MDZ was incubated with human hepatocytes for 120 min. Samples taken at t=0 min, t=60 min and t=120 min were analyzed using an untargeted LC-MS approach (see method section) to identify MDZ derivatives and Table contains the resulting compound list. Extracted ion chromatograms of the detected m/z-values in the t=60 min sample can be found in the SI (Figure ). Compound A (Table ) is recognized as protonated MDZ and the intensity of this compound decreased during incubation, indicating that it is being metabolized. Compound B and C differ by 16 u from the parent drug (A), identifying them as hydroxylation products of MDZ. Compound D has an m/z-value that is 176 u higher than the parent drug, and is therefore likely the product of glucuronidation of MDZ. Compound E has a mass difference of 176 u relative to compounds B and C, and is therefore likely the product of sequential hydroxylation and glucuronidation. We selected the most abundant compounds (A, B, D and E; see Figure in the SI) for further identification using MS/MS and IRIS. It is seen that compound A and B show a rich fragmentation consistent with their structural assignment as protonated MDZ and protonated hydroxy-MDZ. In addition, the MS/MS spectrum of compound B shows an intense waterloss fragment, suggesting that the hydroxyl-group is located on an aliphatic position in the precursor ion. However, no distinction could be made between the two aliphatic positions of MDZ solely based on this fragmentation spectrum. The MS/MS spectra of compounds D and E both show a characteristic glucuronide-loss fragment (-176 u), which confirms their assignment as glucuronides. However, none of the observed fragment ions retains the glucuronide-group, making it impossible to derive the position of the glucuronide-group on the basis of these spectra.