4.5. Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS)

The inhibition of MDZ biotransformation with ENNB1 was analysed by LC-HRMS while using a QExactive^TM Hybrid Quadrupole-Orbitrap mass spectrometer that was equipped with a heated electrospray ion source (HESI-II) and coupled to a Vanquish UHPLC system (Thermo Fisher Scientific). The HESI-II interface was operated at 300 °C in negative and positive ionisation mode while using fast polarity switching in the mass range m/z 100–500, and the parameters were adjusted, as follows: spray voltage 3.2 and 2.8 kV (positive and negative mode, respectively), capillary temperature 280 °C, sheath gas flow rate 35 L/min., auxiliary gas flow rate 10 L/min., and S-lens RF level 55. Data were acquired in the full scan monitoring (FS)/data-dependent MS² (dd-MS²) mode targeting the [M+H]⁺ ions for MDZ (m/z 326.0855), 1-OH-MDZ and 4-OH-MDZ (m/z 342.0834), and 1,4-OH-MDZ (m/z 358.0753). The FS data were acquired at a mass resolution of 70,000 full width half-maximum (FWHM) at m/z 200, while mass resolution was set to 17,500 FWHM at m/z 200 during dd-MS². The automated gain control (AGC) target was set to 1 × 10⁶ ions for a maximum injection time (IT) of 250 ms in the FS mode, whereas the AGC target was 1 × 10⁵ for dd-MS² mode and the IT was 100 ms.

Chromatographic separation was performed at 30 °C on a 150 × 2.1 mm i.d. Kinetex F5 LC column (2.6 μm; Phenomenex) with 0.5 μm × 0.004 in. ID HPLC KrudKatcher Ultra Column In-Line filter (Phenomenex). The mobile phase consisted of water (A) and MeCN (B) containing 0.1% formic acid. Separation was achieved while using a gradient that ranged from 20% B at 1 min. to 80% B at 20 min. and further to 95% B at 21 min. at a flow rate of 0.25 mL/min. After flushing the column for 2 min. with 95% B, the mobile phase composition was returned to the initial conditions, and the column was isocratically eluted for 3.0 min. The total run time was 26.5 min.

Xcalibur software was used for instrument control and calculation of mass errors and elemental compositions. The metabolites were identified based on compound-specific retention times, comparison to a reference standard (4-OH-MDZ), fragmentation patterns, and accurate masses, which were obtained while using a mass window of ± 5 ppm with respect to the theoretical accurate masses. Measured peak areas were used for semi-quantification of the results.