Vanquish horizon uplc

The samples were overall analyzed as in Dall et al. (2021 (link)). In brief, 5 μl was injected on a Vanquish Horizon UPLC (Thermo Fisher Scientific) where a flow of 400 μl/min was used with the following composition of eluent A (0.1% formic acid) and eluent B (0.1% formic acid, acetonitrile): 3% B from 0 to 1.5 min, 3% to 40% B from 1.5 to 3 min, 40% to 95% B from 3 to 5 min, 95% B from 5 to 7.6 min and 95% to 3% B from 7.6 to 8 min before equilibration for 3.5 min with the initial conditions. The flow from the LC coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) for mass spectrometric analysis in both positive and negative ion modes.

The pharmacokinetic assessment was conducted using a total of eight male DIO C57BL/6J mice (n = 4 per subgroup). Mice were administered single s.c. injections with a dose of 100 nmol kg⁻¹ of the experimental compounds. Each subgroup was bled at the following timepoints: subgroup A at 15 min, 45 min, 2 h and 8 h; subgroup B at 30 min, 1 h, 4 h and 24 h.
Mouse plasma containing test substance was crashed in 96-well non-binding plates using liquid–liquid extraction with ethanol containing internal standard. The samples were then centrifuged, and the supernatants were transferred to new wells and diluted with water. The prepared mouse plasma was analysed for the test substance using LC–MS. The system consisted of a TSQ Quantis Triple Quad mass spectrometer (Thermo Fisher Scientific) equipped with a Vanquish Horizon UPLC (Thermo Fisher Scientific). RP-UPLC separation was performed on the Acquity UPLC system (Waters, column: BEH C18 1.7 µm, 2.1 × 50 mm). Mobile phase A was composed of 0.1% formic acid in water and mobile phase B was composed of 0.1% formic acid in acetonitrile. The UPLC flow rate was set to 0.3 ml min⁻¹ at 60 °C using a gradient elution from 10 to 65% B over the course of 4.0 min. The gradient was then ramped from 65% B to 99% B for 0.1 min and held at 99% for 0.9 min. The mass spectrometer was operated in positive-ionization SRM mode.