I class uplc system

Profiling of polar metabolites was done as previously described⁵³ (link) with minor modifications as described below. In brief, the analysis was performed using an Acquity I class UPLC System combined with a mass spectrometer (Thermo Exactive Plus Orbitrap), which was operated in a positive ionization mode using a mass range of 200–800 m/z. The LC separation was done using the SeQuant Zic-pHilic (150 mm × 2.1 mm) with the SeQuant guard column (20 mm × 2.1 mm) (Merck). The mobile phase B was acetonitrile and mobile phase A was 20 mM ammonium carbonate plus 0.1% ammonia hydroxide in water. The flow rate was kept at 200 μl min⁻¹ and the gradient was as follows: 75% B (0–2 min), decrease to 25% B (2–14 min), 25% B (14–18 min), increase back to 75% B (18–19 min), 75% B (19–23 min). Inosine derivatives peaks were identified in the data using MSMS fragmentation, by identifying the inosine base signature as well as phosphates, ribose or deoxyribose. Area under the peak was quantified using MZmine 2.53⁵⁴ (link) with an accepted deviation of 5 ppm.