UHPLC-MS-based Metabolite Separation and Profiling

Metabolites from the metabolomic extraction were separated using a XSelect HSS C18 column (2.1 mm x 150 mm, 3.5 μm particle size) (Waters, Milford, MA) on a Dionex UltiMate 3000 quaternary UHPLC (Thermo Scientific, Waltham, MA) equipped with a refrigerated autosampler (5^oC) and column heater (50^oC). Solvent A was 5 mM DIPEA and 200 mM HFIP and solvent B was 5 mM DIPEA and 200 mM HFIP in methanol. Flow gradient conditions were as follows: 0% B for 2 min at 0.18 ml min⁻¹, increased to 1% B for 2 min at 0.2 ml min⁻¹, increased to 2% B for 4 min, increased to 14% B for 2 min, increased to 70% B for 2 min, increased to 99% B for 1 min, increased flow rate to 0.3 ml min⁻¹ for 0.5 min, increased flow rate to 0.4 ml min⁻¹ for 4 min, then washed by decreasing to 0% B for 2.3 min at 0.3 ml min⁻¹, decreased to 0.2 ml min⁻¹ for 0.2 min, and ending with flow of 0.18 ml min⁻¹. Samples were analyzed using a Q Exactive HF (QE-HF) (Thermo Scientific, Waltham, MA) equipped with a heated electrospray ionization source operated in the negative ion mode. Source parameters were the same as described before (22 (link)). Data acquisition was performed in negative Full Scan mode 240,000 resolutions. Column effluent was diverted to the QE-HF from 1 to 15 min and then to waste for the remaining time of the run.