Untargeted Metabolomic Profiling via UPLC-HRMS

Untargeted metabolic profiling was performed on an ultrahigh performance liquid chromatography-high resolution mass spectrometry (Model: Thermo Ultimate 3000 UPLC and Thermo QExactive mass spectrometer) platform at the University of Florida Southeast Center for Integrated Metabolomics (SECIM). All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 70,000 at m/z 200 as separate injections. Chromatographic separation was attained on an ACE Excel 2 C18 PFP100 × 2.1 mm, a 2 μm particle size column with mobile phase A as 0.1% formic acid in water, and mobile phase B as acetonitrile, at a flow rate of 350 μL/min with a run time of 16.8 min, mass resolution of 35,000 at m/z 200, and mass range of 70–1000 m/z. Injection volume was 4 μL for negative ion mode and 2 μL for positive ion mode. The total run time per sample was 20.5 minutes. Probe (HESI probe) temperature was maintained at 350°C for both positive and negative run with a spray voltage of 3500 V and a capillary temperature of 320 °C [42 (link)].