Chromatography and mass spectrometry for metabolomic profiling

For HILIC analysis, 1 μL of sample was injected onto a Waters ACQUITY UPLC BEH Amide (1.7 μm particle size, 100 × 2.1 mm). Samples underwent a subsequent 1:5 dilution in water for RPLC analysis and 5 μL was injected onto a Waters ACQUITY UPLC HSS T3 column (1.8 μm particle size, 100 × 2.1 mm). Both columns were maintained at 45 °C and mobile phase flow was set to 0.45 mL/min using a Waters ACQUITY UPLC I-Class system (Waters, Milford, MA). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). Mobile phase conditions for each column are described in Table ^S1. Time-of-Flight mass spectrometry was carried out using a Waters Xevo-G2S QTof/MS as previously described^28,29. Briefly, capillary and cone voltage were set at 2 kV and 40 V, respectively. The source temperature was 150 °C and the desolvation temperature was maintained at 600 °C. Nitrogen gas for desolvation and the cone were set at 1200 L/h and 50 L/h, respectively. An MS^E method was used to acquire ions in the range of 50–1200 m/z alternating between MS1 (no collision energy) and MS2 (collision energy ramp of 15–50 V) with a scan time of 0.1 s. Leucine-enkephalin (100 ng/L) was used as a lockmass set to a flow rate of 10 μl/min. The lockmass was acquired every 10 seconds and averaged over 3 scans to ensure mass accuracy throughout the run.