Suspend dried metabolite extracted samples with 100µL of 0.1% Formic acid in water containing 100X diluted Metabolomics QRESS heavy labeled standards.

Vortex, microfuge for 0h 15m 0s at max speed, and take off 50µL into autosampler vial (do not touch bottom of tube).

Make sure there is no air in the bottom of the vial, carefully add the vial into the autosampler of the UPLC Vanquish system.

In the sequence setup view of the Xcalibur software include each sample in a row filling with: file name, sample ID, folder directory to save results, directory for the acquisition method, vial position and injection volume. Normally use 20% of sample.

Speed limits: draw speed 1 uL/s; dispense speed 1 uL/s.

Injection wash parameters: Wash mode: both; Wash time: 10.0s; Wash speed: 10.0 uL/s.

Column temperature:30°C

Run time: 0h 15m 0s

Solvents: %A1: Water, 0.1%FA.

    %A2: Methanol, 0.1% FA.

Upper limit pressure: 1500 bar

Flow = 0.250 ml/min

Equilibration: -3min, 0% B, curve = 5.

Gradient (all steps curve = 5): 0 min 5%B; 8 min

50% B; 8-9 min 98% B; 9-13 98% B;  13-13.1

min 0%B; 13.1-15 min 95%B.

Ion Source Type: H-ESI

Spray Voltage Positive 3500V

Spray Voltage Negative 2500V

Ion Transfer Tube Temperature 275°C, Sheath gas 35, Aux Gas 7, Sweep gas 2, Vaporizer Temp 320°C

Default charge state: 1

Scan 67-1000 m/z, resolution 60K, Quadrupole isolation: True, normalized AGC 25%, RF lens 35%, Max. Inj. Time 50ms, 1 microscan, Data Type Profile, Polarity Positive or Negative (create two methods one for Positive acquistion and another for Negative acquisition)

Min intensity 2E4, Dynamic exclusion - 1 time, 2.5s, 10ppm, isolation 1.5 m/z, resolution 15K, HCD NCE stepped 20, 35, 50%, Max. Inj. Time 25ms, normalized AGC 25%, 1 microscan, Data Type Centroid.