Non-targeted Metabolomic and Lipidomic Analyses by Ultra-High Performance Liquid Chromatography-Mass Spectrometer (UPLC-MS)

Plasma samples (100 μL) were precipitated by addition of 3 volumes of organic solvent [acetonitrile (1:3 v:v)] pre-cooled to −20°C. After vortex mixed for 1 min, overnight at −20°C and then centrifuged at 14,000 g for 20 min. Collected the supernatant and diluted to 50% and UPLC-MS non-targeted metabolomic analyzed. Organic phases were collected and reconstituted in isopropanol/acetonitrile/H₂O (1:1:1 v:v:v) and UPLC-MS non-targeted lipidomic analyzed.

Precipitated samples (non-targeted metabolomic analyzed) were injected onto a Waters HSS T3 column using a Waters Acquity^TM UPLC system equipped with a Waters Xevo^TM G2-XS Qtof. Flow rate was 450 μL/min. The mobile phase A consists of 0.1% formic acid in water and mobile phase B consists of 0.1% formic acid in acetonitrile. After separation by UPLC, mass spectrometry was performed using Waters Xevo^TM G2-XS Qtof. In positive ion-mode, the mass spectrometry of the optimal conditions was as follows:cone voltage at 24 V, capillary voltage 2.5 kV, source temperature was 100°C, cone gas flow was 50 L/h and desolvation gas flow was 800 L/h. Acquisition time was performed from m/z 50 to 1,500 Da. In negative ion mode, the mass spectrometry parameters were: cone voltage at 25 V, capillary voltage 2.5 kV, source temperature was 100°C, cone gas flow at 10 L/h and desolvation gas flow at 600 L/h. Acquisition time was performed from m/z 50 to 1,500 Da.

The extracted samples (organic phase) (non-targeted lipidomic analyzed) were injected onto a Waters CSH C18 column using a Waters Acquity^TM UPLC system equipped with a Waters Xevo^TM G2-XS Qtof. The flow rate was 400 μL/min. The mobile phase A consists of acetonitrile/H₂O (60:40, v:v) mixed with 10 mM ammonium formate and 0.1% formic acid and mobile phase B consists of isopropanol/acetonitrile (90:10, v:v) mixed with 10 mM ammonium formate and 0.1% formic acid. This chromatographic approach allowed an effective separation of the different lipid species. Mass spectrometry was further performed using Waters Xevo^TM G2-XS Qtof. In positive ion-mode, the mass spectrometry of the optimal conditions was as follows: cone voltage at 25 V, capillary voltage 2.5 kV, source temperature was 100°C, cone gas flow was 10 L/h and desolvation gas flow was 600 L/h. Acquisition time was performed from m/z 100 to 2,000 Da. In negative ion mode, the mass spectrometry parameters were: cone voltage at 40 V, capillary voltage 2 kV, source temperature was 100°C, cone gas flow at 50 L/h and desolvation gas flow at 800 L/h. Acquisition time was performed from m/z 100 to 2,000 Da.