384 well microplate

Plasma metabolites were extracted from pre-aliquoted biospecimens (15 μL) with 45 μL of LCMS grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2,000 × g for 10 mins at room temperature. The supernatant (30 μL) was transferred to a 96-well plate, leaving behind the precipitated protein. The supernatant was further diluted with 60 μL of 100 mm ammonium formate, pH3 (Fisher Scientific). For Hydrophilic Interaction Liquid Chromatography (HILIC) positive ion analysis, 15 μL of the supernatant and ammonium formate mix were diluted with 195 μL of 1:3:8:144 water (GenPure ultrapure water system, Thermofisher): LCMS grade methanol (ThermoFisher): 100 mm ammonium formate, pH3 (Fisher Scientific): LCMS grade acetonitrile (ThermoFisher). For C18 analysis, 15 μL of the supernatant and ammonium formate mix were diluted with 90 μL water (GenPure ultrapure water system, ThermoFisher) for positive ion mode. Each sample solution was transferred to 384-well microplate (Eppendorf) for LCMS analysis.