Sample Preparation and UHPLC-MS Analysis

Plasma was collected and stored at −80°C until analysis. Analyses were performed as previously published.^36,37 Prior to LC-MS analysis, samples were placed on ice and diluted with 24 volumes of methanol:acetonitrile:water (5:3:2, v:v). Suspensions were vortexed continuously for 30 min at 4°C. Insoluble material was removed by centrifugation at 10 000 g for 10 min at 4°C and supernatants were isolated for metabolomics analysis by UHPLC-MS. The analytical platform employs a Vanquish UHPLC system (Thermo Fisher Scientific, San Jose, CA) coupled online to a Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Samples were resolved over a Kinetex C18 column, 2.1 × 150 mm, 1.7 µm particle size (Phenomenex, Torrance, CA) equipped with a guard column (SecurityGuard^TM Ultracartridge—UHPLC C18 for 2.1 mm ID Columns—AJO-8782—Phenomenex, Torrance, CA) using an aqueous phase (A) of water and 0.1% formic acid and a mobile phase (B) of acetonitrile and 0.1% formic acid for positive ion polarity mode, and an aqueous phase (A) of water:acetonitrile (95:5) with 1 mmol/L ammonium acetate and a mobile phase (B) of acetonitrile:water (95:5) with 1 mmol/L ammonium acetate for negative ion polarity mode. Samples were eluted from the column using either an isocratic elution of 5% B flowed at 250 µL/min and 25°C or a gradient from 5% to 95% B over 1 min, followed by an isocratic hold at 95% B for 2 min, flowed at 400 µL/min and 45°C. The Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 μscans) from 60 to 900 m/z at 70 000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the Pierce^TM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, ¹³C, and ¹⁵N isotopes were performed using MAVEN (Princeton, NJ).³⁸