Urinary Metabolite Profiling by LC-MS/MS

A panel of 17 urinary metabolites were measured with urine samples collected at baseline and post-treatment from 4 of the 5 study participants. Mass spectrometry (MS) protocols were slightly modified from^{83 (link),84 (link)}. Briefly, for LC/MS/MS we utilized a Thermo Q Exactive HF-X Orbitrap mass spectrometer with a Thermo Vanquish HPLC system, auto-injecting a 5 μL urine sample. For chromatography, we used an Agilent ZORBAX HILIC PLUS column with a mobile phase of components A (10 mM ammonium bicarbonate, 0.05% formic acid in Millipore water, pH=4.2) and B (0.1% formic acid in acetonitrile), with flow rate flow rate of 0.3 mL/min. The gradient ran for 12 minutes. MS settings included a 4300 V spray voltage, nitrogen gas, ion transfer tubes, and auxiliary heater at 320°C and 30°C, respectively. PRM mode was positive polarity. Data were processed using Xcalibur Quant Browser, comparing peak areas to internal standards (A/IS ratio) and a standard curve (0.01–100 μM) for concentration determination.