Targeted Metabolomics Analysis by LC-MS

Metabolite samples were analyzed on a Q-exactive Orbitrap mass spectrometer coupled with a Dionex U3000 high-performance LC (HPLC; Thermo Fisher) with a ZIC-pHILIC column (5 µm, polymeric, 150 × 4.6 mm; SeQuant, Merck). The MS system was operated at 35,000 resolution in both positive and negative electrospray ionization modes with a detection range of 85–1,275 m/z. Column temperature was maintained at 25 C, and the mobile phase consisted of 20 mM of ammonium carbonate (solvent A) and acetonitrile (solvent B). Metabolites were eluted in a step gradient, starting with 80% solvent B at a flow rate of 0.3 ml/min followed by a linear gradient to 50% solvent B over 15 min (Maifiah et al., 2016 (link); Maifiah et al., 2017 (link)). All samples were randomized and analyzed in a single LC-MS batch. Analytical reproducibility was monitored on the basis of pooled QC samples throughout the batch, which were periodically analyzed after groups of six samples. Analyses of a mixture of pure standards containing >250 metabolites were performed to assist in the identification of metabolites.