Metabolites were reconstituted in 100 μL of 0.03% formic acid in analytical grade water, vortexed, and centrifuged to remove insoluble material. Next, 20 μL supernatant was subjected to targeted metabolomics analysis on an AB SCIEX QTRAP 5500 LC/triple quadrupole MS (Applied Biosystems SCIEX) as described (70). Chromatogram review and peak area integration were performed using MultiQuant (version 2.1, Applied Biosystems SCIEX). The peak area for each detected metabolite was normalized against the total ion count of each sample. Normalized peak areas were mean-centered and unit-variance scaled, then evaluated by principal component analysis to visualize clustering and detect outliers using SIMCA-P (version 13.0.1, Umetrics). Partial least-squares discriminant analysis and variable importance in projection analysis, as well as pathway enrichment analysis, were done using Metaboanalyst 3.0 (71).

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