To test the effect of the treatment and age on levels of metabolites, the paired Student's t-test (effect of treatment) and independent Student's t-test (effect of age) were employed, respectively, reflecting the correlation structure of the data. Prior to these tests, a logarithmic transformation (base 10) was applied to the data. Given the presence of zero values within the LC-MS datasets, a uniform increment was added to all measurements before applying the logarithm. In this study, a threshold of p < 0.05 was established for statistical significance. In addition, false discovery rates (FDRs) were performed for the metabolites within multiple comparisons at a significance level of p = 0.1, resulting in corresponding q values for each metabolite.