Fig. 4. Comparison of PCA and MeOH extraction methods for NMN and other metabolites.

a Recovery efficiencies of spiked NMN (M + 14) and regular NMN after PCA and MeOH-chloroform extraction. Indicated concentrations of NMN (M + 14) and regular NMN were added to mouse plasma. After extracting plasma samples with PCA or MeOH-chloroform, each extract was analyzed with dimeLC-MS/MS. Recovery efficiencies were calculated by an iSTD method. Means were obtained from three independent experiments. Two-way ANOVA was conducted to compare the results with Bonferroni’s multiple comparisons post hoc test. Data are presented as mean ± SEM. **p < 0.005; ***p < 0.001. b Comparison of PCA and MeOH extraction methods for primary metabolites in plasma. Plasma samples were extracted by PCA or MeOH-chloroform extraction, and 96 primary metabolites were analyzed using the Primary Metabolites Method Package Ver. 2. Average area ratios from three independent experiments were plotted in a volcano plot. c Area ratios of 10 representative compounds are shown for PCA and MeOH-chloroform extraction methods. Unpaired Student’s t test was conducted to compare the results. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01.