Evaluation of liver tissue extraction protocol for untargeted metabolomics analysis by ultra-high-performance liquid chromatography/tandem mass spectrometry.

The aim of the untargeted metabolomics study is to obtain a global metabolome coverage from biological samples. Therefore, a comprehensive and systematic protocol for tissue metabolite extraction is highly desirable. In this study, we evaluated a comprehensive liver pretreatment strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to obtain more metabolites using four different protocols. These protocols included (A) methanol protein precipitation, (B) two-step extraction of dichloromethane-methanol followed by methanol-water, (C) two-step extraction of methyl tert-butyl ether-methanol followed by methanol-water, and (D) two-step extraction of isopropanol-methanol followed by methanol-water. Our results showed that protocol D was superior to the others due to more extracted features, annotated metabolites, and better reproducibility. And then, the stability and extraction sequence of protocol D were evaluated. The results showed that extraction with isopropanol-methanol followed by methanol-water was the optimum preparation sequence, which offered higher extraction efficiency, satisfactory repeatability, and acceptable stability. Furthermore, the optimal protocol was successfully applied by liver samples of rats after high-fat intervention. In summary, our protocol enabled a comprehensive and systematic evaluation of liver pretreatment to obtain more medium-polar and nonpolar metabolites and was suitable for high-throughput metabolomics analysis.