Evaluation of liver tissue extraction protocol for untargeted metabolomics analysis by ultra‐high‐performance liquid chromatography/tandem mass spectrometry
2021
Liu, Zhipeng | Wang, Peng | Liu, Zengjiao | Wei, Chunbo | Li, Ying | Lau, Nap-yin
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.
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