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In this study, we evaluated a comprehensive liver pretreatment strategy based on ultra\u2010high\u2010performance liquid chromatography coupled with quadrupole time\u2010of\u2010flight mass spectrometry to obtain more metabolites using four different protocols. These protocols included (A) methanol protein precipitation, (B) two\u2010step extraction of dichloromethane\u2010methanol followed by methanol\u2013water, (C) two\u2010step extraction of methyl tert\u2010butyl ether\u2010methanol followed by methanol\u2013water, and (D) two\u2010step extraction of isopropanol\u2010methanol followed by methanol\u2013water. 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\u2010methanol followed by methanol\u2013water 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\u2010fat intervention. In summary, our protocol enabled a comprehensive and systematic evaluation of liver pretreatment to obtain more medium\u2010polar and nonpolar metabolites and was suitable for high\u2010throughput metabolomics analysis.<\/jats:p>","DOI":"10.1002\/jssc.202100051","type":"journal-article","created":{"date-parts":[[2021,6,15]],"date-time":"2021-06-15T08:46:50Z","timestamp":1623746810000},"page":"3450-3461","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Evaluation of liver tissue extraction protocol for untargeted metabolomics analysis by ultra\u2010high\u2010performance liquid chromatography\/tandem mass spectrometry"],"prefix":"10.1002","volume":"44","author":[{"given":"Zhipeng","family":"Liu","sequence":"first","affiliation":[{"name":"National Key Discipline Laboratory Department of Nutrition and Food Hygiene School of Public Health Harbin Medical University  Harbin P. R. 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