Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although they are non-biased and have high coverage, untargeted approaches suffer from unsatisfying repeatability and a requirement for complex data processing. Targeted metabolomics based on triple-quadrupole mass spectrometry (TQMS) could be a complementary tool because of its high sensitivity, high specificity and excellent quantification ability. However, it is usually applicable to known compounds: compounds whose identities are known and/or are expected to be present in the analyzed samples. Pseudotargeted metabolomics merges the advantages of untargeted and targeted metabolomics and can act as an alternative to the untargeted method. Here, we describe a detailed protocol of pseudotargeted metabolomics using UHPLC-TQMS. An in-depth, untargeted metabolomics experiment involving multiple UHPLC-HRMS runs with MS at different collision energies (both positive and negative) is performed using a mixture obtained using small amounts of the analyzed samples. XCMS, CAMERA and Multiple Reaction Monitoring (MRM)-Ion Pair Finder are used to find and annotate peaks and choose transitions that will be used to analyze the real samples. A set of internal standards is used to correct for variations in retention time. High coverage and high-performance quantitative analysis can be realized. The entire protocol takes ~5 d to complete and enables the simultaneously semiquantitative analysis of 800–1,300 metabolites.

非靶向方法通常用于代谢组学研究中小分子有机化合物的检测和发现，而超高效液相色谱-高分辨率质谱 (UHPLC-HRMS) 是非靶向代谢组学最常用的平台之一。尽管它们没有偏见并且具有高覆盖率，但非目标方法的可重复性和复杂数据处理要求不尽如人意。基于三重四极杆质谱 (TQMS) 的靶向代谢组学具有高灵敏度、高特异性和出色的定量能力，可以成为一种补充工具。然而，它通常适用于已知化合物：其身份已知和/或预期存在于分析样品中的化合物。伪靶向代谢组学融合了非靶向和靶向代谢组学的优点，可以作为非靶向方法的替代方法。在这里，我们描述了使用 UHPLC-TQMS 的伪靶向代谢组学的详细方案。一项深入的非靶向代谢组学实验涉及多次 UHPLC-HRMS 在不同碰撞能量（正碰撞能量和负碰撞能量）下使用 MS 运行，使用由少量分析样品获得的混合物。XCMS、CAMERA 和多反应监测 (MRM) - 离子对查找器用于查找和注释峰并选择将用于分析实际样品的离子对。一组内部标准用于校正保留时间的变化。可实现高覆盖率和高性能的定量分析。