The acquisition of high-quality quantitative dataset is the prerequisite for large-scale targeted metabolomics. However, the goal is usually dampened by the complexity of the biological matrices in terms of structural diversity, concentration span, and polarity range. We attempted herein to propose an analytical approach being able to circumvent these technical obstacles, and medicinal bile was employed as a proof of concept. In the liquid chromatography (LC) domain, reversed phase LC and hydrophilic liquid interaction chromatography were coupled in series, namely RPLC-HILIC, to yield appropriate chromatographic behavior for each component regardless of the polarity. In-depth chemical characterization and widely quantitative analysis were successively conducted in the mass spectrometry (MS) domain. Hybrid ion trap-time of flight MS was jointly deployed with hybrid triple quadrupole-linear ion trap MS for metabolite searching because of their orthogonal natures. Afterwards, a tailored MRM strategy that integrated online parameter optimization, ion intensity tailoring, and scheduled monitoring, was subsequently constructed to accomplish comprehensive quantitation although authentic compounds weren't involved and concentration span was quite large. Calibration curve was constructed for each detected identity by preparing and serially diluting the universal metabolome standard (UMS) solution that merged chemical information from all bile samples. Quasi-contents of 164 components including bile acids, sterols, eicosanoids, amino acids, lipids, flavonoids, and so forth, were gained by applying those regressive calibration curves and replaced the role of peak areas to serve as the variables for multivariate statistical analysis. In particular, the concentration ratio between tauroursodeoxycholic acid (TUDCA) and taurohyodeoxycholic acid (THDCA) that were exactly co-eluted as a single peak was calculated from the intensity specific value of m/z 498 > 80 over 498 > 124. Different metabolome occurred among various animal bile samples, and significant variations were also observed for different batches of bear bile powders. Bile acids acted as the indicative components in either discrimination. Overall, RPLC-HILIC-tailored MRM enabled quantitative metabolome profiling of medicinal bile and was thereby a promising analytical tool for large-scale targeted metabolomics.

中文翻译：

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串联耦合反相-亲水相互作用液相色谱-定制的多反应监测，适合药用胆汁大规模靶向代谢组学的工具

获得高质量的定量数据集是大规模靶向代谢组学的先决条件。然而，目标通常会受到生物基质在结构多样性、浓度跨度和极性范围方面的复杂性的阻碍。我们在此尝试提出一种能够绕过这些技术障碍的分析方法，并采用药用胆汁作为概念证明。在液相色谱 (LC) 领域，反相 LC 和亲水性液体相互作用色谱串联耦合，即 RPLC-HILIC，无论极性如何，都能为每个组分产生适当的色谱行为。在质谱 (MS) 领域相继进行了深入的化学表征和广泛的定量分析。由于其正交性质，混合离子阱飞行时间 MS 与混合三重四极杆线性离子阱 MS 联合部署用于代谢物搜索。之后，在不涉及真实化合物且浓度跨度相当大的情况下，随后构建了一个量身定制的 MRM 策略，该策略集成了在线参数优化、离子强度定制和预定监测，以完成综合定量。通过制备和连续稀释通用代谢组标准 (UMS) 溶液，为每个检测到的身份构建校准曲线，该溶液合并了所有胆汁样本的化学信息。胆汁酸、甾醇、类二十烷酸、氨基酸、脂类、黄酮类等164种成分的准含量，通过应用这些回归校准曲线获得，并取代峰面积的作用作为多变量统计分析的变量。特别是，从 m/z 498 > 80 超过 498 > 124 的强度比值计算出作为单峰精确共洗脱的牛磺熊去氧胆酸 (TUDCA) 和牛磺猪去氧胆酸 (THDCA) 之间的浓度比。发生了不同的代谢组在各种动物胆汁样本中，不同批次的熊胆粉也存在显着差异。胆汁酸在任一种区分中都充当指示性成分。总体而言，RPLC-HILIC 定制的 MRM 实现了药用胆汁的定量代谢组分析，因此是大规模靶向代谢组学的有前途的分析工具。