Untargeted metabolomics studies aim to extract a broad coverage of metabolites from biological samples, which largely depends on the sample preparation protocols used for metabolite extraction. The aim of this study was to evaluate a comprehensive sample pretreatment strategy using two-step liquid-liquid extraction to achieve broader metabolome coverage by ultra-high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (UPLC-Q-TOF-MS). We compared four protocols: (A) methanol protein precipitation, (B) Ostro 96-well plates, (C) two-step extraction protocol of CHCL3-MeOH followed by MeOH-H2O, and (D) two-step extraction protocol of CH2CL2-MeOH followed by MeOH-H2O. The number of extracted features, reproducibility and recovery were the major criteria for evaluation. Our results demonstrated that Protocols B, C and D, with approximately similar number of features, extracted more features than Protocol A. Protocols C and D appeared to have similar extraction reproducibility (low coefficient of variation < 30%) and Protocol D enabled an acceptable recovery of serum metabolites. The two-step extraction Protocol D (CH2CL2-MeOH followed by MeOH-H2O) resulted in the greatest improvement in metabolite coverage, satisfactory extraction reproducibility, acceptable recovery and environmental safety. The selected protocol was applied to an obesity metabolomics study to obtain different metabolites between participants with obesity and the controls, and to investigate complex metabolic alterations in obesity during a 2-h oral glucose-tolerance test. Our results suggested that this protocol was useful for analyzing serum metabolome changes in obese individuals in the fasting and postprandial state.

中文翻译：

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UPLC-Q-TOF-MS 对血清样品非靶向代谢分析的两步液-液提取方案进行评估，以实现更广泛的代谢组覆盖

非靶向代谢组学研究旨在从生物样品中提取广泛的代谢物，这在很大程度上取决于用于代谢物提取的样品制备方案。本研究的目的是评估使用两步液-液萃取的综合样品预处理策略，以通过超高效液相色谱结合电喷雾电离飞行时间质谱 (UPLC-Q-飞行时间质谱）。我们比较了四种方案：(A) 甲醇蛋白沉淀，(B) Ostro 96 孔板，(C) CHCL3-MeOH 和 MeOH-H2O 的两步提取方案，以及 (D) CH2CL2 的两步提取方案-MeOH，然后是 MeOH-H2O。提取特征的数量、重现性和回收率是评价的主要标准。我们的结果表明，方案 B、C 和 D 的特征数量大致相似，提取的特征比方案 A 多。方案 C 和 D 似乎具有相似的提取重现性（低变异系数 < 30%），方案 D 实现了可接受的血清代谢物的恢复。两步提取方案 D（CH2CL2-MeOH，然后是 MeOH-H2O）在代谢物覆盖率、令人满意的提取重现性、可接受的回收率和环境安全方面取得了最大的改善。选定的方案被应用于肥胖代谢组学研究，以获得肥胖参与者和对照组之间的不同代谢物，并在 2 小时口服葡萄糖耐量试验期间研究肥胖的复杂代谢改变。