Comprehensive analysis of pig feces metabolome by chromatographic techniques coupled to mass spectrometry in high resolution mode: Influence of sample preparation on the identification coverage
Comprehensive analysis of pig feces metabolome by chromatographic techniques coupled to mass spectrometry in high resolution mode: Influence of sample preparation on the identification coverage
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DOI：
10.1016/j.talanta.2019.02.073
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发表时间：
2019-07-01
期刊：
影响因子：
6.1
通讯作者：
Priego-Capote, F.
中科院分区：
文献类型：
--
作者：
Lopez-Bascon, M. A.;Calderon-Santiago, M.;Priego-Capote, F.
猪粪便是一种有趣的生物样品，可以通过宿主与微生物组之间的相互作用推断出信息，从而在代谢组学实验中实施。然而，猪粪便样本很少受到关注，特别是在非靶向代谢组学研究中。在本研究中，计划了一种分析策略，以最大限度地提高猪粪便样品中发现的代谢物的识别覆盖率。为此，使用两个互补平台，如LC-QTOF MS/MS和GC-TOF/MS。关于样品制备，测试了具有不同极性等级的六种浸提剂溶剂以评价浸提性能，并且在GC-MS的特定情况下，比较了两种衍生化方案。通过所有浸提剂和分析平台的组合，初步鉴定了总共303种化合物。主要鉴定的家族是氨基酸、脂肪酸及其衍生物、碳水化合物和羧酸。对于GC-TOF/MS分析，推荐的提取剂是甲醇，而在衍生化方案中需要甲氧基化，因为该步骤允许检测α-酮酸，其是微生物组状态的直接标志物。关于LC-QTOF MS/MS分析，提出了甲醇（MeOH）或MeOH/水和乙酸乙酯的双重提取方法，以增强极性和非极性代谢产物的检测。
Pig feces is an interesting biological sample to be implemented in metabolomics experiments by virtue of the information that can be deduced from the interaction between host and microbiome. However, pig fecal samples have received scant attention, especially in untargeted metabolomic studies. In this research, an analytical strategy was planned to maximize the identification coverage of metabolites found in pig fecal samples. For this purpose, two complementary platforms such as LC-QTOF MS/MS and GC-TOF/MS were used. Concerning sample preparation six extractant solvents with different polarity grade were tested to evaluate the extraction performance and, in the particular case of GC-MS, two derivatization protocols were compared. A total number of 303 compounds by combination of all the extractants and analytical platforms were tentatively identified. The main identified families were amino acids, fatty acids and derivatives, carbohydrates and carboxylic acids. For GC-TOF/MS analysis, the recommended extractant is methanol, while methoxymation was required in the derivatization protocol since this step allows detecting the a-keto acids, which are direct markers of the microbiome status. Concerning LC-QTOF MS/MS analysis, a dual extraction approach with methanol (MeOH) or MeOH/water and ethyl acetate is proposed to enhance the detection of polar and non-polar metabolites.