Extensive evaluation of sample preparation workflow for gas chromatography-mass spectrometry-based plasma metabolomics and its application in rheumatoid arthritis,Analytica Chimica Acta

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Extensive evaluation of sample preparation workflow for gas chromatography-mass spectrometry-based plasma metabolomics and its application in rheumatoid arthritis
Analytica Chimica Acta ( IF 5.7 ) Pub Date : 2020-09-01 , DOI: 10.1016/j.aca.2020.06.029
Zhuoru He ₁ , Qing Luo ₁ , Zhongqiu Liu ₁ , Lingzhi Gong ₁

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GC-MS platform has been proved to be an important analytical technique for plasma metabolomics, but lack of efficient sample preparation strategies prior to sample injection has limited its wide application. In the present work, twenty two extraction protocols including protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE) and ultrafiltration were simultaneously evaluated using non-spiked and metabolite standards spiked human plasma. Sample-to-extraction solvent ratio and metabolites derivatization conditions were also investigated and optimised. The results demonstrated that stepwise LLE protocol (MeOH-H2O/CHCl3/CHCl3-MeOH) was the most efficient extraction strategy for the combination of untargeted and targeted metabolomics. Sample-to-extraction solvent ratio of 1:3 (v:v) was found to perform better than higher solvent ratios. Also, the maximum derivatization performance was obtained when using 30 μL N-methyl-N-tri-methylsilyltrifluoroacetamide (MSTFA) and 1% trimethyl-chlorosilane (TMCS) incubated at 60 °C for 120 min. In the end, the optimised sample preparation method was applied successfully to plasma untargeted metabolomics of rheumatoid arthritis (RA) and four metabolites (gamma-butyrolactone, MG (0:0/18:0/0:0), tocopherol and oxalic acid) were found to be potential biomarkers for the diagnosis of RA.

中文翻译：

基于气相色谱-质谱的血浆代谢组学样品制备工作流程的广泛评估及其在类风湿性关节炎中的应用

GC-MS 平台已被证明是血浆代谢组学的重要分析技术，但在进样前缺乏有效的样品制备策略限制了其广泛应用。在目前的工作中，包括蛋白质沉淀 (PPT)、液-液萃取 (LLE)、固相萃取 (SPE) 和超滤在内的 22 种提取方案同时使用非加标和代谢物标准加标人血浆进行评估。还研究并优化了样品与萃取溶剂的比例和代谢物衍生条件。结果表明，逐步 LLE 方案 (MeOH-H2O/CHCl3/CHCl3-MeOH) 是结合非靶向和靶向代谢组学的最有效提取策略。样品与萃取溶剂的比例为 1:3 (v: v) 被发现比更高的溶剂比表现更好。此外，当使用 30 μL N-甲基-N-三甲基甲硅烷基三氟乙酰胺 (MSTFA) 和 1% 三甲基-氯硅烷 (TMCS) 在 60 °C 下孵育 120 分钟时，获得了最大的衍生化性能。最后，优化的样品制备方法成功应用于类风湿性关节炎 (RA) 和四种代谢物（γ-丁内酯、MG（0:0/18:0/0:0）、生育酚和草酸）的血浆非靶向代谢组学被发现是诊断 RA 的潜在生物标志物。

更新日期：2020-09-01

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