反相/亲水相互作用双模式交替液相色谱-串联质谱分离系统的构建及其在代谢物全谱研究中的应用  

Construction of an alternating reversed-phase and hydrophilicinteraction liquid chromatography-tandem massspectrometry dual mode chromatography system and itsapplication in comprehensive metabolite profiling analysis

作　　者：黄燕 张兴 蒋婷婷 周雅雯 江鹏 尹红峰 唐涛 HUANG Yan;ZHANG Xing;JIANG Tingting;ZHOU Yawen;JIANG Peng;YIN Hongfeng;TANG Tao(Zhejiang Fuli Analytical Instruments Co.,Ltd.,Taizhou 317500,China)

机构地区：[1]浙江福立分析仪器有限公司,浙江台州317500

出　　处：《色谱》2024年第12期1145-1152,共8页Chinese Journal of Chromatography

基　　金：浙江省重点研发计划项目(2021C01128)~~。

摘　　要：生物样品通常具有基质复杂、种类繁多、理化性质差异极大等特征,采用一种分离模式通常只能得到样品的部分信息,发展多模式结合的分析方法,对于样品的全景特征分析、生物标志物发现皆具有重要意义。本文将HILIC和RPLC两根色谱柱与一台自动进样器、两个输液泵结合,并耦合到质谱仪上采集数据,构建了一种基于单进样器的HILIC/RPLC-MS/MS双模式交替分离系统。构建的系统通过一个两位六通阀和一个两位十通阀使自动进样器始终与质谱仪在同一流路上,但是与HILIC和RPLC两根色谱柱交替相连。当自动进样器与HILIC色谱柱相连时,HILIC运行梯度洗脱,RPLC处于平衡状态,反之亦然,使得总分析时间减少约40%,而且可避免数据集重叠。采用该系统对14种代表性的代谢物(包括有机酸、氨基酸、胆碱和溶血磷脂等)进行了分析,结果表明,相比于RPLC-MS/MS单模式系统和HILIC-MS/MS单模式系统,HILIC/RPLC-MS/MS双模式交替分离系统覆盖的代谢物数量更多。在尿液基质中,14种代谢物的定量线性关系均较好,相关系数(R2)大于0.99,方法的检出限和定量限分别为0.02~42.86 ng/mL和0.08~142.86 ng/mL,在低、中、高3个加标水平下的回收率为85.2%~113.9%,日内、日间精密度(RSD)小于10%。该系统仪器配置简单,操作便捷,实用性强,适合在临床上用于代谢物全谱研究,也可用于蛋白质组学、糖组学的研究,尤其适合大队列样本的组学分析,具有广阔的应用前景。As one of the youngest“omics”disciplines,metabolomics,more specifically,metabolite profiling,enables the comprehensive quantitative analysis of a primary metabolome.At present,comprehensive metabolite profiling analysis is a challenging task owing to the greatly different physicochemical properties of metabolites and the complexity of sample matrices,especially given the wide variety of biological samples.Consequently,the development of a multimodal separation method with higher metabolite coverage than single-mode chromatography is necessary for comprehensive metabolite profiling analysis and biomarker discovery.Reversed-phase liquid chromatography(RPLC)and hydrophilic interaction liquid chromatography(HILIC)are used to analyze nonpolar and polar metabolites,respectively.HILIC and RPLC separation results are commonly combined to achieve comprehensive metabolite profiling analysis.However,this practice leads to overlapping datasets and extended analysis times.Alternatively,multiple LC systems can be coupled to generate combined HILIC and RPLC separation results.In this study,we developed an alternating HILIC/RPLC-tandem mass spectrometry(MS/MS)dual-mode separation system consisting of one autosampler,two high-pressure LC pumps,a HILIC column,an RP column,a 6-port 2-position switching valve,a 10-port 2-position switching valve,and a mass spectrometer for the comprehensive and high-throughput analysis of polar and nonpolar metabolites.In this configuration,the autosampler is always on the same flow path as the mass spectrometer,whereas the HILIC and RP columns are switched in and out of the main flow path.At the initial stage of the analysis,the autosampler is connected to a HILIC column,followed by the mass spectrometer.While gradient elution is performed on the HILIC column,the RP column is equilibrated using the second high-pressure pump.After HILIC separation,the 6-port 2-position and 10-port 2-position switching valves are switched simultaneously.At this stage of the analysis,the autosampler is connected t

关 键 词：反相液相色谱 亲水相互作用液相色谱 代谢物覆盖度 代谢谱分析 双模式分离 

分 类 号：O658[理学—分析化学]