Quantitative Metabolite Profiling Utilizing Parallel Column Analysis for Simultaneous Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Separations Combined with Tandem Mass Spectrometry
Quantitative Metabolite Profiling Utilizing Parallel Column Analysis for Simultaneous Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Separations Combined with Tandem Mass Spectrometry
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DOI：
10.1021/ac5003454
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发表时间：
2014-05-06
影响因子：
7.4
通讯作者：
Koellensperger, Gunda
中科院分区：
文献类型：
--
作者：
Klavins, Kristaps;Drexler, Hedda;Koellensperger, Gunda
在这项工作中，建立了一种全自动平行LC柱方法，以便在一次分析运行中进行正交亲水相互作用色谱（HILIC）和反相（RPLC）色谱，用于中心碳代谢产物的靶向定量质谱测定。以这种方式，与先前建立的涉及两个单独的分析运行的双重分离工作流程相比，可以显著提高分析通量。将两份样品等分试样同时进样到使用十端口阀的双柱设置柱上，并进行平行分离。两种分离方法均采用粒径小于2 μ m的固定相。柱后合并HILIC和RPLC洗脱液，然后进行ESI-MS/MS检测。优化了正交分离条件，使反相分离在5.5min内完成，总分离时间为2个保留时间段;总运行时间为15 min。该装置用于定量30种主要细胞间代谢物，包括氨基酸，有机酸，和核苷酸，其使用完全C-13标记的酵母提取物进行内标化。与HILIC MS/MS和RPLC MS/MS在单独的分析运行中的比较表明，通过平行LC柱方法实现了优异的分析性能。实验重复性（N = 5）平均为5%。此外，新方法的检测限范围为0.002-15 μ M，与在单独的HILIC MS/MS和RPLC MS/MS运行中获得的检测限（范围为0.01-44 μ M）一致。
In this work, a fully automated parallel LC column method was established in order to perform orthogonal hydrophilic interaction chromatography (HILIC) and reversed-phase (RPLC) chromatography within one analytical run for targeted quantitative mass spectrometric determination of metabolites from central carbon metabolism. In this way, the analytical throughput could be significantly improved compared to previously established dual separation work flows involving two separate analytical runs. Two sample aliquots were simultaneously injected onto a dual column setup columns using a ten-port valve, and parallel separations were carried out. Sub 2 pm particle size stationary phases were employed for both separation methods. HILIC and RPLC eluents were combined post column followed by ESI-MS/MS detection. The orthogonal separations were optimized, aiming at an overall separation with 2 retention time segments, while reversed-phase separation was accomplished within 5.5 min; metabolites on the HILIC phase were retained for a minimum time of 6 min. The overall run time was 15 min. The setup was applied to the quantification of 30 primary intercellular metabolites, including amino acids, organic acids, and nucleotides employing internal standardization by a fully C-13-labeled yeast extract. The comparison with HILIC MS/MS and RPLC MS/MS in separate analytical runs revealed that an excellent analytical performance was achieved by the parallel LC column method. The experimental repeatability (N = 5) was on average 5%). Moreover, limits of detection for the new approach ranging from 0.002-15 mu M were in a good agreement with ones obtained in separate HILIC MS/MS and RPLC MS/MS runs (ranging from 0.01-44 mu M).