Miniaturized Two-Dimensional Heart Cutting for LC–MS-Based Metabolomics

Liquid chromatography–mass spectrometry (LC–MS)-based
metabolomics usually combines hydrophilic interaction liquid chromatography
(HILIC) and reversed-phase (RP) chromatography to cover a wide range
of metabolomes, requiring both significant sample consumption and
analysis time for separate workflows. We developed an integrated workflow
enabling the coverage of both polar and nonpolar metabolites with
only one injection of the sample for each ionization mode using heart-cutting
trapping to combine HILIC and RP separations. This approach enables
the trapping of some compounds eluted from the first chromatographic
dimension for separation later in the second dimension. In our case,
we applied heart-cutting to non-retained metabolites in the first
dimension. For that purpose, two independent miniaturized one-dimensional
HILIC and RP methods were developed by optimizing the chromatographic
and ionization conditions using columns with an inner diameter of
1 mm. They were then merged into one two-dimensional micro LC–MS
method by optimization of the trapping conditions. Equilibration of
the HILIC column during elution on the RP column and vice versa reduced
the overall analysis time, and the multidimensionality allows us to
avoid signal measurements during the solvent front. To demonstrate
the benefits of this approach to metabolomics, it was applied to the
analysis of the human plasma standard reference material SRM 1950,
enabling the detection of hundreds of metabolites without the significant
loss of some of them while requiring an injection volume of only 0.5
μL.

基于液相色谱-质谱联用（Liquid chromatography–mass spectrometry, LC–MS）的代谢组学通常结合亲水相互作用液相色谱（hydrophilic interaction liquid chromatography, HILIC）与反相（reversed-phase, RP）色谱，以覆盖宽泛的代谢物谱，但需分别运行两套独立流程，导致样品消耗量与分析时长均显著增加。本研究开发了一套集成化分析流程，借助中心切割捕集技术整合HILIC与RP分离模式，仅需针对每种电离模式单次进样，即可同时覆盖极性与非极性代谢物。该方法可将第一维色谱洗脱的部分组分捕集，待后续在第二维色谱中完成分离。在本研究中，我们将中心切割技术应用于第一维色谱中的非保留代谢物。为此，我们通过优化内径为1 mm的色谱柱的色谱与电离参数，分别建立了两套独立的微型一维HILIC与RP分析方法；随后通过优化捕集条件，将两套方法整合为一套二维微型LC–MS分析方法。在RP色谱柱洗脱过程中同步平衡HILIC色谱柱，反之亦然，该操作缩短了整体分析时长；同时多维分离策略可避免在溶剂前沿进行信号采集。为验证该方法在代谢组学研究中的应用优势，我们将其应用于人类血浆标准参考物质SRM 1950的分析，实现了数百种代谢物的检出，且未显著丢失目标代谢物，同时仅需0.5 μL的进样体积。