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（LC-MS）的代谢组学通常结合亲水相互作用液相色谱（HILIC）和反相色谱（RP）分离技术，以覆盖广泛的代谢物谱，这要求在单独的工艺流程中消耗大量的样品和分析时间。本研究开发了一种集成工作流程，通过使用心切割捕获技术，仅通过一次进样即可覆盖极性和非极性代谢物，分别针对不同的电离模式。该方法允许在第二维度的分离过程中捕获从第一维度的色谱中洗脱出的某些化合物。在我们的案例中，我们对第一维度的非保留代谢物进行了心切割处理。为此，我们开发了两种独立的微型一维HILIC和RP方法，通过优化色谱和电离条件，并使用内径为1毫米的柱子。然后，通过优化捕获条件，将它们合并为一种二维微液相色谱-质谱联用方法。在反相色谱柱洗脱过程中对HILIC柱进行平衡，反之亦然，从而减少了整体分析时间，多维度特性使我们能够在溶剂前沿避免信号测量。为了展示该方法在代谢组学中的应用优势，我们对人体血浆标准参考物质SRM 1950进行了分析，实现了数百种代谢物的检测，同时显著减少了其中一些代谢物的损失，且仅需0.5微升的进样量。