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 mu L.

Automated summary

We developed an integrated workflow combining hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography to cover polar and nonpolar metabolites with only one sample injection. This approach involves trapping compounds eluted from the first chromatographic dimension for later separation in the second dimension. Through optimization of chromatographic and ionization conditions, we developed two independent miniaturized one-dimensional HILIC and RP methods, which were then merged into a two-dimensional micro LC-MS method by optimizing the trapping conditions. This approach reduces analysis time and allows for the detection of hundreds of metabolites without significant loss.