# Comparative evaluation of reversed-phase and hydrophilic interaction liquid chromatography columns for untargeted profiling of bioactive compounds in Hypericum perforatum

## TL;DR

This study compares reversed-phase and hydrophilic interaction liquid chromatography columns for analyzing bioactive compounds in Hypericum perforatum, aiming to improve metabolite profiling.

## Contribution

The novelty lies in the comparative evaluation of RPLC and HILIC columns for untargeted profiling of diverse metabolites in plant extracts.

## Key findings

- RPLC and HILIC columns showed complementary performance in resolving isobaric compound pairs.
- Integration of RPLC and HILIC data improved the comprehensive characterization of metabolites in Hypericum perforatum.
- Different stationary phase chemistries significantly impacted chromatographic performance and compound coverage.

## Abstract

The achievement of a comprehensive profiling of plant metabolites has long represented a challenge, not only due to their wide-ranging abundances but also as a result of their considerable chemical diversity. Recent advances in highly sensitive liquid chromatographic (LC) techniques, particularly when coupled with high-resolution mass spectrometry (HRMS), have established metabolomics as a key approach for the analysis of thousands of non-volatile metabolites in crude natural extracts. Nevertheless, the different polarities of primary and secondary metabolites often limit the efficacy of conventional reversed-phase liquid chromatography (RPLC) in providing exhaustive compound coverage. To address this limitation, orthogonal separation techniques such as hydrophilic interaction liquid chromatography (HILIC) should be employed as a complement to RPLC. In this work, four columns with identical geometrical specifications but with different stationary phase chemistry (one reversed-phase C18 and three different HILIC adsorbents) were employed for the untargeted analysis of bioactive compounds contained in Hypericum perforatum. The columns were evaluated not only in terms of chromatographic performance but also based on their ability to resolve challenging isobaric compound pairs of isobaric compounds. Finally, the integration of RPLC and HILIC data enabled a more comprehensive characterization of the metabolites associated with the plant.

The online version contains supplementary material available at 10.1007/s00216-025-06030-8.