# The benefits of multi-2D LC × LC compared to LC × LC for the analysis of European herbal remedies

## TL;DR

This paper compares advanced chromatography methods to analyze complex compounds in European medicinal plants, showing improved results with a new multi-2D approach.

## Contribution

The study introduces a novel multi-2D LC × LC platform that significantly enhances separation efficiency for analyzing herbal remedy metabolites.

## Key findings

- The multi-2D LC × LC method achieved 91% higher peak capacity compared to traditional LC × LC.
- It improved orthogonality by 8.2% and peak distribution by 30%.
- The method enabled detailed chemical characterization of phenolic compounds in five medicinal plants.

## Abstract

Due to the demand in alternative medicine to treat various diseases, medicinal plants as ancient remedies gained more recent interest for the discovery and isolation of active compounds (Int J Environ Res Public Health. 2020;17(10):3376, J Ethnopharmacol. 2017;199:161-167). As these plant matrices contain an enormous range of metabolites, the challenge lies in the selection and application of separation techniques capable of resolving such complexity. For this reason, comprehensive two-dimensional liquid chromatography (LC × LC), due to the increased peak capacity compared to one-dimensional LC, was firstly selected for the chemical characterization of five European medicinal plants (Angelica archangelica, Angelica sylvestris, Agrimonia eupatoria, Sambucus ebulus, and Sambucus nigra). For the method development, complimentary columns were chosen, optimized, and compared regarding peak capacity, orthogonality, and peak distribution. Moreover, a platform with even higher separation power was chosen using a PFP column in the first dimension and two complimentary columns in the second dimension, ZIC-HILIC from 0 to 20 min and polar C18 from 20 min until 60 min at the end, called multi-2D LC × LC. The optimized method resulted in a gain of 91% for the peak capacity, 8.2% for orthogonality, and 30% better peak distribution compared to the LC × LC methods. The hyphenation to high-resolution mass spectrometry enabled the chemical characterization of phenolic compounds and their distribution among the plants as well as potential marker substances for authentication purposes, providing the first comparative study of these five European herbal remedies.

The online version contains supplementary material available at 10.1007/s00216-025-06278-0.