LC-MS metabolomics and chemotaxonomy of Ilex species

Ilex is a cosmopolitan monotypic genus in the Aquifoliaceae with centers of diversity in Asia and South America. Several species have been used traditionally in the preparation of medicinal teas for treating and preventing various ailments, while others are consumed primarily for their caffeine content. In order to assess the evolution of metabolites in caffeine-containing Ilex species, leaf samples of 41 Ilex species and hybrids were collected, processed, and extracted for analyses by UPLC-TQD and UPLC-qToF mass spectrometry. Multiple reaction monitoring of compounds and their major mass fragments were developed to detect methylxanthines in leaf extracts at lower limits of detection between 1 – 10 ng/mL. Three methylxanthines (caffeine, theobromine, and theophylline) and the methyluric acid, theacrine, were quantified in I. guayusa, I. paraguariensis, and I. vomitoria. Ilex guayusa showed the highest content of caffeine, followed by I. paraguariensis and I. vomitoria. Though there have been reports of caffeine content in I. cassine, we were not able to detect caffeine nor other methylxanthines using both UPLC-TQD-MS and UPLC-qToF-MS. Chlorogenic acid and three of its isomers were quantified to compare total chlorogenic acid content across all species in this study. The results showed that all caffeine-containing Ilex species also had high concentrations of chlorogenic acids. Ilex guayusa, I. paraguariensis, and I. vomitoria were compared with Ilex cassine using UPLC-qToF-MS and the data analyzed using PCA, OPLS-DA, and heat-maps to visualize and identify differences in metabolite profiles among these species. The results revealed triterpenoid saponins as chemotaxonomically informative marker compounds in caffeine-containing Ilex species.