Abstract
Dicaffeoylquinic acids (diCQAs) are plant metabolites and undergo trans-cis-isomerization when exposed to UV irradiation. As such, diCQAs exist in both trans- and cis-configurations and amplify the already complex plant metabolome. However, analytical differentiation of these geometrical isomers using mass spectrometry (MS) approaches has proven to be extremely challenging. Exploring the chromatographic space to develop possible conditions that would aid in differentially separating and determining the elution order of these isomers is therefore imperative. In this study, simple chromatographic parameters, such as column chemistry (phenyl versus alkyl), mobile phase composition (methanol or acetonitrile), and column temperature, were investigated to aid in the separation of diCQA geometrical isomers. The high-performance liquid chromatography photodiode array (HPLC-PDA) chromatograms revealed four isomers post UV irradiation of diCQA authentic standards. The elution profile/order was seen to vary on different reverse-phase column chemistries (phenyl versus alkyl) using different mobile phase composition. Here, the elution profile/order on the phenyl-derived column matrices (with methanol as the mobile phase composition) was observed to be relatively reproducible as compared to the alkyl (C18) columns. Chromatographic resolution of diCQA geometrical isomers can be enhanced with an increase in column temperature. Lastly, the study highlights that chromatographic elution order/profile cannot be relied upon to fathom the complexity of isomeric plant metabolites.
Highlights
Dicaffeoylquinic acids are plant secondary metabolites that are part of the family of bioactive metabolites called chlorogenic acids
It is reported that hydroxycinnamic acid (HCA) derivatives such as caffeic acid are initially synthesized in the transconfiguration through the phenylpropanoid pathway [3]
Positional isomers of Dicaffeoylquinic acids (diCQAs) (Scheme 1) were UV irradiated, and the resulting samples were analyzed under reverse-phase chromatographic conditions using nine different column chemistries
Summary
Dicaffeoylquinic acids (diCQAs) are plant secondary metabolites that are part of the family of bioactive metabolites called chlorogenic acids. Authentic standards of most of the HCA derivatives (trans-isomers), such as diCQAs, are commercially available, and these standards can be used to produce their ciscounterparts through the process of photoisomerization [4, 6, 12, 14, 15] Identification of these related geometrical compounds using analytical techniques such as liquid chromatography linked to mass spectrometry (LC-MS) has proven impossible as they produce similar/identical MS fragmentation patterns [4]. Insight on the chromatographic elution order of these metabolites (both transand cis-isomers) will contribute to ongoing efforts in designing analytical methods for differential identification of isomers contributing to plant sample dimensionality Such chromatographic separation efforts will further allow collection (LC fractionation) of these peaks (metabolites) to study their respective bioactivity differences
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