Revising Reverse-Phase Chromatographic Behavior for Efficient Differentiation of Both Positional and Geometrical Isomers of Dicaffeoylquinic Acids

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. e high-performance liquid chromatography photodiode array (HPLC-PDA) chromatograms revealed four isomers post UV irradiation of diCQA authentic standards. e 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 (C 18 ) 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.

Profiling of Chlorogenic Acids from Bidens pilosa and Differentiation of Closely Related Positional Isomers with the Aid of UHPLC-QTOF-MS/MS-Based In-Source Collision-Induced Dissociation

“…Differences observed in the intensities of fragment ions can be ascribed to variances in energy distribution which cause the structurally similar isomers to behave differently under the described MS conditions. On a reverse-phase column the elution order of di-CQA regio-isomers is expected to be as follows: 3,4-di-CQA, 3,5-di-CQA, followed by 4,5-di-CQA eluting the latest, validating the annotation of di-CQAs shown in Table 1 [21,29]. These di-CQA were differentially present throughout tissues and cell cultures of B. pilosa.…”

“…Metabolite (10) was identified as CA (Figure S7B) by its molecular ion [M-H] -, at m/z 473 which fragmented to give product ions at m/z 311 [CTA-H]due to the loss of a second caffeic acid residue. Other daughter ions at m/z 149 [TA-H]and m/z 135 [CFA-CO 2 ]were also observed [29]. Profiling of the tartaric esters revealed that they are only present in the aerial parts of the plants and absent in the roots.…”

“…Identification and quantification of structurally related compounds is challenging and may require the use of reliable advanced analytical techniques. In previous studies, geometrical isomers of CGAs have been differentiated using elution order, but this has been shown to be unreliable [23], mainly due to differences in column chemistries [29]. Discrimination between geometrical isomers has been achieved by liquid chromatography-mass spectrometry (LC-MS) through the use of in-source collision induced dissociation (ISCID), where cis-isomers of di-CQAs were shown to preferentially bind to alkali metals (sodium, lithium, and potassium adduct-MS), differentiating them from trans-isomers [30].…”

Synchronized Survey Scan Approach Allows for Efficient Discrimination of Isomeric and Isobaric Compounds during LC-MS/MS Analyses

“…Plants produce a myriad of organic compounds referred to as secondary metabolites (natural products) which differ in their structure and biosynthetic origins. These metabolites undergo chemical modifications, such as conjugation [ 1 , 2 ], and isomerization (positional and geometrical) [ 3 – 6 ], which further contribute to the high complexity of the plant metabolome. For instance, secondary metabolites such as hydroxycinnamic acid (HCA) derivatives have the potential to form conjugates with organic acids such as isocitric acid [ 1 ], tartaric acid [ 2 , 7 – 9 ], and quinic acid [ 4 , 10 , 11 ], thus forming hydroxycinnamoyl-isocitric acid [ 1 ], hydroxycinnamoyl-tartaric acid [ 2 , 7 – 9 ], and hydroxycinnamoyl-quinic acid [ 4 , 10 , 11 ], respectively.…”

“…In addition, these HCA derivatives undergo isomerization to produce positional isomers such as di-acylated hydroxycinnamoyl-quinic acid derivatives like 1,3-dicaffeoylquinic acid (1,3-diCQA) or 1,5-diCQA [ 3 – 6 ]. As such, some HCA conjugates have been found to result in isobaric compounds which produce similar mass spectrometry (MS) fragmentation patterns [ 1 ], and this renders identification challenging from an analytical perspective.…”