Highlighting mass spectrometric fragmentation differences and similarities between hydroxycinnamoyl-quinic acids and hydroxycinnamoyl-isocitric acids

Masike et al. Chemistry Central Journal (2017) 11:29 DOI 10.1186/s13065-017-0262-8 PRELIMINARY COMMUNICATION Open Access Highlighting mass spectrometric fragmentation differences and similarities between hydroxycinnamoyl‑quinic acids and hydroxycinnamoyl‑isocitric acids Keabetswe Masike1, Msizi I. Mhlongo1, Shonisani P. Mudau1, Ofentse Nobela1, Efficient N. Ncube1, Fidele Tugizimana1, Mosotho J. George1,2 and Ntakadzeni E. Madala1* Abstract Background: Plants contain a myriad of metabolites which exhibit diverse biological activities. However, in-depth analyses of these natural products with current analytical platforms remains an undisputed challenge due to the multidimensional chemo-diversity of these molecules, amplified by both isomerization and conjugation. In this study, we looked at molecules such as hydroxyl-cinnamic acids (HCAs), which are known to exist as positional and geometrical isomers conjugated to different organic acids namely quinic- and isocitric acid. Objective: The study aimed at providing a more defined distinction between HCA conjugates from Amaranthus viridis and Moringa oleifera, using mass spectrometry (MS) approaches. Methods: Here, we used a UHPLC–MS/MS targeted approach to analyze isobaric HCA conjugates extracted from the aforementioned plants. Results: Mass spectrometry results showed similar precursor ions and fragmentation pattern; however, distinct differences were seen with ions at m/z 155 and m/z 111 which are associated with isocitric acid conjugates. Conclusion: Our results highlight subtle differences between these two classes of compounds based on the MS fingerprints, enabling confidence differentiation of the compounds. Thus, these findings provide a template reference for accurate and confident annotation of such compounds in other plants. Keywords: Amaranthus viridis, Hydroxyl-cinnamic acid, Hydroxycinnamoyl-isocitric acid, Hydroxycinnamoyl-quinic acid, Mass spectrometry, Moringa oleifera Background Plants are a source of various natural compounds with a wide spectrum of bioactivities. These compounds are categorized into primary and secondary metabolites, where the former are involved in housekeeping functions and the latter are used by plants in interactions with their environment [1]. The most dominant of the secondary metabolites are phenylpropanoids, a class of compounds *Correspondence: 1 Department of Biochemistry, University of Johannesburg, Auckland Park, P.O. Box 524, Johannesburg 2006, South Africa Full list of author information is available at the end of the article that bear a 3-carbon (C-3) chain linked to 6-carbon (C-6) aromatic ring [2–5]. The diversification of phenylpropanoids in different plant species has previously been attributed to the presence or absence of active enzymes involved in their biosynthetic pathway [2, 6]. Some of the known phenylpropanoids include flavonoids, isoflavonoids, coumarins, anthocyanins, stilbenes, benzoic acids, benzaldehyde derivatives, phenylpropenes and hydroxyl-cinnamic acid (HCA) derivatives, among others [2, 7, 8]. HCA derivatives form one of the largest classes of phenylpropanoid-derived plant compounds [9, 10], and include caffeic-, ferulic- and p-coumaric acids. These © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Masike et al. Chemistry Central Journal (2017) 11:29 metabolites contribute to the abundance of plant natural products as they form conjugates with different molecules such as sugars, polyamines and organic acids [9, 11–15]. The most common example of HCAs conjugated to organic acids are chlorogenic acids (CGAs), which are formed from an esterification reaction between the organic acid, quinic acid (QA) and one to four (identical or different) residues of HCA derivatives [12]. In nature, mono-acyl CGAs commonly occur as three regio-isomers where C3, C4 and C5 hydroxides on the QA are esterified giving rise to three positional isomers [16–18]. However, 1-acyl CGA has occasionally been noted in some plant species [19, 20]. Lastly, geometrical isomerization (trans and cis) of the different HCA derivatives seals the final diversification of these molecules [14–17, 21–24]. Another example of HCA derivatives forming conjugates with organic acids includes the esterification between isocitric acid (IA) and one of the HCA derivatives to form hydroxycinnamoyl-isocitric acid [25] as shown in Scheme 1. Unlike QA with four possible esterification positions, this esterification of IA moiety can occur at position 2 (C2). In addition, the diversification of hydroxycinnamoyl-isocitric acid only includes the conjugation of different HCA derivatives to the organic acid and the geometrical isomerization thereof. The botanical distribution of hydroxycinnamoyl-isocitric acid derivatives is not well documented. This is possibly due to the misidentification as mono-acyl CGAs since both respective group of compounds have a molecular mass of 354 Da for caffeoyl-, 338 Da for p-coumaroyl- and 368 Da for feruloyl conjugates [16, 25]. Page 2 of 7 In recent years, liquid chromatography (LC)–MS has become one of the most common techniques for annotation of plant metabolites as well as discerning between different positional isomers of mono-, di- and tri-acyl CGAs [14–16, 22, 23, 26, 27]. However, very little has been done for geometrical isomers of CGAs [28, 29]. Despite the remarkable analytical developments and methodologies, there are still some common misrepresentation in annotation of these two classes of compounds. This could be due to their similar MS fragmentation patterns leading to poor resolution and undifferentiation of these molecules thereafter. Herein we, demonstrate the unique and similar chromatographic and mass spectrometric characteristics of hydroxycinnamoyl-quinic- and hydroxycinnamoyl-isocitric acids using LC–MS experiments. Authentic standards and plant extracts of Moringa oleifera and Amaranthus viridis, were employed to demonstrate the common elements that bring confusion. These two plant species are reported to respectively accumulate/produce these compounds in abundance [24, 30]. Methods Chemical and reagents Authentic standards of caffeic acid-derived chlorogenic acids (3-, 4- and 5-caffeoylquinic acid) were purchased from Phytolab (Vestenbergsgreuth, Germany). Analytical-grade methanol and acetonitrile were purchased from Romil Pure Chemistry (Cambridge, UK). Formic acid was obtained from Sigma-Aldrich (St (...truncated)