Highlighting Mass Spectrometric Fragmentation Differences And

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 mul- tidimensional 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 that bear a 3-carbon (C-3) chain linked to 6-carbon (C-6) Plants are a source of various natural compounds with a aromatic ring [2–5]. The diversification of phenylpro- wide spectrum of bioactivities. These compounds are cat- panoids in different plant species has previously been egorized into primary and secondary metabolites, where attributed to the presence or absence of active enzymes the former are involved in housekeeping functions and involved in their biosynthetic pathway [2, 6]. Some of the latter are used by plants in interactions with their the known phenylpropanoids include flavonoids, isofla- environment [1]. The most dominant of the secondary vonoids, coumarins, anthocyanins, stilbenes, benzoic metabolites are phenylpropanoids, a class of compounds acids, benzaldehyde derivatives, phenylpropenes and hydroxyl-cinnamic acid (HCA) derivatives, among others [2, 7, 8]. HCA derivatives form one of the largest classes *Correspondence: [email protected] 1 Department of Biochemistry, University of Johannesburg, Auckland of phenylpropanoid-derived plant compounds [9, 10], Park, P.O. Box 524, Johannesburg 2006, South Africa and include caffeic-, ferulic- andp -coumaric acids. These Full list of author information is available at the end of the article © 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 Page 2 of 7 metabolites contribute to the abundance of plant natu- In recent years, liquid chromatography (LC)–MS ral products as they form conjugates with different mol- has become one of the most common techniques for ecules such as sugars, polyamines and organic acids [9, annotation of plant metabolites as well as discerning 11–15]. The most common example of HCAs conjugated between different positional isomers of mono-, di- and to organic acids are chlorogenic acids (CGAs), which tri-acyl CGAs [14–16, 22, 23, 26, 27]. However, very lit- are formed from an esterification reaction between the tle has been done for geometrical isomers of CGAs [28, organic acid, quinic acid (QA) and one to four (identical 29]. Despite the remarkable analytical developments or different) residues of HCA derivatives [12]. and methodologies, there are still some common mis- In nature, mono-acyl CGAs commonly occur as three representation in annotation of these two classes of regio-isomers where C3, C4 and C5 hydroxides on the compounds. This could be due to their similar MS frag- QA are esterified giving rise to three positional isomers mentation patterns leading to poor resolution and un- [16–18]. However, 1-acyl CGA has occasionally been differentiation of these molecules thereafter. Herein we, noted in some plant species [19, 20]. Lastly, geometrical demonstrate the unique and similar chromatographic isomerization (trans and cis) of the different HCA deriva- and mass spectrometric characteristics of hydroxycin- tives seals the final diversification of these molecules namoyl-quinic- and hydroxycinnamoyl-isocitric acids [14–17, 21–24]. Another example of HCA derivatives using LC–MS experiments. Authentic standards and forming conjugates with organic acids includes the ester- plant extracts of Moringa oleifera and Amaranthus vir- ification between isocitric acid (IA) and one of the HCA idis, were employed to demonstrate the common ele- derivatives to form hydroxycinnamoyl-isocitric acid [25] ments that bring confusion. These two plant species are as shown in Scheme 1. Unlike QA with four possible reported to respectively accumulate/produce these com- esterification positions, this esterification of IA moiety pounds in abundance [24, 30]. can occur at position 2 (C2). In addition, the diversification of hydroxycinnamoyl-isocitric acid only includes the Methods conjugation of different HCA derivatives to the organic Chemical and reagents acid and the geometrical isomerization thereof. The Authentic standards of caffeic acid-derived chlorogenic botanical distribution of hydroxycinnamoyl-isocitric acid acids (3-, 4- and 5-caffeoylquinic acid) were purchased derivatives is not well documented. This is possibly due from Phytolab (Vestenbergsgreuth, Germany). Analyt- to the misidentification asmono -acyl CGAs since both ical-grade methanol and acetonitrile were purchased respective group of compounds have a molecular mass of from Romil Pure Chemistry (Cambridge, UK). Formic 354 Da for caffeoyl-, 338 Da for p-coumaroyl- and 368 Da acid was obtained from Sigma-Aldrich (St. Louis, MO, for feruloyl conjugates [16, 25]. USA). Scheme 1 Structures of mono-acylated HCA conjugates of quinic and isocitric acid Masike et al. Chemistry Central Journal (2017) 11:29 Page 3 of 7 Metabolite extraction at various collision energies (5–35 eV) to mimic MSE The dried leaves ofM. oleifera and A. viridis were pul- experiments. verized using a clean and dry quartz mortar and pestle. For extraction, the respective amounts of powdered leaf Results and discussion material (0.2 g) were mixed with 2 mL of 50% aqueous Compound annotation methanol and these extracts were placed (with the lids of As one of the main aspects of the present study, we the tubes closed to avoid evaporation) in a heating block compare hydroxycinnamoyl-quinic- and hydroxycin- at 60 °C for 2 h. The samples were sonicated for 30 min namoyl-isocitric acid derivatives and show how both using an ultrasonic bath and then centrifuged at 9740×g chromatography and mass spectrometry can be used to for 10 min at 4 °C. The resulting supernatants for both distinguish these isobaric compounds. Single ion moni- plant samples were then subjected to UV-irradiation for toring (SIM) chromatograms of hydroxycinnamoyl- induction of geometrical isomerization [21]. Coffee bean- quinic- and hydroxycinnamoyl-isocitric acid from M. and pineapple extracts to be used as surrogate standards oleifera and A. viridis leaf extracts are shown respec- were prepared by extracting 0.2 g of these materials in tively in Fig. 1. The mass spectra and retention times of 1 mL of 50% methanol. the compounds under study were compared with those of available standards (i.e. 3-CQA, 4-CQA and 5-CQA). Ultra‑high performance liquid chromatography mass Coffee bean extracts have been previously reported to be spectrometry (UHPLC–MS/MS) analysis remarkably rich in a variety of CGAs, including feruloyl A Shimadzu Nexera 30 UHPLC (Kyoto Japan) fitted with and ρ-coumaroyl derivatives [9, 13, 27]. Furthermore, a Viva C18 analytical column (3.0 µm, 2.1 × 100 mm; a study by Steingass et al. [31] revealed the presence of Restek, USA) was used with the following settings: an hydroxycinnamoyl isocitric acids in pineapple extracts. injection volume of 2 µL, column oven temperature of Hence in this study, coffee bean- and pineapple extracts 40 °C, a binary solvent mixture consisting of MilliQ water were analyzed using the same optimized method and the containing 0.1% formic acid (eluent A) and methanol results obtained therefore served as surrogate standards containing 0.1% formic acid (eluent B) with a constant for feruloyl and

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