Highlighting mass spectrometric fragmentation differences and similarities between hydroxycinnamoyl-quinic acids and hydroxycinnamoyl-isocitric acids
© The Author(s) 2017
Received: 14 November 2016
Accepted: 28 March 2017
Published: 4 April 2017
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.
The study aimed at providing a more defined distinction between HCA conjugates from Amaranthus viridis and Moringa oleifera, using mass spectrometry (MS) approaches.
Here, we used a UHPLC–MS/MS targeted approach to analyze isobaric HCA conjugates extracted from the aforementioned plants.
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.
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.
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 . The most dominant of the secondary metabolites are phenylpropanoids, a class of compounds 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 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 .
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 un-differentiation 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].
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. Louis, MO, USA).
The dried leaves of M. oleifera and A. viridis were pulverized using a clean and dry quartz mortar and pestle. For extraction, the respective amounts of powdered leaf material (0.2 g) were mixed with 2 mL of 50% aqueous methanol and these extracts were placed (with the lids of the tubes closed to avoid evaporation) in a heating block at 60 °C for 2 h. The samples were sonicated for 30 min using an ultrasonic bath and then centrifuged at 9740×g for 10 min at 4 °C. The resulting supernatants for both plant samples were then subjected to UV-irradiation for induction of geometrical isomerization . Coffee bean- and pineapple extracts to be used as surrogate standards were prepared by extracting 0.2 g of these materials in 1 mL of 50% methanol.
Ultra-high performance liquid chromatography mass spectrometry (UHPLC–MS/MS) analysis
A Shimadzu Nexera 30 UHPLC (Kyoto Japan) fitted with a Viva C18 analytical column (3.0 µm, 2.1 × 100 mm; Restek, USA) was used with the following settings: an injection volume of 2 µL, column oven temperature of 40 °C, a binary solvent mixture consisting of MilliQ water containing 0.1% formic acid (eluent A) and methanol containing 0.1% formic acid (eluent B) with a constant flow rate of 0.4 mL/min. The gradient elution was used with the following conditions: 5% eluent B maintained for 3 min, followed by a linear increase to 45% of eluent B at 25 min, then a further increase to 90% at 30 min, conditions were held constant for 2 min before being decreased to the initial conditions at 34 min followed by a 6 min isocratic wash at 5% to re-equilibrate the column. The total chromatographic run time was 40 min. The data were acquired using a UV detector set at 325 nm.
The chromatographic effluent was further introduced to an MS detector and ionized by electrospray (ESI). The ionized ions were further analyzed by a triple quadrupole (QqQ) mass spectrometer operating under the following settings: the interface voltage was set at 3.5 kV (in negative ESI mode), the source temperature was 300 °C, nitrogen was used as the drying gas at the flow rate of 15.00 L/min and argon used as a nebulizing gas at a flow rate of 3.00 L/min, argon was also used as a collision gas with a pressure of approximately 230 kPa in the collision cell. For each run, the MS spectra at the mass range 100–1000 Da was collected continuously with a scan time of 1 s. For targeted analyses, the product scan MS mode was used to monitor the fragmentation patterns of the following ions: m/z 353 for caffeoyl-quinic acid and caffeoyl-isocitric acid, m/z 337 for coumaroyl-quinic acid and coumaroyl-isocitric acid and finally m/z 367 for feruloyl-quinic acid and feruloyl-isocitric acid. Exhaustive MS fragmentation was achieved by collecting data at various collision energies (5–35 eV) to mimic MSE experiments.
Results and discussion
Characterization of hydroxyl-cinnamic acid conjugates from M. oleifera and A. viridus
191, 179, 161, 135
191, 173, 163, 119
193, 191, 173, 149, 134
191, 179, 173, 135
191, 173, 155, 111
173, 163, 137, 119
173, 155, 111
193, 173, 134
173, 155, 111
Furthermore, in a chromatographic space, it was interestingly observed that IA derivatives elute later than the QA counterparts (Fig. 1 and Table 1). For example, all three CQA regio-isomers eluted at retention times (Rt) 4.7 min for 3-CQA, 8.3 min for 5-CQA and 9.3 min for 4-CQA (Fig. 1a) compared to caffeoyl-isocitric acid (CIA) which eluted at Rt 9.6 min (Fig. 1d). Similarly, the same elution order was also consistent for p-coumaroyl-quinic acid (Fig. 1b) and feruloyl-quinic acid (Fig. 1c) with respect to their isocitric acid counterparts (Fig. 1e, f). Our results are consistent with the reported elution order observed elsewhere , where caffeoyl-quinic acids are seen to elute earlier than caffeoyl-isocitric acids on a C18 column. This suggests that in a reverse phase chromatographic space, the elution of IA conjugates is more retarded than the respective QA conjugates, an indication that IA derivatives are less polar than QA derivatives. This postulation can be explained by structural differences and stereochemistry of these compounds, resulting in differences in polarities. For instance, the QA possess more hydroxyl (–OH) groups (four in total), thus rendering it more polar relative to IA with only a single –OH group. Moreover, the IA has more C=O groups in close proximity which may led to the formation of intramolecular hydrogen bonds resulting in higher hydrophobicity. According to the experimentally determined LogPo/w values shown elsewhere (http://www.chemspider.com), quinic acid is evidently more polar as it has a value of −2.01 whereas isocitric acid has a value of −1.47.
Proposed fragmentation/structural re-arrangements of quinic- and isocitric acid
The results from MS analyses of hydroxycinnamoyl-quinic and hydroxycinnamoyl-isocitric acid show both QA and IA to be readily lost as product ions at m/z 191. However, the downstream MS fragmentation of these organic acids are different (Scheme 2). For instance, QA produces intense ions at m/z 191 [QA–H]− and m/z 173, the latter resulting from the subsequent loss of water (−18 Da) [QA–H–H2O]− (Scheme 2). Similarly, IA at m/z 191 also undergoes dehydration to give an ion at m/z 173 [IA–H–H2O]−. Consequently, the IA moiety undergoes further structural rearrangement when the ion at m/z 173 sequentially loses water (−18 Da) to give a unique ion at m/z 155 [IA–H–2H2O]−. The resulting product ion is further decarboxylated (−44 Da) to give another unique product ion at m/z 111 [IA–H–2H2O–CO2]− (Scheme 2b). From the above it can be noted that the ions at m/z 155 and 111 characteristic for IA conjugates, which allows reliable distinction from QA derivatives.
In conclusion, this work confirms the presence of hydroxycinnamoyl-isocitrates in A. viridis and hydroxycinnamoyl-quinates in M. oleifera, respectively. Although these compounds share similar MS molecular fingerprints, this work highlights the mass spectrometric fragmentation differences between the two related groups of compounds. Herein, the minor variations/differences with regard to the respective diagnostic peaks allow for the unambiguous annotation. As such, these findings illustrate the combinatorial and efficient ability of LC–MS to unequivocally distinguish between hydroxycinnamoyl-isocitrates and hydroxycinnamoyl-quinates. Furthermore, these findings are expected to provide a template reference for annotation of these compounds in other plants.
NEM conceived the study. KM, SPM, ENN, MIM and ON conducted the experiment. KM, SPM, NEM and MIM analysed the data. KM, SPM, ENN, MIM and ON wrote the manuscript. NEM, TF and MJG participated in critical reading of the manuscript. NEM and MJG supervised the study. All authors read and approved the final manuscript.
The authors would like to thank the University of Johannesburg and the NRF for the financial support. The authors would also like to thank RESTEK for the RASP grant used purchase the chromatographic columns. Dr. Riaan Meyer and Mr. Darryl Harris from Shimandzu South Africa are thanked for their technical assistance. Dr. Lizelle Piater is also thanked for reading the final copy of this paper.
The authors declare that they have no competing interests.
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