Browsing by Author "Masike, Keabetswe"
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- ItemThe development of standardized ion mobility and mass spectrometry metabolomics methods for the characterization of plant phenolics(Stellenbosch : Stellenbosch University, 2021-04) Masike, Keabetswe; Stander, Marietjie; De Villiers, Andre; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Plants and plant-derived products contain a plethora of phenolic compounds, with a broad range of health benefits and useful applications such as in drug design. These phenolic compounds are often amplified by conjugation and rearrangements, thus producing isobaric and isomeric species. The standard analytical method for the identification and characterization of plant phenolics, liquid chromatography (LC) hyphenated to photo diode array (PDA) and/or high-resolution mass spectrometry (HR-MS) detectors, is not able to discriminate isomeric species in complex plant samples. The incorporation of ion mobility spectrometry (IMS) to LC-PDA-HR-MS workflows is being recognized as an additional orthogonal dimension of separation to HR-MS; where ions are separated through a drift region, filled with gas, based on their size, shape, and charge. The attractive component of IMS is the determination of the collisional cross section (CCS/Ω) values, which describes the unique rotationally averaged surface area of the ion, as it interacts and travels through the gas-filled drift region. The CCS as a feature, can be beneficial in the development of an in-house phenolics compound library in analytical laboratories, which can help expedite the characterization of phenolic compounds in varying research fields, such as in plant metabolomics and food science. The goal of the work reported in this thesis was, therefore, to develop LC-PDA-IM-HR-MS methods capable of structurally characterizing plant phenolics, found in South African indigenous herbal teas and plant species, based on a range of structural descriptors (e.g., tR, spectroscopic data, mass spectral information (including high resolution and MS/MS data), and CCS value). In the first part of the study, the phenolic profiles of Protea pure and hybrid cultivars were characterised by LC-PDA-IM-HR-MS for the first time in detail. Whereby, IMS in conjunction with other structure elucidation techniques, namely tandem MS data, UV-vis spectroscopy, nuclear magnetic resonance (NMR) were used for characterization of 67 metabolites. With the aid of NMR an undescribed hydroxycinnamic acid-polygalatol ester, caffeoyl-O-polygalatol (1,5-anhydro-[6-O-caffeoyl]-sorbitol(glucitol)) was isolated and characterised for the first time. Furthermore, positional isomers with similar MS/MS profiles were resolved by the IMS-dimension and consequently could be distinguished by their differences in CCS values. The CCS values obtained using two IMS platforms (drift-time ion mobility spectrometry (DTIMS) and travelling wave IMS (TWIMS)) were compared, and it was observed that the CCS values obtained with a TWIMS instrument were underestimated for compounds with CCS values below 200 Å2. Conversely, good agreement was obtained between both instruments for compounds with higher CCS values. Poor calibration of the TWIMS platform was attributed to the underestimation of the CCS values below 200 Å2. In the second part of the study, a detailed comparison of phytochemical profiles of a much larger set of Protea species, selections, and cultivars was reported. Using metabolomics tools and the data collected and documented (in the previous study) of phenolic compounds characterised based on their UPLC-PDA-IM-HR-MS profiles, plant metabolites associated with a post-harvesting disorder, leaf blackening, in Protea were identified/and annotated. Species, selections, and cultivars susceptible to leaf blackening contained features identified as benzenetriol- and/or hydroquinone-glycoside derivatives. On the other hand, stems not prone to blackening were linked to phenolic compounds with known protective properties against biotic and abiotic stressors. CCS values of the metabolites with protective features against leaf blackening, and those for compounds that instigate the process, were determined. Such observations serve as preliminary insights that can help accelerate plant improvement and aid in the selection of trait-specific markers in plant metabolomics. In the final portion of the study, using direct injection-IM-MS (DI-IM-MS) and descriptive chemometrics, differences between adulterated herbal teas (rooibos and honeybush) were observed. The diagnostic value of marker compounds in distinguishing the three commercialised honeybush species (Cyclopia intermedia, C. genistoides and C. subternata) for the quality control purpose were observed. To derive CCS values using the TWIMS instrument two calibrants, poly-DL-alanine and poly-L-malic acid, were compared. Poly-DL- alanine is a widely used TWIMS calibrant which results in an underestimation for compounds with CCS values below 200 Å2 (initial study), when compared to poly-L-malic acid an improvement in the underestimation of CCS values below 200 Å2 was observed. DI-IM-MS proved to be a useful tool for quality control purposes, particularly considering the analysis time is 1 minute per sample.
- ItemA metabolomics-guided exploration of the phytochemical constituents of Vernonia fastigiata with the aid of pressurized hot water extraction and liquid chromatography-mass spectrometry(MDPI, 2017) Masike, Keabetswe; Khoza, Bradley S.; Steenkamp, Paul A.; Smit, Elize; Dubery, Ian A.; Madala, Ntakadzeni E.Vernonia fastigiata is a multi-purpose nutraceutical plant with interesting biological properties. However, very little is known about its phytochemical composition and, thus the need for its phytochemical characterization. In the current study, an environmentally friendly method, pressurized hot water extraction (PHWE), was used to extract metabolites from the leaves of V. fastigiata at various temperatures (50 °C, 100 °C, 150 °C and 200 °C). Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-qTOF-MS) analysis in combination with chemometric methods, particularly principal component analysis (PCA) and liquid/gas chromatography mass spectrometry (XCMS) cloud plots, were used to descriptively visualize the data and identify significant metabolites extracted at various temperatures. A total of 25 different metabolites, including hydroxycinnamic acid derivatives, clovamide, deoxy-clovamide and flavonoids, were noted for the first time in this plant. Overall, an increase in extraction temperature resulted in an increase in metabolite extraction during PHWE. This study is the first scientific report on the phytochemical composition of V. fastigiata, providing insight into the components of the chemo-diversity of this important plant.
- ItemPatterns of variation and chemosystematic significance of phenolic compounds in the genus cyclopia (fabaceae, podalyrieae)(MDPI, 2019) Stander, Maria. A.; Redelinghuys, Herman; Masike, Keabetswe; Long, Helen; Van Wyk, Ben-ErikENGLISH ABSTRACT: As a contribution towards a better understanding of phenolic variation in the genus Cyclopia (honeybush tea), a collection of 82 samples from 15 of the 23 known species was analysed using liquid-chromatography–high resolution mass spectrometry (UPLC-HRMS) in electrospray ionization (ESI) negative mode. Mangiferin and isomangiferin were found to be the main compounds detected in most samples, with the exception of C. bowiena and C. buxifolia where none of these compounds were detected. These xanthones were found to be absent from the seeds and also illustrated consistent differences between species and provenances. Results for contemporary samples agreed closely with those based on analysis of a collection of ca. 30-year-old samples. The use of multivariate tools allowed for graphical visualizations of the patterns of variation as well as the levels of the main phenolic compounds. Exclusion of mangiferin and citric acid from the data was found to give better visual separation between species. The use of UPLC-HRMS generated a large dataset that allowed for comparisons between species, provenances and plant parts (leaves, pods, flowers and seeds). Phenetic analyses resulted in groupings of samples that were partly congruent with species but not with morphological groupings within the genus. Although different provenances of the same species were sometimes found to be very variable, Principle Component Analysis (PCA) indicated that a combination of compounds have some (albeit limited) potential as diagnostic characters at species level. 74 Phenolic compounds are presented, many of which were identified for the first time in Cyclopia species, with nine of these being responsible for the separation between samples in the PCAs.