The development of standardized ion mobility and mass spectrometry metabolomics methods for the characterization of plant phenolics
| dc.contributor.advisor | Stander, Marietjie | en_ZA |
| dc.contributor.advisor | De Villiers, Andre | en_ZA |
| dc.contributor.author | Masike, Keabetswe | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Biochemistry. | en_ZA |
| dc.date.accessioned | 2021-05-31T09:22:54Z | |
| dc.date.available | 2021-05-31T09:22:54Z | |
| dc.date.issued | 2021-04 | |
| dc.description | Thesis (PhD)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Plante en plantaardige produkte bevat 'n magdom van fenoliese verbindings, met 'n wye verskeidenheid gesondheidsvoordele en ander nuttige toepassings byvoorbeeld die ontwikkeling van geneesmiddels. Hierdie fenoliese verbindings word dikwels verander deur konjugasie en herrangskikkingsreaksies, en sodoende word isobariese spesies en isomere gevorm. Die standaard analitiese metode vir die identifisering en karakterisering van plantfenole, vloeistofchromatografie (LC) wat aan fotodiode-reeks (PDA) en / of hoë-resolusie massa spektrometrie (HR-MS) detektore gekoppel is, kan nie altyd isomere in komplekse plantmonsters onderskei nie. Die integrasie van ioon mobiliteit spektrometrie (IMS) in LC- PDA-HR-MS- werkstrome word erken as 'n aanvullende ortogonale dimensie van skeiding vir HR-MS; waar ione geskei word deur 'n dryfgebied, gevul met gas, gebaseer op hul grootte, vorm en lading. Die voordeel van IMS is die bepaling van die botsingsdeursnee (CCS / Ω) waarde, wat die unieke rotasiegemiddelde oppervlakarea van die ioon beskryf. CCS is voordeling vir die ontwikkeling van 'n biblioteek met fenoliese verbindings in analitiese laboratoriums, wat kan help om die karakterisering van fenoliese verbindings in verskillende navorsingsvelde, soos plantmetabolika en voedselwetenskap, te vergemaklik. Die doel van die werk wat in hierdie proefskrif oor verslag gedoen word, was dus om LC-PDA- IM-HR-MS-metodes te ontwikkel wat in staat is om die strukture van plantfenoliese stowwe, wat in Suid-Afrikaanse inheemse kruietees en plantspesies voorkom, te karakteriseer gebaseer op 'n reeks strukturele beskrywers (bv. tR, spektroskopiese data, massa spektrale inligting (insluitend hoë resolusie en MS/MS data) en CCS waarde). In die eerste gedeelte van die studie is die fenoliese profiele van Protea suiwer en baster kultivars vir die eerste keer in detail gekarakteriseer deur LC-PDA-IM-HR-MS. Waarvolgens IMS saam met ander struktuur-opklaringsstegnieke, naamlik tandem MS data, UV-vis spektroskopie, kernmagnetiese resonansie (NMR) gebruik is vir die karakterisering van 67 metaboliete. Met behulp van NMR is 'n onbeskrewe hidroksie kaneelsuur-poligalatolester, kaffeoïel-O- polygalatol (1,5-anhidro-[6-O-kaffeoïel]-sobitol(glukitol) geïsoleer en vir die eerste keer gekarakteriseer. Verder is posisionele isomere met soortgelyke MS/MS-profiele geïdentifiseer deur die IMS-dimensie en kan gevolglik onderskei word deur hul verskille in CCS-waardes. Die CCS-waardes wat verkry is met behulp van twee IMS-platforms (dryftyd ioon mobiliteit spektrometrie (DTIMS) en bewegende golf-IMS (TWIMS)) is vergelyk, en daar is waargeneem dat die CCS-waardes wat met 'n TWIMS-instrument verkry is, onderskat is vir verbindings met CCS-waardes onder 200 Å2. Andersins is goeie ooreenkoms tussen beide instrumente verkry vir verbindings met hoër CCS-waardes. Swak kalibrasie van die TWIMS-platform is toegeskryf aan die onderskatting van die CCS-waardes onder 200 Å2. In die tweede deel van die studie word 'n gedetailleerde vergelyking van fitochemiese profiele van 'n veel groter stel Protea-spesies, seleksies en kultivars gerapporteer. Met behulp van metabolomika-tegnieke en die data wat versamel en gedokumenteer is (in die vorige studie) van fenoliese verbindings op grond van hul UPLC-PDA-IM-HR-MS-profiele, die plantmetaboliete wat verband hou met 'n na-oes probleem, blaarverswarting in Protea, geïdentifiseer/en geannoteer. Spesies, seleksies en kultivars wat vatbaar is vir blaarverswarting bevat benseentriol- en / of hidrokinoon-glikosiedderivate. Daarteenoor bevat stingels wat nie geneig is tot verswarting nie, weer meer fenoliese verbindings met bekende beskermende eienskappe teen biotiese en abiotiese stresfaktore. CCS-waardes van die metaboliete met beskermende eienskappe teen blaarverswarting, en die waardes vir verbindings wat die proses begin, is bepaal. Sulke waarnemings voeg waarde toe tot bestaande inligting wat kan help om plantverbetering te versnel en te help met die keuse van eienskapspesifieke merkers in plantmetabolomika. In die laaste gedeelte van die studie, is daar met behulp van direkte inspuiting-IMS (DI-IM- MS) en chemometrie, is verskille waargeneem tussen vervalsde kruietee (rooibos en heuningbos). Merkerverbindings om tussen die drie kommersiële heuningbosspesies (Cyclopia intermedia, C. genistoides en C. subternata) te onderskei is waargeneem. Om CCS- waardes met behulp van die TWIMS-instrument af te lei, is twee kalibrante, poli-DL-alanien en poli-L-appelsuur, vergelyk. Poli-DL-alanien is die algemeen gebruikte TWIMS kalibrant wat lei tot 'n onderskatting van verbindings met CCS waardes onder 200 Å2 (aanvanklike studie), in vergelyking met poly-L-appelsuur, 'n verbetering in die onderskatting van CCS waardes onder 200 Å2. DI-IM-MS blyk 'n nuttige hulpmiddel te wees vir doeleindes van gehaltebeheer, veral aangesien die ontledingstyd 1 minuut per monster is. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.format.extent | xv, 196 pages : illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/110518 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Plant phenolics | en_ZA |
| dc.subject | Plants -- Effect of phenol on | en_ZA |
| dc.subject | Phenolic compounds | en_ZA |
| dc.subject | Indigenous plants -- South Africa | en_ZA |
| dc.subject | Ion mobility mass spectrometry | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | The development of standardized ion mobility and mass spectrometry metabolomics methods for the characterization of plant phenolics | en_ZA |
| dc.type | Thesis | en_ZA |