13 research outputs found

    Synchronized survey scan approach allows for efficient discrimination of isomeric and isobaric compounds during LC-MS/MS analyses

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    Abstract: Liquid chromatography-mass spectrometry- (LC-MS-) based multiple reaction monitoring (MRM) methods have been used to detect and quantify metabolites for years. ,ese approaches rely on the monitoring of various fragmentation pathways of multiple precursors and the subsequent corresponding product ions. However, MRM methods are incapable of confidently discriminating between isomeric and isobaric molecules and, as such, the development of methods capable of overcoming this challenge has become imperative. Due to increasing scanning rates of recent MS instruments, it is now possible to operate MS instruments both in the static and dynamic modes. One such method is known as synchronized survey scan (SSS), which is capable of acquiring a product ion scan (PIS) during MRM analysis. ,e current study shows, for the first time, the use of SSS-based PIS approach as a feasible identification feature of MRM. To achieve the above, five positional isomers of dicaffeoylquinic acids (diCQAs) were studied with the aid of SSS-based PIS method. Here, theMRMtransitions were automatically optimized using a 3,5-diCQA isomer by monitoring fragmentation transitions common to all five isomers. Using the mixture of these isomers, fragmentation spectra of the five isomers achieved with SSS-based PIS were used to identify each isomer based on previously published hierarchical fragmentation keys. ,e optimized method was also used to detect and distinguish between diCQA components found in Bidens pilosa and their isobaric counterparts found in Moringa oleifera plants. ,us, the method was shown to distinguish (by differences in fragmentation patterns) between diCQA and their isobars, caffeoylquinic acid (CQA) glycosides. In conclusion, SSS allowed the detection and discrimination of isomeric and isobaric compounds in a single chromatographic run by producing a PIS spectrum, triggered in the automatic MS/MS synchronized survey scan mode

    A metabolomics-guided exploration of the phytochemical constituents of Vernonia fastigiata with the aid of pressurized hot water extraction and liquid chromatography-mass spectrometry

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    CITATION: Masike, K., et al. 2017. A metabolomics-guided exploration of the phytochemical constituents of Vernonia fastigiata with the aid of pressurized hot water extraction and liquid chromatography-mass. Molecules, 22(8):1200, doi:10.3390/molecules22081200.The original publication is available at http://www.mdpi.comVernonia 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.http://www.mdpi.com/1420-3049/22/8/1200Publisher's versio

    The development of standardized ion mobility and mass spectrometry metabolomics methods for the characterization of plant phenolics

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    Thesis (PhD)--Stellenbosch University, 2021.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.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.Doctora

    Investigating the metabolomic dimensions of plant samples through optimization of extraction and chromatographic techniques

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    M.Sc. (Biochemistry)Abstract: Please refer to full text to view abstrac

    sj-docx-1-ems-10.1177_14690667231164096 - Supplemental material for Exploratory data fusion of untargeted multimodal LC-HRMS with annotation by LCMS-TOF-ion mobility: White wine case study

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    Supplemental material, sj-docx-1-ems-10.1177_14690667231164096 for Exploratory data fusion of untargeted multimodal LC-HRMS with annotation by LCMS-TOF-ion mobility: White wine case study by Mpho Mafata, Maria Stander, Keabetswe Masike and Astrid Buica in European Journal of Mass Spectrometry</p

    Revising Reverse-Phase Chromatographic Behavior for Efficient Differentiation of Both Positional and Geometrical Isomers of Dicaffeoylquinic Acids

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    Dicaffeoylquinic acids (diCQAs) are plant metabolites and undergo trans-cis-isomerization when exposed to UV irradiation. As such, diCQAs exist in both trans- and cis-configurations and amplify the already complex plant metabolome. However, analytical differentiation of these geometrical isomers using mass spectrometry (MS) approaches has proven to be extremely challenging. Exploring the chromatographic space to develop possible conditions that would aid in differentially separating and determining the elution order of these isomers is therefore imperative. In this study, simple chromatographic parameters, such as column chemistry (phenyl versus alkyl), mobile phase composition (methanol or acetonitrile), and column temperature, were investigated to aid in the separation of diCQA geometrical isomers. The high-performance liquid chromatography photodiode array (HPLC-PDA) chromatograms revealed four isomers post UV irradiation of diCQA authentic standards. The elution profile/order was seen to vary on different reverse-phase column chemistries (phenyl versus alkyl) using different mobile phase composition. Here, the elution profile/order on the phenyl-derived column matrices (with methanol as the mobile phase composition) was observed to be relatively reproducible as compared to the alkyl (C18) columns. Chromatographic resolution of diCQA geometrical isomers can be enhanced with an increase in column temperature. Lastly, the study highlights that chromatographic elution order/profile cannot be relied upon to fathom the complexity of isomeric plant metabolites

    Detailed phenolic characterization of Protea pure and hybrid cultivars by liquid chromatography–ion mobility–high resolution mass spectrometry (LC-IM-HR-MS)

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    In this study we report a detailed investigation of the polyphenol composition of pure ( and ) and hybrid cultivars (Black beauty and Limelight). Aqueous methanol extracts of leaf and bract tissues were analyzed by ultrahigh pressure liquid chromatography hyphenated to photodiode array and ion mobility-high resolution mass spectrometric (UHPLC-PDA-IM-HR-MS) detection. A total of 67 metabolites were characterized based on their relative reversed phase (RP) retention, UV-vis spectra, low and high collision energy HR-MS data, and collisional cross section (CCS) values. These metabolites included 41 phenolic acid esters and 25 flavonoid derivatives, including 5 anthocyanins. In addition, an undescribed hydroxycinnamic acid-polygalatol ester, caffeoyl--polygalatol (1,5-anhydro-[6--caffeoyl]-sorbitol(glucitol)) was isolated and characterized by 1D and 2D NMR for the first time. This compound and its isomer are shown to be potential chemo-taxonomic markers

    Patterns of variation and chemosystematic significance of phenolic compounds in the genus cyclopia (fabaceae, podalyrieae)

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    CITATION: Stander, M. A., et al. 2019. Patterns of variation and chemosystematic significance of phenolic compounds in the genus cyclopia (fabaceae, podalyrieae). Molecules, 24(13):2352, doi:10.3390/molecules24132352.The original publication is available at http://www.mdpi.comPublication of this article was funded by the Stellenbosch University Open Access Fund.ENGLISH 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.https://www.mdpi.com/1420-3049/24/13/2352Publisher's versio

    Computational Metabolomics Tools Reveal Metabolic Reconfigurations Underlying the Effects of Biostimulant Seaweed Extracts on Maize Plants under Drought Stress Conditions

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    Drought is one of the major abiotic stresses causing severe damage and losses in economically important crops worldwide. Drought decreases the plant water status, leading to a disruptive metabolic reprogramming that negatively affects plant growth and yield. Seaweed extract-based biostimulants show potential as a sustainable strategy for improved crop health and stress resilience. However, cellular, biochemical, and molecular mechanisms governing the agronomically observed benefits of the seaweed extracts on plants are still poorly understood. In this study, a liquid chromatography–mass spectrometry-based untargeted metabolomics approach combined with computational metabolomics strategies was applied to unravel the molecular ‘stamps’ that define the effects of seaweed extracts on greenhouse-grown maize (Zea mays) under drought conditions. We applied mass spectral networking, substructure discovery, chemometrics, and metabolic pathway analyses to mine and interpret the generated mass spectral data. The results showed that the application of seaweed extracts induced alterations in the different pathways of primary and secondary metabolism, such as phenylpropanoid, flavonoid biosynthesis, fatty acid metabolism, and amino acids pathways. These metabolic changes involved increasing levels of phenylalanine, tryptophan, coumaroylquinic acid, and linolenic acid metabolites. These metabolic alterations are known to define some of the various biochemical and physiological events that lead to enhanced drought resistance traits. The latter include root growth, alleviation of oxidative stress, improved water, and nutrient uptake. Moreover, this study demonstrates the use of molecular networking in annotating maize metabolome. Furthermore, the results reveal that seaweed extract-based biostimulants induced a remodeling of maize metabolism, subsequently readjusting the plant towards stress alleviation, for example, by increasing the plant height and diameter through foliar application. Such insights add to ongoing efforts in elucidating the modes of action of biostimulants, such as seaweed extracts. Altogether, our study contributes to the fundamental scientific knowledge that is necessary for the development of a biostimulants industry aiming for a sustainable food security
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