Patterns of Variation and Chemosystematic Significance of Phenolic Compounds in the Genus Cyclopia (Fabaceae, Podalyrieae).

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.

A Metabolomics-Guided Exploration of the Phytochemical Constituents of Vernonia fastigiata with the Aid of Pressurized Hot Water Extraction and Liquid Chromatography-Mass Spectrometry.

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.

Exploratory data fusion of untargeted multimodal LC-HRMS with annotation by LCMS-TOF-ion mobility: White wine case study.

Applied sciences have increased focus on omics studies which merge data science with analytical tools. These studies often result in large amounts of data produced and the objective is to generate meaningful interpretations from them. This can sometimes mean combining and integrating different datasets through data fusion techniques. The most strategic course of action when dealing with products of unknown profile is to use exploratory approaches. For omics, this means using untargeted analytical methods and exploratory data analysis techniques. The current study aimed to perform data fusion on untargeted multimodal (negative and positive mode) liquid chromatography-high-resolution mass spectrometry data using multiple factor analysis. The data fusion results were interpreted using agglomerative hierarchical clustering on biplot projections. The study reduced the thousands of spectral signals processed to less than a hundred features (a primary parameter combination of retention time and mass-to-charge ratios, RT_m/z). The correlations between cluster members (samples and features from) were calculated and the top 10% highly correlated features were identified for each cluster. These features were then tentatively identified using secondary parameters (drift time, ion mobility constant and collision cross-section values) from the ion mobility spectra. These ion mobility (secondary) parameters can be used for future studies in wine chemical analysis and added to the growing list of annotated chemical signals in applied sciences.

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

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.

Recent applications of ion mobility spectrometry in natural product research.

Ion mobility spectrometry (IMS) is a rapid separation technique capable of extracting complementary structural information to chromatography and mass spectrometry (MS). IMS, especially in combination with MS, has experienced inordinate growth in recent years as an analytical technique, and elicited intense interest in many research fields. In natural product analysis, IMS shows promise as an additional tool to enhance the performance of analytical methods used to identify promising drug candidates. Potential benefits of the incorporation of IMS into analytical workflows currently used in natural product analysis include the discrimination of structurally similar secondary metabolites, improving the quality of mass spectral data, and the use of mobility-derived collision cross-section (CCS) values as an additional identification criterion in targeted and untargeted analyses. This review aims to provide an overview of the application of IMS to natural product analysis over the last six years. Instrumental aspects and the fundamental background of IMS will be briefly covered, and recent applications of the technique for natural product analysis will be discussed to demonstrate the utility of the technique in this field.

Application of Metabolomics Tools to Determine Possible Biomarker Metabolites Linked to Leaf Blackening in Protea.

The postharvesting disorder leaf blackening is the main cause of product rejection in Protea during export. In this study, we report an investigation into metabolites associated with leaf blackening in Protea species. Methanol extracts of leaf and involucral bract tissue were analyzed by liquid chromatography hyphenated to photodiode array and high-resolution mass spectrometry (LC-PDA-HRMS), where 116 features were annotated. Analytical data obtained from 37 Protea species, selections, and hybrids were investigated using metabolomics tools, which showed that stems susceptible to leaf blackening cluster together and contained features identified as benzenetriol- and/or hydroquinone-derived metabolites. On the other hand, species, selections, and cultivars not prone to blackening were linked to metabolites with known protective properties against biotic and abiotic stressors. During the browning process, susceptible cultivars also produce these protective metabolites, yet at innately low levels, which may render these species and cultivars more vulnerable to blackening. Metabolites that were found to be correlated to the instigation of the browning process, all comprising benzenetriol- and hydroquinone-glycoside derivatives, are highlighted to provide preliminary insights to guide the development of new Protea cultivars not susceptible to leaf blackening.

Detailed Phenolic Characterization of Protea Pure and Hybrid Cultivars by Liquid Chromatography-Ion Mobility-High Resolution Mass Spectrometry (LC-IM-HR-MS).

In this study we report a detailed investigation of the polyphenol composition of Protea pure (P. cynaroides and P. neriifolia) 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-O-polygalatol (1,5-anhydro-[6-O-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.

Synchronized Survey Scan Approach Allows for Efficient Discrimination of Isomeric and Isobaric Compounds during LC-MS/MS Analyses.

Liquid chromatography-mass spectrometry- (LC-MS-) based multiple reaction monitoring (MRM) methods have been used to detect and quantify metabolites for years. These 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. The 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, the MRM transitions 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. The 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. Thus, 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.

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

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.

Deciphering the influence of column chemistry and mass spectrometry settings for the analyses of geometrical isomers of L-chicoric acid.

Resolving the chemo-diversity of plant extract samples is an essential step for in-depth analyses of natural products which often exhibit promising biological activities. One of the challenges in this endeavor has been the confident differentiation of geometrical isomers. In this study, we investigated these aspects in chromatography (column chemistry and mobile phase composition) and mass spectrometry settings with regards to better differentiation of geometrical isomers. A standard of a hydroxycinnamic acid (HCA) derivative, L-chicoric acid (L-CA) - a di-acylated caffeoyltartaric acid ester found in a number of plant families - was used. Geometrical isomers of L-CA were formed by exposing the compound to ultraviolet (UV) radiation, to mimic the natural environment. The high performance liquid chromatography photo-diode array (HPLC-PDA) and ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) platforms were used to analyze the trans and cis geometrical isomers of L-CA. The HPLC-PDA results confirmed the generation of two cis geometrical isomers following UV exposure of the authentic trans-L-CA standard. Furthermore, the HPLC-PDA analyses demonstrated that the changes in both column chemistry (reverse-phase: C18, biphenyl, phenyl-hexyl and pentafluorophenyl propyl) and mobile phase composition (aqueous acetonitrile and aqueous methanol) affect the chromatographic elution profiles of the L-CA isomers. The MS results, on the other hand, revealed undisputed fragmentation differences between the geometrical isomers of L-CA. Thus, this study demonstrates that the identification of the L-CA isomers can be achieved more efficiently and confidently with good chromatography coupled to well-optimized mass spectrometry conditions, a requirement which has been proven impossible with other types of HCA derivatives. Moreover, differences in the binding modes of L-CA geometrical isomers to the HIV type 1 integrase enzyme were observed, suggesting a synergistic anti-HIV-1 activity of these isomers.

Highlighting mass spectrometric fragmentation differences and similarities between hydroxycinnamoyl-quinic acids and hydroxycinnamoyl-isocitric acids.

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 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. 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.