High-Resolution UPLC-MS Profiling of Anthocyanins and Flavonols of Red Cabbage (Brassica oleracea L. var. capitata f. rubra DC.) Cultivated in Egypt and Evaluation of Their Biological Activity

In this paper, biological investigations and a high-resolution UPLC-PDA-ESI-qTOF-HRMS technique were employed for Brassica oleracea L. var. capitata f. rubra DC. (red cabbage) of the family Brassicaceae (Cruciferae), cultivated in Egypt, for the first time. The positive ionization mode is usually performed to identify anthocyanins. However, this technique cannot differentiate between anthocyanins and corresponding non-anthocyanin polyphenols. Thus, the negative ionization mode was also used, as it provided a series of characteristic ions for the MS analysis of anthocyanins. This helped in identifying five kaempferol derivatives for the first time in red cabbage, as well as nine—previously reported—anthocyanins. For the biological investigations, the acidified methanolic extract of fresh leaves and the methanolic extract of air-dried powdered leaves were examined for their antioxidant, antimicrobial, and anticancer activities. The freshly prepared phenolic extract was proven to be more biologically potent. Statistical significance was determined for its anticancer activity in comparison with standard doxorubicin.

An optimized LC-HRMS untargeted metabolomics workflow for multi-matrices investigations in the three-spined stickleback

Environmental metabolomics has become a growing research field to understand biological and biochemical perturbations of organisms in response to various abiotic or biotic stresses. It focuses on the comprehensive and systematic analysis of a biologic system’s metabolome. This allows the recognition of biochemical pathways impacted by a stressor, and the identification of some metabolites as biomarkers of potential perturbations occurring in a body. In this work, we describe the development and optimization of a complete reliable methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) for untargeted metabolomics studies within a fish model species, the three-spined stickleback (Gasterosteus aculeatus). We evaluated the differences and also the complementarities between four different matrices (brain, gills, liver and whole fish) to obtain metabolome information. To this end, we optimized and compared sample preparation and the analytical method, since the type and number of metabolites detected in any matrix are closely related to these latter. For the sample preparation, a solid-liquid extraction was performed on a low quantity of whole fish, liver, brain, or gills tissues using combinations of methanol/water/heptane. Based on the numbers of features observed in LC-HRMS and on the responses of analytical standards representative of different metabolites groups (amino acids, sugars…), we discuss the influence of the nature, volume, and ratio of extraction solvents, the sample weight, and the reconstitution solvent. Moreover, the analytical conditions (LC columns, pH and additive of mobile phases and ionization modes) were also optimized so as to ensure the maximum metabolome coverages. Thus, two complementary chromatographic procedures were combined in order to cover a broader range of metabolites: a reversed phase separation (RPLC) on a C18 column followed by detection with positive ionization mode (ESI+) and a hydrophilic interaction chromatography (HILIC) on a zwitterionic column followed by detection with negative ionization mode (ESI-). This work provides information on brain, gills, liver, vs the whole body contribution to the stickleback metabolome. These information would help to guide ecotoxicological and biomonitoring studies.

Enhancing Metabolomic Coverage in Positive Ionization Mode Using Dicationic Reagents by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

Mass spectrometry imaging is a powerful tool to analyze a large number of metabolites with their spatial coordinates collected throughout the sample. However, the significant differences in ionization efficiency pose a big challenge to metabolomic mass spectrometry imaging. To solve the challenge and obtain a complete data profile, researchers typically perform experiments in both positive and negative ionization modes, which is time-consuming. In this work, we evaluated the use of the dicationic reagent, 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride (abbreviated to [C5(bpyr)2]F2) to detect a broad range of metabolites in the positive ionization mode by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI MSI). [C5(bpyr)2]F2 at 10 µM was doped in 50% MeOH/H2O (v/v) electrospray solvent to form +1 charged adducted ions with anionic species (−1 charged) through post-electrospray ionization. This method was demonstrated with sectioned rat liver and hen ovary. A total of 73 deprotonated metabolites from rat liver tissue sections were successfully adducted with [C5(bpyr)2]2+ and putatively identified in the adducted positive ionization polarity, along with 164 positively charged metabolite ions commonly seen in positive ionization mode, which resulted in 44% increased molecular coverage. In addition, we were able to generate images of hen ovary sections showing their morphological features. Following-up tandem mass spectrometry (MS/MS) indicated that this dicationic reagent [C5(bpyr)2]2+ could form ionic bonds with the headgroup of glycerophospholipid ions. The addition of the dicationic reagent [C5(bpyr)2]2+ in the electrospray solvent provides a rapid and effective way to enhance the detection of metabolites in positive ionization mode.

Development of a UPLC–MS/MS method for determination of a dual EZH1/2 inhibitor UNC1999 in rat plasma

Aim: We aimed to establish and validate a simple and sensitive UPLC–MS/MS method for the determination of UNC1999, a dual inhibitor against EZH1 and EZH2 in plasma samples. Materials & methods: UNC1999 in rat plasma was processed with protein precipitation method and then separated on a C18 column and detected under positive ionization mode. The method presented good linearity over the range of 1.0–2000 ng/ml with good accuracy and precision. UNC1999 was absorbed slowly and achieved a maximum concentration of 118.8 ± 12.0 ng/ml 1.5 h after oral administration. Conclusion: The method provides a favorable character in selectivity, linearity, accuracy, precision, recovery, matrix effects and stabilities and was suitable for describing the pharmacokinetic profile of UNC1999.

Physicochemical Characterization and Paper Spray Mass Spectrometry Analysis of Myrciaria floribunda (H. West ex Willd.) O. Berg Accessions

Myrciaria floribunda, also known as rumberry, is a tree native to the Brazilian Atlantic Forest, where its fruits have the potential for commercial use. This study evaluates the antioxidant potential, determines the phytochemical profile, and chemically characterizes the rumberry fruit. Accessions were sampled from the Rumberry Active Germplasm Bank of the Federal University of Alagoas, Brazil. Physical characteristics, chemical characteristics, and phenolic compound content were analyzed. Chemical profile characterization was carried out using PSMS. Accessions had an average weight of 0.86 g. Accession AC137 presented a higher pulp yield (1.12 g). AC132 and AC156 had larger fruits, AC137 showed greater firmness (5.93 N), and AC160 had a higher and total phenolic content ratio (279.01 ± 11.11). Orange-colored accessions scored higher in evaluated parameters, except for AC160 for phenolic content. Thirty-two compounds were identified on positive ionization mode and 42 compounds on negative ionization mode using PSMS. Flavonoids, followed by the derivatives of benzoic acid, sugars, and phenylpropanoids, were the most prominent. Myricitrin, quercitrin, and catechin stand out as flavonoids that have been reported in previous studies with antioxidant and antimicrobial properties, in addition to health and therapeutical benefits, demonstrating the potential of the rumberry fruit.

Simultaneous estimation of ZY-19489 and its active metabolite ZY-20486 in human plasma using LC–MS/MS, a novel antimalarial compound

Aim: ZY-19489 is a new antimalarial drug candidate and selective LC–MS/MS method was established for estimation of ZY-19489 and its metabolite in human plasma. Materials & methods: LLE was employed for extraction, mass spectrometric quantification performed using positive ionization mode and DCP-IMP was used as an internal standard. The chromatographic separation was achieved using mobile phase 5 mM ammonium formate in water and 0.1% v/v ammonia solution in methanol: acetonitrile (90: 10%, v/v) and column Agilent Zorbex Extended C18, 3.5 μm, 100 × 4.6 mm with a 6-min run time. Results: The calibration curve of ZY-19489 was linear over range 1–500 ng/ml and 2–200 ng/ml for metabolite. Assay was reproducible, selective and devoid of matrix effect. Conclusion: The validated assay was implemented for clinical sample analysis derived from healthy human subjects and parasitemia-induced subjects.

Conversion of Organic Dyes into Pigments: Extraction of Flavonoids from Blackberries (Rubus ulmifolius) and Stabilization

The blackberry’s color is composed mainly of natural dyes called anthocyanins. Their color is red–purple, and they can be used as a natural colorant. Anthocyanins are flavonoids, which are products of plants, and their colors range from orange and red to various shades of blue, purple and green, according to pH. In this study, the chemical composition of an extract obtained from blackberries was defined by LC-ESI/LTQOrbitrap/MS in positive and negative ionization mode. Furthermore, we investigated the adsorption process of blackberry extract using several inorganic fillers, such as metakaolin, silica, Lipari pumice, white pozzolan and alumina. The pigments exhibit different colors as a function of their interactions with the fillers. The analysis of the absorption data allowed the estimation of the maximum adsorbing capacity of each individual filler tested. Through thermogravimetric measurements (TGA), the thermal stability and the real adsorption of the organic extract were determined.

UHPLC-Orbitrap-MS Tentative Identification of 51 Oleraceins (Cyclo-Dopa Amides) in Portulaca oleracea L. Cluster Analysis and MS2 Filtering by Mass Difference

Oleraceins are a class of indoline amide glycosides found in Portulaca oleracea L. (Portulacaceae), or purslane. These compounds are characterized by 5,6-dihydroxyindoline-2-carboxylic acid N-acylated with cinnamic acid derivatives, and many are glucosylated. Herein, hydromethanolic extracts of the aerial parts of purslane were subjected to UHPLC-Orbitrap-MS analysis, in negative ionization mode. Diagnostic ion filtering (DIF), followed by diagnostic difference filtering (DDF), were utilized to automatically filter out MS data and select plausible oleracein structures. After an in-depth MS2 analysis, a total of 51 oleracein compounds were tentatively identified. Of them, 26 had structures, matching one of the already known oleracein, and the other 25 were new, undescribed in the literature compounds, belonging to the oleracein class. Moreover, based on selected diagnostic fragment ions, clustering algorithms and visualizations were utilized. As we demonstrate, clustering methods provide valuable insights into the mass fragmentation elucidation of natural compounds in complex mixtures.

Implications of sample treatment on characterization of the riverine environmental metabolome

High-resolution mass spectrometry techniques are widely used in the environmental sciences to characterize natural organic matter and, when utilizing these instruments, researchers must make multiple decisions regarding sample pre-treatment and the instrument ionization mode. To identify how these choices alter organic matter characterization and resulting conclusions, we analyzed a collection of 17 riverine samples from East River, CO (USA) under four PPL-based Solid Phase Extraction (SPE) treatment and electrospray ionization polarity (e.g., positive and negative) combinations: SPE (+), SPE (-), non-SPE (-), and non-SPE (+). The greatest number of formula assignments were achieved with SPE-treated samples due to the removal of compounds that could interfere with ionization. Furthermore, the SPE (-) treatment captured the most formulas across the widest chemical compound diversity. In addition to a reduced number of assigned formulas, the non-SPE datasets resulted in altered thermodynamic interpretations that could cascade into incomplete assumptions about the availability of organic matter pools for microbial heterotrophic respiration. Thus, we infer that the SPE (-) treatment is the best single method for characterizing environmental organic matter pools unless the focus is on lipid-like compounds, in which case we recommend a combination of SPE (-) and SPE (+) to adequately characterize these molecules.