scholarly journals An RP-LC-UV-TWIMS-HRMS and Chemometric Approach to Differentiate between Momordicabalsamina Chemotypes from Three Different Geographical Locations in Limpopo Province of South Africa

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1896
Author(s):  
Pieter Venter ◽  
Kholofelo Malemela ◽  
Vusi Mbazima ◽  
Leseilane J. Mampuru ◽  
Christo J. F. Muller ◽  
...  
Keyword(s):  
Molecular Species ◽  
High Resolution Mass Spectrometry ◽  
Parent Compound ◽  
Travelling Wave ◽  
Reversed Phase ◽  
Limpopo Province ◽  
Leaf Extracts ◽  
Triterpenoid Glycosides ◽  
Ht 29 ◽  
Geographical Locations

Momordica balsamina leaf extracts originating from three different geographical locations were analyzed using reversed-phase liquid chromatography (RP-LC) coupled to travelling wave ion mobility (TWIMS) and high-resolution mass spectrometry (HRMS) in conjunction with chemometric analysis to differentiate between potential chemotypes. Furthermore, the cytotoxicity of the three individual chemotypes was evaluated using HT-29 colon cancer cells. A total of 11 molecular species including three flavonol glycosides, five cucurbitane-type triterpenoid aglycones and three glycosidic cucurbitane-type triterpenoids were identified. The cucurbitane-type triterpenoid aglycones were detected in the positive ionization mode following dehydration [M + H − H2O]+ of the parent compound, whereas the cucurbitane-type triterpenoid glycosides were primarily identified following adduct formation with ammonia [M + NH4]+. The principle component analysis (PCA) loadings plot and a variable influence on projection (VIP) analysis revealed that the isomeric pair balsaminol E and/or karavilagen E was the key molecular species contributing to the distinction between geographical samples. Ultimately, based on statistical analysis, it is hypothesized that balsaminol E and/or karavilagen E are likely responsible for the cytotoxic effects in HT-29 cells.

A Fast and Sensitive Method Combining Reversed-Phase Chromatography with High Resolution Mass Spectrometry to Quantify 2-Fluoro-2-Deoxyglucose and Its Phosphorylated Metabolite for Determining Glucose Uptake

2019 ◽  
Author(s):  
Ashley Williams ◽  
Deborah Muoio ◽  
Guofang Zhang
Keyword(s):  
Glucose Uptake ◽  
Biological Samples ◽  
High Resolution Mass Spectrometry ◽  
Reversed Phase ◽  
Sodium Adduct ◽  
Negative Mode ◽  
Glucose Analogues ◽  
Positive Mode ◽  
Q Exactive ◽  
Glucose Analogue

Quantative measurements of the glucose analogue, 2-deoxyglucose (2DG), and its phosphorylated metabolite (2-deoxyglucose-6-phosphate (2DG-6-P)) are critical for the measurement of glucose uptake. While the field has long identified the need for sensitive and reliable assays that deploy non-radiolabled glucose analogues to assess glucose uptake, no analytical MS-based methods exist to detect trace amounts in complex biological samples. In the present work, we show that 2DG is poorly suited for MS-based methods due to interfering metabolites. We therefore developed and validated an alternative C18-based LC-Q-Exactive-Orbitrap-MS method using 2-fluoro-2-deoxyglucose (2FDG) to quantify both 2FDG and 2FDG-6-P by measuring the sodium adduct of 2FDG in the positive mode and deprotonation of 2FDG-6-P in the negative mode. The low detection limit of this method can reach 81.4 and 48.8 fmol for both 2FDG and 2FDG-6-P, respectively. The newly developed method was fully validated via calibration curves in the presence and absence of biological matrix. The present work is the first successful LC-MS method that can quantify trace amounts of a nonradiolabeled glucose analogue and its phosphorylated metabolite and is a promising analytical method to determine glucose uptake in biological samples.

Confirmation of Long Term Excreted Metabolites of Stanozolol by Gas Chromatography Coupled with High Resolution Mass Spectrometry (GC/HRMS)

2008 ◽  
Vol 59 (7) ◽  
Author(s):  
Ileana Vajiala ◽  
Ruxandra Subasu ◽  
Mirela Zorio ◽  
Rodica Picu
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Parent Compound ◽  
Urine Specimen ◽  
Decision Making Process ◽  
Purification Procedure ◽  
Specific Identification ◽  
Immunoaffinity Purification ◽  
In Doping ◽  
Resolution Mass

Upon screening identification of Stanozolol, GC/HRMS confirmation of the suspicious sample is done by reanalysis of the urine specimen, where a specific immunoaffinity purification procedure is used to selectively isolate the long term excreted metabolites of Stanozolol. By meeting the specific identification criteria for more than one metabolite of the same parent compound, additional evidence could be obtained in the decision making process in doping control.

scholarly journals A combined flow injection/reversed phase chromatography – high resolution mass spectrometry workflow for accurate absolute lipid quantification with 13C- internal standards

The Analyst ◽  
2021 ◽  
Author(s):  
Harald Schoeny ◽  
Evelyn Rampler ◽  
Yasin El Abiead ◽  
Felina Hildebrand ◽  
Olivia Zach ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Flow Injection ◽  
High Resolution Mass Spectrometry ◽  
Reversed Phase ◽  
Internal Standards ◽  
Lipid Quantification ◽  
High Resolution Mass ◽  
Resolution Mass

We propose a fully automated novel workflow for lipidomics based on flow injection- followed by liquid chromatography high resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome...

scholarly journals Pro-Apoptotic Activity of Artichoke Leaf Extracts in Human HT-29 and RKO Colon Cancer Cells

2021 ◽  
Vol 18 (8) ◽  
pp. 4166
Author(s):  
Milena Villarini ◽  
Mattia Acito ◽  
Raffaella di Vito ◽  
Samuele Vannini ◽  
Luca Dominici ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Colon Cancer ◽  
Chlorogenic Acid ◽  
Cancer Cells ◽  
Herbaceous Plant ◽  
Colon Cancer Cells ◽  
Leaf Extracts ◽  
Caffeoylquinic Acids ◽  
Induction Of Apoptosis ◽  
Ht 29

(1) Background: Cynara cardunculus L. subsp. scolymus (L.) Hegi, popularly known as artichoke, is an herbaceous plant belonging to the Asteraceae family. Artichoke leaf extracts (ALEs) have been widely used in traditional medicine because of their hepatoprotective, cholagogic, hypoglycaemic, hypolipemic and antibacterial properties. ALEs are also recognized for their antioxidative and anti-inflammatory activities. In this study, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as effect on cell growth of ALEs on human colon cancer HT-29 and RKO cells. HT-29 and RKO cells exhibit a different p53 status: RKO cells express the wild-type protein, whereas HT-29 cells express a p53-R273H contact mutant. (2) Methods: Four different ALEs were obtained by sequential extraction of dried artichoke leaves; ALEs were characterized for their content in chlorogenic acid, cynaropicrin, and caffeoylquinic acids. HT-29 and RKO cells were used for in vitro testing (i.e., cytotoxicity and genotoxicity assessment, cell cycle analysis, apoptosis induction). (3) Results: Two out of the four tested ALEs showed marked effects on cell vitality toward HT-29 and RKO tumour cells. The effect was accompanied by a genotoxic activity exerted at a non-cytotoxic concentrations, by a significant perturbation of cell cycle (i.e., with increase of cells in the sub-G1 phase), and by the induction of apoptosis. (4) Conclusions: ALEs rich in cynaropicrin, caffeoylquinic acids, and chlorogenic acid showed to be capable of affecting HT-29 and RKO colon cancer cells by inducing favourable biological effects: cell cycle perturbation, activation of mitochondrial dependent pathway of apoptosis, and the induction of genotoxic effects probably mediated by the induction of apoptosis. Taken together, these results weigh in favour of a potential cancer chemotherapeutic activity of ALEs.

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260354
Author(s):  
Emmanuelle Lebeau-Roche ◽  
Gaëlle Daniele ◽  
Aurélie Fildier ◽  
Cyril Turies ◽  
Odile Dedourge-Geffard ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Gasterosteus Aculeatus ◽  
High Resolution Mass Spectrometry ◽  
Reversed Phase ◽  
Research Field ◽  
Untargeted Metabolomics ◽  
Whole Body ◽  
Systematic Analysis ◽  
Fish Model ◽  
Ionization Mode

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.

scholarly journals Acetaminophen Metabolism Revisited Using Non-Targeted Analyses: Implications for Human Biomonitoring

2020 ◽  
Author(s):  
Arthur David ◽  
Jade Chaker ◽  
Thibaut Léger ◽  
Raghad Al-Salhi ◽  
Marlene Danner Dalgaard ◽  
...  
Keyword(s):  
Phase Ii ◽  
Enterohepatic Circulation ◽  
High Resolution Mass Spectrometry ◽  
Parent Compound ◽  
Human Biomonitoring ◽  
Relevant Information ◽  
Point Of View ◽  
Internal Exposure ◽  
Current Standard ◽  
Excretion Rates

The analgesic paracetamol (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is criticical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key biomonitoring issues with APAP using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. Using non-targeted analyses based on high-resolution mass spectrometry, we identified in a controlled longitudinal exposure study with male volunteers, unrecognized APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, the conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods provide an option to decrease potential underestimation of APAP use and challenges the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of non-persistant chemical such as APAP. <br>

scholarly journals Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Constantin Blöchl ◽  
Christof Regl ◽  
Christian G. Huber ◽  
Petra Winter ◽  
Richard Weiss ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Polyclonal Antibodies ◽  
Reversed Phase ◽  
Igg Subclasses ◽  
Intact Protein ◽  
Protein Subunit ◽  
Bacterial Protease ◽  
Grass Pollen Allergen ◽  
Functional Antibodies ◽  
Therapeutic Monoclonal Antibodies

Abstract In recent years, advanced HPLC-MS strategies based on intact protein (“top-down”) or protein subunit (“middle-up/middle-down”) analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.

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