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Keywords = HILIC-HPLC

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19 pages, 2690 KiB  
Article
Development and Validation of Targeted Metabolomics Methods Using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) for the Quantification of 235 Plasma Metabolites
by Kangkang Xu, Franz Berthiller, Barbara U. Metzler-Zebeli and Heidi E. Schwartz-Zimmermann
Molecules 2025, 30(3), 706; https://doi.org/10.3390/molecules30030706 - 5 Feb 2025
Viewed by 2885
Abstract
Plasma contains metabolites with diverse physicochemical properties, ranging from highly polar to highly apolar, and concentrations spanning at least nine orders of magnitude. Plasma metabolome analysis is valuable for monitoring health and evaluating medical interventions but is challenging due to the metabolome’s diversity [...] Read more.
Plasma contains metabolites with diverse physicochemical properties, ranging from highly polar to highly apolar, and concentrations spanning at least nine orders of magnitude. Plasma metabolome analysis is valuable for monitoring health and evaluating medical interventions but is challenging due to the metabolome’s diversity and complexity. This study aims to develop and validate targeted LC-MS/MS methods for quantifying 235 mammalian metabolites from 17 compound classes in porcine plasma without prior derivatization. Utilizing reversed-phase and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, each analyte is identified and quantified using two selected reaction monitoring (SRM) transitions. Fast polarity switching and scheduled SRM enhance the metabolome coverage and throughput, enabling the analysis of one sample in about 40 min. A simple “dilute and shoot” sample preparation protocol was employed, with samples injected at two dilution levels to align metabolite concentrations within calibration curve ranges. Validation in porcine plasma included assessments of carryover, linearity, detection and quantification limits, repeatability and recovery. The method was further applied to plasma samples from various animal species, demonstrating its applicability to human and animal studies. This study establishes two robust LC-MS/MS methods for comprehensive porcine plasma metabolome quantification, advancing large-scale targeted metabolomics in biomedical research. Full article
(This article belongs to the Special Issue The Application of LC-MS in Pharmaceutical Analysis)
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14 pages, 1399 KiB  
Article
Development of a Validated HPLC-UV Method for the Determination of Panthenol, Hesperidin, Rutin, and Allantoin in Pharmaceutical Gel-Permeability Study
by Sofia Almpani, Pavlina-Ioanna Agiannitou, Paraskevi Kyriaki Monou, Georgios Kamaris and Catherine K. Markopoulou
Separations 2025, 12(2), 19; https://doi.org/10.3390/separations12020019 - 22 Jan 2025
Cited by 1 | Viewed by 1641
Abstract
A pressure ulcer is the necrosis of the skin and tissues due to prolonged pressure. Its prevention and treatment are of great importance not only for the health but also for the patient’s quality of life and are considered the highest priority. In [...] Read more.
A pressure ulcer is the necrosis of the skin and tissues due to prolonged pressure. Its prevention and treatment are of great importance not only for the health but also for the patient’s quality of life and are considered the highest priority. In the present study, a reliable analytical method is developed for the quantitative determination of panthenol, hesperidin, rutin, and allantoin by HPLC and UV detectors. The substances were formulated into a pharmaceutical gel, with healing and regenerative properties recommended for first- and second-degree bedsores. Their separation was achieved with a ZIC-Hilic column (150 × 4.6 mm), 5 μm, and a gradient elution system (Solvent A: CH3CN-H2O, 90:10 v/v/v and Solvent B: CH3CN-H2O, 10:90 v/v). The method was evaluated based on the required specifications (%RSD < 2, % Recovery > 96.7%) and was applied for the quantitative extraction of the active substances in the gel. The purification of the samples was carried out using experimental design and Cross-D-Optimal methodology (%RSD < 2.2, % Recovery > 96.9%). Subsequently, the gel was studied in terms of the transdermal permeation of the active pharmaceutical ingredients (APIs) through vertical Franz cells and their behavior (Papp values) was compared with a similar aqueous suspension product (reference formulation). The samples were reconstituted by lyophilization and extraction with methanol. According to the results, the drugs exhibit satisfactory penetration, ensuring the healing of problems that may occur in the skin and dermis. Full article
(This article belongs to the Collection State of the Art in Separation Science)
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22 pages, 4719 KiB  
Article
Faecal Metabolome Profiles in Individuals Diagnosed with Hyperplastic Polyps and Conventional Adenomas
by Alberto Valdés, Sergio Ruiz-Saavedra, Nuria Salazar, Alejandro Cifuentes, Adolfo Suárez, Ylenia Díaz, Carmen González del Rey, Sonia González and Clara G. de los Reyes-Gavilán
Int. J. Mol. Sci. 2024, 25(24), 13324; https://doi.org/10.3390/ijms252413324 - 12 Dec 2024
Cited by 1 | Viewed by 1203
Abstract
Colorectal cancer (CRC) development is a gradual process in which progressive histological alterations of the intestinal mucosa damage occur over years. This process can be influenced by modifiable external factors such as lifestyle and diet. Most CRC cases (>80%) originate from conventional adenomas [...] Read more.
Colorectal cancer (CRC) development is a gradual process in which progressive histological alterations of the intestinal mucosa damage occur over years. This process can be influenced by modifiable external factors such as lifestyle and diet. Most CRC cases (>80%) originate from conventional adenomas through the adenomatous pathway and usually harbour dysplastic cells, whereas the serrated pathway is less frequent (<20% cases) and comprises hyperplastic polyps and other polyps containing dysplastic cells. The aim of the present work was to shed light on alterations of the faecal metabolome associated with hyperplastic polyps and conventional adenomas. Metabolites were analysed by Reversed-Phase High-Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (RP/HPLC-Q/TOF-MS/MS) and Hydrophilic Interaction Liquid Chromatography–Quadrupole-Time of Flight Mass Spectrometry (HILIC-Q/TOF-MS/MS) and the results were integrated. Comparisons were performed between controls without mucosal lesions and the polyps’ group, hyperplastic polyps versus conventional adenomas, and hyperplastic polyps or conventional adenomas versus controls. Alterations of metabolites in specific biochemical modules differentiated hyperplastic polyps and conventional adenomas. The metabolome of the hyperplastic polyps was characterized by an enrichment in glycerophospholipids and an altered metabolism of the degradation pathways of xanthines/purines and pyrimidines, whereas the enrichment in some phenolic compounds and disaccharides, all of them from exogenous origin, was the main differential faecal signature of conventional adenomas. Further research could help to elucidate the contribution of diet and the intestinal microbiota to these metabolomics alterations. Full article
(This article belongs to the Section Molecular Biology)
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14 pages, 1320 KiB  
Article
Development of a Simple and Validated LC–MS/MS Method for Quantitative Determination of Ketotifen in Beagle Dog Plasma and Its Application to Bioequivalence Study of Ketotifen Syrup Dosage Form
by Eunseo Song, Wang-Seob Shim, Doowon Choi, Yuna Song, Hyeong Geun Jo, Soobok Lee, Suk Han Jung, Yeo Jin Choi and Kyung-Tae Lee
Molecules 2024, 29(18), 4505; https://doi.org/10.3390/molecules29184505 - 23 Sep 2024
Viewed by 1678
Abstract
A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer [...] Read more.
A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer in multiple reaction monitoring (MRM) mode in electrospray ionization (ESI) positive ionization mode. The transitions were monitored at m/z 310.2 → 96.0 for KTF and m/z 313.2 → 99.1 for IS. KTF and IS were extracted from plasma using liquid-liquid extraction with methyl tertiary-butyl ether and then analyzed for 3 min with extracted samples (7 µL) into the LC–MS/MS system. Analytes were separated on a Luna® Hilic column (50 × 2.0 mm i.d., 3 μm) using the Nexera X2 HPLC. The mobile phase A consisted of 10 mmol/L ammonium formate (pH 3.0), while mobile phase B consisted of 0.05% formic acid in acetonitrile. The ratio of mobile phase was 5:95 (v/v) at a flow rate of 0.2 mL/min. The method has been thoroughly validated in accordance with the bioanalytical method validation guidelines established by the Ministry of Food and Drug Safety in Korea and the U.S. Food and Drug Administration, addressing selectivity, lower limit of quantification, linearity, carryover, precision, accuracy, recovery, matrix effect, and stability. The developed LC–MS/MS method was effectively utilized for the bioequivalence assessment of ketotifen in Beagle dog plasma following the oral administration of ketotifen syrup. Full article
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12 pages, 1620 KiB  
Article
Green HPLC Enantioseparation of Chemopreventive Chiral Isothiocyanates Homologs on an Immobilized Chiral Stationary Phase Based on Amylose tris-[(S)-α-Methylbenzylcarbamate]
by Francesca Romana Mammone, Alessia Panusa, Roberta Risoluti and Roberto Cirilli
Molecules 2024, 29(12), 2895; https://doi.org/10.3390/molecules29122895 - 18 Jun 2024
Cited by 3 | Viewed by 1674
Abstract
Sulforaphane is a chiral phytochemical with chemopreventive properties. The presence of a stereogenic sulfur atom is responsible for the chirality of the natural isothiocyanate. The key role of sulfur chirality in biological activity is underscored by studies of the efficacy of individual enantiomers [...] Read more.
Sulforaphane is a chiral phytochemical with chemopreventive properties. The presence of a stereogenic sulfur atom is responsible for the chirality of the natural isothiocyanate. The key role of sulfur chirality in biological activity is underscored by studies of the efficacy of individual enantiomers as chemoprotective agents. The predominant native (R) enantiomer is active, whereas the (S) antipode is inactive or has little or no biological activity. Here we provide an enantioselective high-performance liquid chromatography (HPLC) protocol for the direct and complete resolution of sulforaphane and its chiral natural homologs with different aliphatic chain lengths between the sulfinyl sulfur and isothiocyanate group, namely iberin, alyssin, and hesperin. The chromatographic separations were carried out on the immobilized-type CHIRALPAK IH-3 chiral stationary phase with amylose tris-[(S)-methylbenzylcarbamate] as a chiral selector. The effects of different mobile phases consisting of pure alcoholic solvents and hydroalcoholic mixtures on enantiomer retention and enantioselectivity were carefully investigated. Simple and environmentally friendly enantioselective conditions for the resolution of all chiral ITCs were found. In particular, pure ethanol and highly aqueous mobile phases gave excellent enantioseparations. The retention factors of the enantiomers were recorded as the water content in the aqueous-organic modifier (methanol, ethanol, or acetonitrile) mobile phases progressively varied. U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography retention mechanism on the CHIRALPAK IH-3 chiral stationary phase. Finally, experimental chiroptical studies performed in ethanol solution showed that the (R) enantiomers were eluted before the (S) counterpart under all eluent conditions investigated. Full article
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10 pages, 6594 KiB  
Communication
Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography
by Shimba Kawasue, Kyoko Kuniyoshi, Masashi Uema and Naomasa Oshiro
Toxins 2024, 16(6), 260; https://doi.org/10.3390/toxins16060260 - 4 Jun 2024
Cited by 3 | Viewed by 1720
Abstract
Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives [...] Read more.
Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX. Full article
(This article belongs to the Special Issue Analytical Chemistry Techniques in Toxin Detection)
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14 pages, 1611 KiB  
Article
Analysis of the Ibotenic Acid, Muscimol, and Ergosterol Content of an Amanita Muscaria Hydroalcoholic Extract with an Evaluation of Its Cytotoxic Effect against a Panel of Lung Cell Lines In Vitro
by Alexander Dushkov, Zuzana Vosáhlová, Alexander Tzintzarov, Květa Kalíková, Tomáš Křížek and Iva Ugrinova
Molecules 2023, 28(19), 6824; https://doi.org/10.3390/molecules28196824 - 27 Sep 2023
Cited by 15 | Viewed by 7729
Abstract
The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented cases of use of A. muscaria-containing preparations against various diseases, including cancer, [...] Read more.
The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented cases of use of A. muscaria-containing preparations against various diseases, including cancer, to no apparent ill effect. The search for compounds that can be used to treat cancer among various plants and fungi has been intensifying in recent years. In light of this, we describe an HPLC HILIC analytical method for the evaluation of the content of the anticancer compound ergosterol (ERG) and the neuroactive alkaloids ibotenic acid (IBO) and muscimol (MUS) that contribute significantly to the unpleasant physiological syndrome associated with A. muscaria consumption. A ‘homemade’ A. muscaria tincture made using 80-proof rye vodka as the solvent, an A. muscaria extract made with a standardized water–ethanol solution as the solvent, and fractions obtained from the second extract via liquid–liquid extraction with nonpolar solvents were analyzed. The study also presents the results of capillary zone electrophoresis with contactless conductivity detection and UHPLC-MS/MS analyses of the IBO and MUS content of the two native A. muscaria extracts and an evaluation of the standardized extract’s cytotoxic effect against a small panel of lung cell cultures in vitro. Our results show that the standardized extract has a significant cytotoxic effect and does not contain the compounds of interest in any significant quantity. Full article
(This article belongs to the Section Natural Products Chemistry)
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22 pages, 29803 KiB  
Article
Generation and Characterization of Native and Sialic Acid-Deficient IgE
by Alex J. McCraw, Richard A. Gardner, Anna M. Davies, Daniel I. R. Spencer, Melanie Grandits, Gerd K. Wagner, James M. McDonnell, Sophia N. Karagiannis, Alicia Chenoweth and Silvia Crescioli
Int. J. Mol. Sci. 2022, 23(21), 13455; https://doi.org/10.3390/ijms232113455 - 3 Nov 2022
Cited by 6 | Viewed by 3286
Abstract
Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The [...] Read more.
Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs. Full article
(This article belongs to the Special Issue Molecular Mechanisms of Allergy and Asthma 2.0)
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8 pages, 273 KiB  
Article
Quantification of a Sulfated Marine-Inspired Antifouling Compound in Several Aqueous Matrices: Biodegradation Studies and Leaching Assays from Polydimethylsiloxane Coatings
by Cátia Vilas-Boas, Virgínia Gonçalves, Paolo De Marco, Emília Sousa, Madalena Pinto, Elisabete R. Silva, Maria Elizabeth Tiritan and Marta Correia-da-Silva
Mar. Drugs 2022, 20(9), 548; https://doi.org/10.3390/md20090548 - 25 Aug 2022
Cited by 3 | Viewed by 2206
Abstract
The development of marine-inspired compounds as non-toxic antifouling (AF) agents has been pursued in the last years. Sulfur is the third most common element in seawater. Sulfur is present in oxygenated seawater as sulfate anion (SO42−), which is the most [...] Read more.
The development of marine-inspired compounds as non-toxic antifouling (AF) agents has been pursued in the last years. Sulfur is the third most common element in seawater. Sulfur is present in oxygenated seawater as sulfate anion (SO42−), which is the most stable combination of sulfur in seawater, and several promising AF secondary metabolites with sulfate groups have been described. However, sulfated compounds proved to be an analytical challenge to quantify by HPLC. Taking these facts into consideration, this work presents the development and validation of a method for the quantification of gallic acid persulfate (GAP) in seawater and ultrapure water matrix, based on hydrophilic interaction liquid chromatography (HILIC). This method was used to evaluate GAP stability following several abiotic and biotic degradation assays, and to quantify its release in seawater from room-temperature-vulcanizing polydimethylsiloxane commercial coating. GAP was very stable in several water matrices, even at different pH values and in the presence/absence of marine microorganisms and presented a leaching value lower than 0.5%. This work discloses HILIC as an analytical method to overcome the difficulties in quantifying sulfated compounds in water matrices and highlights the potential of GAP as a promising long-lasting coating. Full article
(This article belongs to the Special Issue Marine Natural Products with Antifouling Activity, 2nd Edition)
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12 pages, 1853 KiB  
Article
Separation of Monosaccharide Anomers on Photo-Click Cysteine-Based Stationary Phase: The α/β Interconversion Process Studied by Dynamic Hydrophilic Liquid Chromatography
by Andrea Calcaterra, Simone Manetto, Fabio Buonsenso, Antonio Francioso, Marco Pierini and Claudio Villani
Separations 2022, 9(8), 203; https://doi.org/10.3390/separations9080203 - 5 Aug 2022
Cited by 3 | Viewed by 3015
Abstract
In High-Performance Liquid Chromatography (HPLC), the separation of reducing sugars can typically show three possible typologies of chromatographic profiles (i.e., single peak, two resolved peaks and two peaks interconnected by a plateau) due to the rate at which the relevant α/β anomers interconversion [...] Read more.
In High-Performance Liquid Chromatography (HPLC), the separation of reducing sugars can typically show three possible typologies of chromatographic profiles (i.e., single peak, two resolved peaks and two peaks interconnected by a plateau) due to the rate at which the relevant α/β anomers interconversion (anomerization) can take place in relation to their elution-time. By analyzing these chromatographic profiles, thermodynamic and kinetic properties of anomerization phenomenon can be extrapolated. In this work we studied the anomerization of some monosaccharides by using a recently developed photo-click cysteine-based stationary phase through dynamic hydrophilic interaction liquid chromatography (D-HILIC) conditions. In the 5–25 °C temperature range, the ΔG#α→β and ΔG#β→α barriers were found to achieve values within the interval 21.1/22.2 kcal/mol for glucose, with differences between α→β and β→α reactions of about 0.4 kcal/mol. For xylose, in the same temperature range, the ΔG#α→β and ΔG#β→α barriers are between 20.7 to 21.5 kcal/mol, with differences between α→β and β→α reactions of about 0.2 kcal/mol. The experimental data are in agreement with those reported in literature, confirming the this new stationary phase using HILIC conditions is a robust platform to measure kinetic and thermodynamic properties of the isomerization reaction. Full article
(This article belongs to the Section Chromatographic Separations)
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26 pages, 6933 KiB  
Article
Concepts for New Rapid Simple HPLC Method for Quantification of Fosfomycin Trometamol in Pharmaceutical Dosage Forms with Direct UV Detection
by Marjan Piponski, Tanja Bakovska Stoimenova, Tetiana Melnyk, Sergiy Kovalenko, Elena Lazarevska Todevska, Marjan Velkovski, Sami El Deeb, Yuriy Mysula and Liliya Logoyda
Sci. Pharm. 2022, 90(2), 35; https://doi.org/10.3390/scipharm90020035 - 24 May 2022
Cited by 4 | Viewed by 9999
Abstract
Two different concepts for developing direct HPLC-UV methods for quantifying fosfomycin trometamol were developed without any derivatization and modification of the analyte. In the first concept, without the use of alkylamines as ion-pairs in the mobile phase, by using cyanopropyl CN and a [...] Read more.
Two different concepts for developing direct HPLC-UV methods for quantifying fosfomycin trometamol were developed without any derivatization and modification of the analyte. In the first concept, without the use of alkylamines as ion-pairs in the mobile phase, by using cyanopropyl CN and a strong anion-exchanger column, we investigated the possibility of their highly polar and anion-exchanging forces and mechanisms to retain, separate and detect trometamol without the help of additional agents or modifiers. In the second concept, the most frequent reversed-phase C18 columns with different characteristics and vendors were tested in combination with different length-based alkylamines with 3–10 C atoms in their chains. In our research, we found that the ion-pairing of fosfomycin with 6–10 C-atom-based alkyl-length of aliphatic chains manifested the most appropriate strength of interactions between alkyl-paired trometamol molecules and octadecylsilane or C18 bonded RP column to achieve optimal retention, selectivity and peak shape on chromatograms, with the possibility for the fine-tuning of elution time. The simplicity of our method concept omits the need for expensive and sophisticated columns like HILIC, C30 graphite carbon, and mixed-mode-based columns for easier retaining, separation, and determination of fosfomycin, and for its quantification purposes, especially in high-throughput analyses in regular quality-control laboratories. Full article
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17 pages, 1614 KiB  
Article
Sulfated Phenolic Substances: Preparation and Optimized HPLC Analysis
by Lucie Petrásková, Kristýna Káňová, Katerina Brodsky, Anastasiia Hetman, Barbora Petránková, Helena Pelantová, Vladimír Křen and Kateřina Valentová
Int. J. Mol. Sci. 2022, 23(10), 5743; https://doi.org/10.3390/ijms23105743 - 20 May 2022
Cited by 5 | Viewed by 3550
Abstract
Sulfation is an important reaction in nature, and sulfated phenolic compounds are of interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be prepared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work was to obtain [...] Read more.
Sulfation is an important reaction in nature, and sulfated phenolic compounds are of interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be prepared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work was to obtain a large library of sulfated phenols, phenolic acids, flavonoids, and flavonolignans and optimize their HPLC (high performance liquid chromatography) analysis. Four new sulfates of 2,3,4-trihydroxybenzoic acid, catechol, 4-methylcatechol, and phloroglucinol were prepared and fully characterized using MS (mass spectrometry), 1H, and 13C NMR. The separation was investigated using HPLC with PDA (photodiode-array) detection and a total of 38 standards of phenolics and their sulfates. Different stationary (monolithic C18, C18 Polar, pentafluorophenyl, ZICpHILIC) and mobile phases with or without ammonium acetate buffer were compared. The separation results were strongly dependent on the pH and buffer capacity of the mobile phase. The developed robust HPLC method is suitable for the separation of enzymatic sulfation reaction mixtures of flavonoids, flavonolignans, 2,3-dehydroflavonolignans, phenolic acids, and phenols with PDA detection. Moreover, the method is directly applicable in conjunction with mass detection due to the low flow rate and the absence of phosphate buffer and/or ion-pairing reagents in the mobile phase. Full article
(This article belongs to the Collection Feature Papers in Bioactives and Nutraceuticals)
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12 pages, 1142 KiB  
Article
Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma
by Gwendolyn van Gorkom, Birgit Gijsbers, Erik-Jan Ververs, Ahmed El Molla, Cindy Sarodnik, Celine Riess, Will Wodzig, Gerard Bos and Catharina Van Elssen
Antioxidants 2022, 11(1), 134; https://doi.org/10.3390/antiox11010134 - 7 Jan 2022
Cited by 11 | Viewed by 3703
Abstract
Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, [...] Read more.
Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood. Full article
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12 pages, 3012 KiB  
Article
Analytical Problems in Separation of Selenomethionine and Its Oxidative Product in HILIC HPLC
by Aleksandra Sentkowska and Krystyna Pyrzynska
Molecules 2021, 26(16), 5073; https://doi.org/10.3390/molecules26165073 - 21 Aug 2021
Cited by 7 | Viewed by 2478
Abstract
Selenomethionine (SeMet) is one of the main selenium forms in foods and supplements. Determining its presence in natural food samples creates difficulties due to possible oxidation processes. The objective of this study was to evaluate the possible degradation of SeMet in water extracts [...] Read more.
Selenomethionine (SeMet) is one of the main selenium forms in foods and supplements. Determining its presence in natural food samples creates difficulties due to possible oxidation processes. The objective of this study was to evaluate the possible degradation of SeMet in water extracts of green teas, one of the most consumed beverages worldwide. Such a medium has not been investigated at this time. The HILIC-HPLC MS/MS method with different stationary phases was used to achieve the satisfactory separation of SeMet and selenomethionine oxide (SeMetO). The addition of dithiothreitol and β-mercaptoethanol, recommended to ensure that SeMet is kept in the reduced form, was also evaluated. The best separation was achieved using the zwitterionic HILIC stationary phase coupled to mass spectrometry and MeOH with water (85/15, v/v) as the eluent. Extraction was done with hot water with the addition of β-mercaptoethanol. The infusions prepared from Lung-Ching teas (from the Zhejiang Province in China) contained the highest concentration of selenium in a typical cup of tea (12.5–17.3 µg L−1). For other tested teas it decreased in the following order: Yunnan > Dilmah > Lipton. For Lung-Ching teas, the sum of concentrations of SeMet and SeMetO corresponded to about 46–63% of the total selenium in their extracts. Full article
(This article belongs to the Collection Advances in Food Analysis)
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13 pages, 1639 KiB  
Article
Determination of 1-Deoxynojirimycin (1-DNJ) in Leaves of Italian or Italy-Adapted Cultivars of Mulberry (Morus sp.pl.) by HPLC-MS
by Lucia Marchetti, Alessio Saviane, Antonella dalla Montà, Graziella Paglia, Federica Pellati, Stefania Benvenuti, Davide Bertelli and Silvia Cappellozza
Plants 2021, 10(8), 1553; https://doi.org/10.3390/plants10081553 - 28 Jul 2021
Cited by 21 | Viewed by 4867
Abstract
Recently, 1-DNJ has been widely studied by scientists for its capacity to inhibit α-glucosidase and reduce postprandial blood glucose and fat accumulation. To the best of our knowledge, this is the first analytical determination of 1-DNJ in Morus sp.pl. leaves carried out on [...] Read more.
Recently, 1-DNJ has been widely studied by scientists for its capacity to inhibit α-glucosidase and reduce postprandial blood glucose and fat accumulation. To the best of our knowledge, this is the first analytical determination of 1-DNJ in Morus sp.pl. leaves carried out on Italian crops, and it could be used as a reference to assess the quality of the plant material in comparison to Far Eastern Asia cultivations. The effects of two thermal treatments were compared to test the incidence of the drying process on the 1-DNJ extractability. In addition, two harvesting seasons in the same year (2017) and two subsequent harvesting years (2017–2018) were considered. The amount of 1-DNJ herein found was comparable to that reported in the scientific literature for Asian cultivations. The increase in 1-DNJ along the summer and the higher level of this compound in the apical leaves also complies with previous findings. However, a strong implication for the climatic conditions in the different years and a significant interaction between climate and genotypes suggest exploring very carefully the agronomic practices and selecting cultivars according to different environmental conditions with a view to standardize the 1-DNJ amount in leaves. Full article
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