Nuevas contribuciones de la cromatografía electrocinética con detección UV y de espectrometría de masas en el campo de las separaciones quirales

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Nuevas contribuciones de la cromatografía electrocinética con detección UV y de espectrometría de masas en el campo de las separaciones quirales

Premio Extraordinario de Doctorado 201

A chiral electrokinetic chromatography method for the separation and quantitation of licarbazepine and licarbazepine acetate in pharmaceutical formulations and urine samples

S-Licarbazepine acetate is a new antiepileptic that is quickly metabolized to S-licarbazepine which is the active principle. In this study, an enantioselective methoddology enabling the simultaneous separation of licarbazepine acetate and licarbazepine by Electrokinetic Chromatography has been developed for the first time. After evaluating the potential of different chiral selectors, including bile salts and cyclodextrins, and selecting carboxymethyl-?-cyclodextrin as the most appropriate, a Box-Behnken experimental design was effectively applied for the optimization of the experimental separation conditions. Employing the best conditions, the four enantiomers were simultaneously separated (resolution values > 2.4) in less than 7 min. The evaluation of the figures of merit of the developed methodology showed to be suitable to determine both compounds. Finally, the EKC method was successfully applied in three different studies: (i) the quality control of the enantiopure pharmaceutical formulation, (ii) the monitoring of the stability and gastrointestinal digestion of the pharmaceutical formulation through a hydrolysis study, and (iii) the determination of licOH enantiomers in urine samples

Enantiomeric determination of econazole and sulconazole by electrokinetic chromatography using hydroxypropyl-b-cyclodextrin combined with ionic liquids based on L-lysine and Ly-glutamic acid

Two analytical methodologies based on the combined use of hydroxypropyl-beta-cyclodextrin and two different amino acid-based chiral ionic liquids (tetrabutylammonium-L-lysine or tetrabutylammonium-L-glutamic acid) in electrokinetic chromatography were developed in this work to perform the enantios-elective determination of econazole and sulconazole in pharmaceutical formulations. The influence of different experimental variables such as buffer concentration, applied voltage, nature and concentration of the ionic liquid, temperature and injection time, on the enantiomeric separation was investigated. The combination of hydroxypropyl-beta-cyclodextrin and tetrabutylammonium-L-lysine under the optimized conditions enabled to achieve the enantiomeric determination of both drugs with high enantiomeric resolution (3.5 for econazole and 2.4 for sulconazole). The analytical characteristics of the developed methodologies were evaluated in terms of linearity, precision, LOD, LOQ and recovery showing good performance for the determination of both drugs which were successfully quantitated in pharmaceutical formulations. This work reports the first analytical methodology enabling the enantiomeric determination of sulconazole in pharmaceutical formulations

High resolution liquid chromatography tandem mass spectrometry for the separation and identification of peptides in coffee silverskin protein hydrolysates

An analytical methodology was developed for the first time in this work to investigate the peptide composition of coffee silverskin protein hydrolysates. Coffee silverskin is the only by-product produced in the coffee roasting process and it contains a relatively high amount of proteins (16.2-19.0%). Different extraction procedures were tested to obtain protein extracts from coffee silverskin samples which were subsequently submitted to enzymatic digestion using different enzymes. Protein hydrolysates from Arabica coffee silverskin obtained using three roasting degrees (light, medium and dark) were considered in order to evaluate the influence of this process on peptide composition. Antioxidant and hypocholesterolemic activities were investigated for these hydrolysates. A method based on the use of liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer was developed enabling the separation and identification of different short chain peptides in the coffee silverskin hydrolysates using de novo sequencing tool. Different peptides, with a number of amino acids ranging from 4 to 12, were identified in the coffee silverskin analyzed. Peptides obtained were different depending on the enzymatic hydrolysis employed. As general trend, the results obtained showed that peptide composition in coffee silverskin protein hydrolysates was not significantly affected by the coffee roasting process

Chiral micellar electrokinetic chromatography

The potential of Micellar Electrokinetic Chromatography to achieve enantiomeric separations is reviewed in this article. The separation principles and the most frequently employed separation strategies to achieve chiral separations by Micellar Electrokinetic Chromatography are described. The use of chiral micellar systems alone or combined with other micellar systems or chiral selectors, as well as of mixtures of achiral micellar systems with chiral selectors is discussed together with the effect of different additives present in the separation medium. Indirect methods based on the derivatization of analytes with chiral derivatizing reagents and the use of achiral micelles are also considered. Preconcentration techniques employed to improve sensitivity and the main approaches developed to facilitate the coupling with Mass Spectrometry are included. The most recent and relevant methodologies developed by chiral Micellar Electrokinetic Chromatography and their applications in different fields are presented

Enantiomeric separation of non-protein amino acids by Electrokinetic Chromatography

New analytical methodologies enabling the enantiomeric separation of a group of non-protein amino acids of interest in the pharmaceutical and food analysis fields were developed in this work using Electrokinetic Chromatography. The use of FMOC as derivatization reagent and the subsequent separation using acidic conditions (formate buffer at pH 2.0) and anionic cyclodextrins as chiral selectors allowed the chiral separation of eight from the ten non-protein amino acids studied. Pyroglutamic acid, norvaline, norleucine, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, and selenomethionine were enantiomericaly separated using sulfated-alpha-CD while sulfated-gamma-CD enabled the enantiomeric separation of norvaline, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, selenomethionie, citrulline, and pipecolic acid. Moreover, the potential of the developed methodologies was demonstrated in the analysis of citrulline and its enantiomeric impurity in food supplements. For that purpose, experimental and instrumental variables were optimized and the analytical characteristics of the proposed method were evaluated. LODs of 2.1 x 10(-7) and 1.8 x 10(-7) M for D-and L-citrulline, respectively, were obtained. D-Cit was not detectable in any of the six food supplement samples analyzed showing that the effect of storage time on the racemization of citrulline was negligible. (C) 2016 Elsevier B.V. All rights reserved

Isolation of proteins from spent coffee grounds. Polyphenol removal and peptide identification in the protein hydrolysates by RP-HPLC-ESI-Q-TOF

Several works have been focused on the extraction of polysaccharides, polyphenols and caffeine from spent coffee grounds (SCG) and their application in food formulations, but the peptide bioactivity from SCG protein hydrolysates has never been addressed. In the present work and for the first time, two different methods to isolate proteins from SCG have been compared, demonstrating that a urea-based extraction buffer provides a higher yield. This extraction method was then applied to compare the protein content in SCG from different coffee-brewing preparations, showing a higher protein content in SCG from espresso coffee machines. In addition, a polyphenol extraction step to remove interferences has been evaluated and the hydrolysis of the extracted proteins using alcalase and thermolysin enzymes has been compared. The effect of roasting degree on the antioxidant and in vitro angiotensin-converting enzyme (ACE)-inhibitory activity has been evaluated. The results show that the ACE-inhibitory activity is higher when SCG proteins are obtained from medium and dark roasted coffees and then hydrolyzed with thermolysin. Finally, the peptides contained in these hydrolysates have been identified by reversed-phase high-performance liquid chromatography coupled via electrospray ionization to a quadrupole time-of-flight mass spectrometer (RP-HPLC-ESI-Q-TOF)

Capillary electrophoresis determination of non-protein amino acids as quality markers in foods

Non-protein amino acids mainly exist in food as products formed during food processing, as metabolic intermediates or as additives to increase nutritional and functional properties of food. This fact makes their analysis and determination an attractive field in food science since they can give interesting information on the quality and safety of foods. This article presents a comprehensive review devoted to describe the latest advances in the development of (achiral and chiral) analytical methodologies by capillary electrophoresis and microchip capillary electrophoresis for the analysis of non-protein amino acids in a variety of food samples. Most relevant information related to sample treatment, experimental separation and detection conditions, preconcentration strategies and limits of detection will be provided.Universidad de Alcal

A rapid electrokinetic chromatography method using short-end injection for the enantioselective separation of tryptophan

A rapid enantioselective methodology for the analysis of tryptophan was developed in this work by electrokinetic chromatography using short-end injection and an anionic cyclodextrin as chiral selector. No previous derivatization of tryptophan was necessary. The influence of different experimental variables on the enantiomeric separation was investigated. The use of a 100 mM formate buffer (pH 2.2) containing 1.25% sulfated-?-cyclodextrin with an uncoated fused-silica capillary of 50 µm inner diameter with a total length of 48.5 cm (effective length of 8.5 cm), and an injection by applying a pressure of -50 mbar (short-end injection) for 4 s, enabled the enantiomeric separation of tryptophan within 2.5 min with a resolution of 7.4. As desirable, the enantiomeric impurity, D-tryptophan, was the first-migrating enantiomer. The analytical characteristics of the developed methodology were evaluated in terms of linearity, precision, accuracy, and limits of detection and quantification, showing its good performance to be applied to the analysis of tryptophan-based dietary supplements. A relative limit of detection of 0.1% was obtained for the enantiomeric impurity, D-tryptophan, in the presence of the L-enantiomer. Results showed that the developed methodology is an interesting alternative for the enantioselective analysis of tryptophan enabling the rapid quality control of dietary supplements