Cristina Montealegre

Potential of vancomycin for the enantiomeric resolution of FMOC‐amino acids by capillary electrophoresis‐ion‐trap‐mass spectrometry

The potential of the antibiotic vancomycin (VC) as chiral selector for the enantiomeric separation of amino acids by CE‐ESI‐MS/MS2 was investigated for the first time in this work. Derivatization of amino acids with FMOC‐Cl was carried out to enable their interaction with VC as well as the formation of precursor ions with larger m/z which were employed in MS2 experiments. The partial filling of a coated capillary was employed to avoid the loss in MS sensitivity originated by the introduction of VC in the ionization source. Under optimized conditions, the simultaneous enantiomeric separation and unequivocal identification of 17 amino acids (two of them being nonprotein amino acids) took place in about 20 min with LODs in the micromolar range.

Analysis of olive allergens

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Besides this, some cases of contact dermatitis and food allergy to the olive fruit and olive oil have been also described. Several scientific studies dealing with olive allergens has been reported, being the information available about them constantly increasing. Up to date, twelve allergens have been identified in olive pollen while just one allergen has been identified in olive fruit. This review article describes considerations about allergen extraction and production, also describing the different methodologies employed in the physicochemical and immunological characterization of olive allergens. Finally, a revision of the most relevant studies in the analysis of both olive pollen and olive fruit allergens is carried out.

Development of chiral methodologies by capillary electrophoresis with ultraviolet and mass spectrometry detection for duloxetine analysis in pharmaceutical formulations.

Two chiral methodologies were developed by capillary electrophoresis (CE) with UV and mass spectrometry (MS) detection to ensure the quality control of the drug duloxetine, commercialized as a pure enantiomer. Both methods were optimized to achieve a high baseline enantioresolution (Rs>2) and an acceptable precision (RSD values <5% for instrumental repeatability and <10% for intermediate precision). In addition to allow the unequivocal identification of duloxetine enantiomers, the CE-MS method improved the sensitivity with respect to the use of CE-UV (LOD 200 ng/mL by CE-UV and 20 ng/mL by CE-MS) enabling to detect 0.02% of duloxetine enantiomeric impurity. This is the lowest LOD value ever reported for this drug, being this work the first one enabling to accomplish with the ICH guidelines requirements. The developed methods were validated and applied for the first time to the analysis of four pharmaceutical formulations. The content of R-duloxetine in all these samples was below the detection limit and the amount of S-duloxetine was in good agreement with the labeled content, obtaining results by the two methods that did not differ significantly (p-values >0.05).

Determination and characterization of glycerophospholipids in olive fruit and oil by nonaqueous capillary electrophoresis with electrospray-mass spectrometric detection.

A nonaqueous capillary electrophoresis method with electrospray-mass spectrometric detection was developed to study the glycerophospholipid fraction in olive fruit and olive oil samples. In olive fruits, where the information available about the phospholipid fraction was very scarce, results obtained in this work allowed us to complete and improve this knowledge. The glycerophospholipid fraction of the olive fruit samples analyzed was composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (lyso-PE), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidic acid (lyso-PA), and phosphatidylglycerol (PG). Differences in the relative abundance of the glycerophospholipid classes determined were observed as a function of the botanical and geographical origin of the olive fruits analyzed. Interestingly, the olive stone and pulp analyzed also showed different glycerophospholipid compositions. For olive oil, five glycerophospholipids (lyso-PA, PC, PE, lyso-PE, and PG) were detected. Finally, identification of the main molecular species in the different glycerophospholipid classes for the olive fruit samples analyzed was accomplished by tandem mass spectrometric experiments and information from the literature.

Molecular characterization of phospholipids by high-performance liquid chromatography combined with an evaporative light scattering detector, high-performance liquid chromatography combined with mass spectrometry, and gas chromatography combined with a flame ionization detector in different oat varieties.

Oat (Avena sativa L.) is an important crop produced in various regions of Europe and North America. Oat lipids are a heterogeneous mixture of acyl lipids and unsaponifiable components. The neutral lipids are mainly triacylglycerols and account for 50-60% of total oat lipids. Oat oil is also rich in polar lipids, that is, phospholipids and glycolipids. Characterization of oat polar lipids has largely been performed by thin-layer chromatography (TLC), but the composition of phospholipid classes has been poorly studied. The aim of our work was the determination of different phospholipids in Romanian oat samples. For that purpose, one commercial sample (Comun) and four pure varieties (Jeremy, Lovrin 1, Lovrin 27-T, and Mures) were used. High-performance liquid chromatography combined with an evaporative light scattering detector results allowed us to establish that phosphatidylethanolamine was the most representative phospholipid in all of the oat samples. In addition, high-performance liquid chromatography combined with electrospray ionization mass spectrometry analysis showed that C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:0, and C20:1 were the fatty acids bound to the glycerol backbone. Using first-preparative TLC and later gas chromatography, it was demonstrated that linoleic acid (C18:2) was the main fatty acid of the phospholipid fraction in all of the samples.

Separation of proteins from olive oil by CE: an approximation to the differentiation of monovarietal olive oils.

The separation of proteins from olive oils by CE has been achieved for the first time in this work. Based on the protein profiles obtained using UV detection, a novel approach has also been proposed enabling the differentiation of monovarietal olive oils. An extraction procedure based on the precipitation of proteins in cold acetone and their isolation and solubilization in a hydro‐organic medium prior to their in‐capillary preconcentration in CE was a critical step in method development since proteins are minor components of olive oils. Then, a CE separation using a basic medium in a dynamically coated capillary originated electrophoretic profiles with multiple peaks from which seven of them were identified as proteins. When different monovarietal olive oils were injected, three different regions (zones I, II, and III) were identified. Zone III, called as differential zone, enabled to distinguish easily between extra virgin olive oils made from the Arbequina variety and the Picual and Hojiblanca varieties. This is the first time that protein profiles have been employed for the differentiation of botanical varieties of olive oils showing an enormous potential as traceability markers for these highly appreciated oils.

Analysis of antibiotics by CE and their use as chiral selectors: An update

The widespread use of antibiotics in medicine and as growth‐promoting agents has increased the demand for suitable analytical techniques for their analysis. Analytical methods based on CE or miniaturized CE systems have proved over the years their ability for the analysis of antibiotics. Since our last review (Electrophoresis 2014, 35, 28–49) several new CE methodologies have been reported for antibiotic analysis. This review presents an update of the literature published from June 2013 to June 2015 for the analysis of antibiotics by CE. UV continues being the most used detection system for antibiotics analysis by CE. Strategies to improve sensitivity as the use of sensitive detection systems and the application of preconcentration techniques appear to be the major developments. Furthermore, the use of portable and miniaturized devices for antibiotic analysis is presented in detail. Applications of the developed methodologies to the determination of residues of antibiotics in biological, food, and environmental samples are carefully described. Finally, new developments and applications of antibiotics as chiral selectors in CE are also included.

On‐line two‐dimensional capillary electrophoresis with mass spectrometric detection using a fully electric isolated mechanical valve

CE is becoming more and more important in many fields of bioanalytical chemistry. Besides optical detection, hyphenation to ESI‐MS detection is increasingly applied for sensitive identification purposes. Unfortunately, many CE techniques and methods established in research and industry are not compatible to ESI‐MS since essential components of the background electrolyte interfere in ES ionization. In order to identify unknown peaks in established CE methods, here, a heart‐cut 2D‐CE separation system is introduced using a fully isolated mechanical valve with an internal loop of only 20 nL. In this system, the sample is separated using potentially any non‐ESI compatible method in the first separation dimension. Subsequently, the portion of interest is cut by the internal sample loop of the valve and reintroduced to the second dimension where the interfering compounds are removed, followed by ESI‐MS detection. When comparing the separation efficiency of the system with the valve to a system using a continuous capillary only a slight increase in peak width is observed. Ultraviolet/visible detection is integrated in the first dimension for switching time determination, enabling reproducible cutting of peaks of interest. The feasibility of the system is successfully demonstrated by a 2D analysis of a BSA tryptic digest sample using a nonvolatile (phosphate based) background electrolyte in the first dimension.

Peanut Allergens: An Overview

Peanut is recognized as a potent food allergen producing one of the most frequent food allergies. This fact has originated the publication of an elevated number of scientific reports dealing with peanut allergens and, especially, the prevalence of peanut allergy. For this reason, the information available on peanut allergens is increasing and the debate about peanut allergy is always renewed. This article reviews the information currently available on peanut allergens and on the techniques used for their chemical characterization. Moreover, a general overview on the current biotechnological approaches used to reduce or eliminate peanut allergens is also provided.

Separation of olive proteins by capillary gel electrophoresis.

Olive proteins are not well known and there are still a lot of unknown information requiring further studies focused on the determination and characterization of these proteins. Despite the widely use of gel electrophoresis, this is the first time that capillary gel electrophoresis (CGE) is applied to separate proteins extracted from olive fruits. Seven common peaks were identified in the twenty olive varieties studied in this work. According to their migration times, these seven peaks could correspond to molecules with molecular masses of 11.0±0.4, 13.9±0.5, 16.3±0.8, 22.1±0.6, 30±1, 48±1, and 53±2 kDa. All of the determined molecular masses could be attributed to proteins and four of them have been previously observed by SDS-PAGE. The electrophoretic profiles were also evaluated for their capability to differentiate olive varieties according to their presumed geographical origin. Results demonstrated that this method could successfully classify the studied olive varieties by its combination with multivariate chemometrics tools.