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In this study, new chiral ionic liquids based on the non-protein amino acid L-carnitine as cationic chiral counterpart and several anions (bis(trifluoromethane)sulfonimide (NTf2 -), L-lactate- or Cl-) as counterions were synthesized. Moreover, three different salts based on L-carnitine were also synthesized and the other three were commercially acquired and used for comparison. The synthesized ionic liquids and salts were characterized by nuclear magnetic resonance, fourier transform infrared spectroscopy, highperformance liquid chromatography-mass spectrometry, and elemental analysis. Subsequently, they were used as additives to establish a gamma-CD-based dual system for the enantiomeric separation of cysteine and homocysteine (previously derivatized with fluorenylmethoxycarbonyl chloride) by capillary electrokinetic chromatography. The effect of the nature of the anionic counterions and the presence of different substituents on the L-carnitine molecule on the chiral separation of both amino acids was investigated. The enantioseparations obtained with each dual system studied were compared in terms of the enantiomer effective mobilities (mu eff) and the effective electrophoretic selectivity (alpha eff). Practically, all the dual systems evaluated exhibited substantially improved enantioseparations for the two amino acids compared with the single gamma-CD system

https://www.sciencedirect.com/science/article/pii/S002196731930247X?via%3Dihub, Diabetes mellitus is a major health concern nowadays. It is estimated that 40 % of diabetics are affected by diabetic nephropathy, one of the complications derived from high glucose blood levels which can lead to chronic loss of kidney function. It is now clear that the renal proximal tubule plays a critical role in the progression of diabetic nephropathy but research focused on studying the molecular mechanisms involved is still needed. The aim of this work was to develop a liquid chromatography-mass spectrometry platform to carry out, for the first time, the untargeted metabolomic analysis of high glucose-induced changes in cultured human proximal tubular HK-2 cells. In order to find the metabolites which were affected by high glucose and to expand the metabolite coverage, intra- and extracellular fluid from HK-2 cells exposed to high glucose (25 mM), normal glucose (5.5 mM) or osmotic control (5.5 mM glucose + 19.5 mM mannitol) were analyzed by two complementary chromatographic modes: hydrophilic interaction and reversed-phase liquid chromatography. Non-supervised principal components analysis showed a good distribution among the three groups of samples. Statistically significant variables were chosen for further metabolite identification. Different metabolic pathways were affected mainly those derived from amino acidic, polyol, and nitrogenous bases metabolism.

There are hundreds of nonprotein amino acids whose importance in food and biological matrices is still unknown. Many of these compounds can be found in food as products formed during the processing, as metabolic intermediates or because they are added to increase functional and nutritional properties of food. Moreover, this kind of amino acids have also demonstrated to play relevant roles in the pharmaceutical and clinical fields since they may be used therapeutically in the treatment of some pathologies and their levels may be related with some diseases. These facts imply that the analysis of nonprotein amino acids can be useful to obtain relevant information in the food and biological fields. This article reviews the most recent advances in the development of analytical methodologies employing capillary electrophoresis for the achiral and chiral analysis of nonprotein amino acids in food and biological samples. With this aim, the most relevant information concerning the separation and detection of these compounds by capillary electrophoresis is discussed and detailed experimental conditions under which their determination was achieved in food and biological samples are given covering the period of time from 2015 to 2018.

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease. Although hyperglycaemia has been determined as the most important risk factor, hypoxia also plays a relevant role in the development of this disease. In this work, a comprehensive metabolomic study of the response of HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells, to hyperglycemic, hypoxic diabetic-like milieu has been performed. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O2) were compared to cells in control conditions (5.5 mM glucose/18.6% O2) at 48 h. The combination of advanced metabolomic platforms (GC-TOF MS, HILIC- and CSH-QExactive MS/MS), freely available metabolite annotation tools, novel databases and libraries, and stringent cut-off filters allowed the annotation of 733 metabolites intracellularly and 290 compounds in the extracellular medium. Advanced bioinformatics and statistical tools demonstrated that several pathways were significantly altered, including carbohydrate and pentose phosphate pathways, as well as arginine and proline metabolism. Other affected metabolites were found in purine and lipid metabolism, the protection against the osmotic stress and the prevention of the activation of the β-oxidation pathway. Overall, the effects of the combined exposure of HK-cells to high glucose and hypoxia are reasonably compatible with previous in vivo works.

During diabetes, renal proximal tubular cells (PTC) are exposed to a combination of high glucose and hypoxic conditions, which plays a relevant role in the development of diabetic kidney disease (DKD). In this work, a time-series proteomic study was performed to analyse the effect of a diabetic-like microenvironment induced changes on HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O-2) were compared to cells in control conditions for up to 48 h. Diabetic conditions increased the percentage of death cells after 24 and 48 h, but no differences in the protein/cell ratio were found. The relative protein quantification using dimethyl-labeling and UHPLC-MS/MS analysis allowed the identification of 317, 296 and 259 proteins at 5, 24 and 48 h, respectively. The combination of statistical and time expression profile analyses indicated an increased expression of proteins involved in glycolysis, and a decrease of cytoskeletal-related proteins. The exposure of HK-2 cells to high glucose and hypoxia reproduces some of the effects of diabetes on PTC and, with the limitations inherent toin vitrostudies, propose new mechanisms and targets to be considered in the management of DKD.

The separation of the four isomers of tetramethrin was performed for the first time using a cyclodextrin-micellar electrokinetic chromatography methodology. Using sodium deoxycholate and 2-hydroxypropyl-β-CD as chiral selectors, tetramethrin isomers were separated with resolution values of 1.7 and 1.1 for trans- and cis-isomers, respectively, in analysis times lower than 12.5 min. Once developed and optimized, the analytical method was applied to the analysis of an antiparasitic commercial formulation and to the evaluation of the stability and ecotoxicity of tetramethrin. Using measured concentrations, the stability was assessed at enantiomeric level and the ecotoxicological parameters on Daphnia magna were determined. Tetramethrin presents toxicity on aquatic microinvertebrates, with EC50 (t = 72 h) of 1.8 mg/L. The acute toxicity of tetrametrin was attributed to the trans-1 enantiomer. The first evidence of oxidative stress-mediated mode of action for tetramethrin on Daphnia magna is reported in the present work.

A capillary micellar electrokinetic chromatography (MEKC) method was developed enabling the stereoselective separation of the insecticide bioallethrin. The use of sodium deoxycholate bile salt and acetyl-beta-cyclodextrin (acetyl-beta-CD) made possible the separation of bioallethrin stereoisomers with a high enantioresolution (7.4) in a short analysis time (6.5 min). The analytical characteristics of the developed method were evaluated in terms of linearity, accuracy, precision, and limits of detection (LOD) and quantitation (LOQ) showing a good performance for the quantitation of bioallethrin stereoisomers with LODs of 0.2 and 0.3 mg/L. The developed method was applied to the stereoselective analysis of a commercial bioallethrin pediculicide formulation and to evaluate the toxicity of bioallethrin stereoisomers on the growth of the unicellular freshwater green alga Pseudokirchneriella subcapitata and on the germination of the higher plant Sorghum bicolor (non-target organisms). The analysis of the commercial pediculicide showed a good agreement between the contents determined for the two stereoisomers and those labelled in the commercial samples. Different toxic responses and biodegradation profiles were found for each stereoisomer in ecotoxicity assays. The mixture of SIR stereoisomers of bioallethrin resulted more toxic than S-bioallethrin for green algae, with EC50 values of 1.10 +/- 0.06 for the mixture and of 1.73 +/- 0.05 mg/L for the pure S-biallethrin (esbiol). Germination of plants seeds was also affected. (C) 2017 Elsevier B.V. All rights reserved.

Two new chiral ionic liquids based on the ester of the non-protein amino acid carnitine as cationic counterpart were synthesized, characterized and evaluated as chiral selectors for the enantiomeric separation of homocysteine and cysteine derivatized with 9-fluorenylmethoxycarbonyl chloride by electrokinetic chromatography. The use of both ionic liquids, l -carnitine methyl ester bis(trifluoromethane)sulfonimide and l -carnitine methyl ester l -(+)-lactate, as sole chiral selectors in the separation buffer, did not allow the enantiomeric separation of the studied amino acids. However, the combined use of l -carnitine methyl ester bis(trifluoromethane)sulfonimide ionic liquid with γ-CD forming a dual system led to the enantiomeric separation of both analytes and showed the existence of a strong synergistic effect. On the contrary, the dual system γ-CD plus l -carnitine methyl ester l -(+)-lactate did not improve the enantiomeric separations with respect to those obtained using the CD alone. The influence of different experimental variables such as buffer composition and pH, and ionic liquid concentration was investigated. Also, the nature of the anionic moiety was evaluated by comparing the results obtained with both ionic liquids when combined with γ-CD. The use of 2 mM γ-CD combined with 5 mM l -carnitine methyl ester bis(trifluoromethane)sulfonimide in a 50 mM phosphate buffer at pH 7.0 enabled to achieve the simultaneous enantiomeric separation of homocysteine and cysteine with high resolution values (>6.0) in analysis times close to 12 min.

The implications of chirality in different environments are already well known and reported extensively in the literature. Capillary Electrophoresis, a separation technique that only requires few nanoliters of sample, has demonstrated its potential for chiral analysis in the past years. The aim of this article is to provide an overview on the fundamentals and characteristics of Chiral Capillary Electrophoresis as well as the main advances and trends in this topic. Special attention is paid to the most recent technological and methodological developments achieved mainly in the most employed separation mode (Electroki-netic Chromatography). The most noteworthy and recent applications reported on the enantiomeric separation and determination of compounds in pharmaceutical, food, biomedical, environmental or forensic samples will also be critically overviewed. The characteristics of the developed methodologies will be detailed in Tables and future trends will also be discussed. (C) 2020 Elsevier B.V. All rights reserved.

Diabetic nephropathy is characterized by the chronic loss of kidney function due to high glucose renal levels. HK-2 proximal tubular cells are good candidates to study this disease. The aim of this work was to study an in vitro model of high glucose-induced metabolic alterations in HK-2 cells to contribute to the pathogenesis of this diabetic complication. An untargeted metabolomics strategy based on CE-MS was developed to find metabolites affected under high glucose conditions. Intracellular and extracellular fluids from HK-2 cells treated with 25 mM glucose (high glucose group), with 5.5 mM glucose (normal glucose group), and with 5.5 mM glucose and 19.5 mM mannitol (osmotic control group) were analyzed. The main changes induced by high glucose were found in the extracellular medium where increased levels of four amino acids were detected. Three of them (alanine, proline, and glutamic acid) were exported from HK-2 cells to the extracellular medium. Other affected metabolites include Amadori products and cysteine, which are more likely cause and consequence, respectively, of the oxidative stress induced by high glucose in HK-2 cells. The developed CE-MS platform provides valuable insight into high glucose-induced metabolic alterations in proximal tubular cells and allows identifying discriminative molecules of diabetic nephropathy.

A chiral methodology was developed for the first time to ensure the quality control of ivabradine, a novel anti-ischemic and heart rate lowering drug commercialized as a pure enantiomer. With this aim, electrokinetic chromatography (EKC) was employed and the enantiomeric separation of ivabradine was investigated using different anionic and neutral cyclodextrins (CDs) and amino acid-based chiral ionic liquids (CILs) as sole chiral selectors. Baseline separation was only achieved with sulfated CDs, and the best enantiomeric resolution was obtained with sulfated-γ-CD. Under the optimized conditions, ivabradine enantiomers were separated in 6 min with a resolution of 2.7. Nuclear magnetic resonance experiments showed a 1:1 stoichiometry for the enantiomer-CD complexes and apparent and averaged equilibrium constants were determined. The combined use of sulfated-γ-CD and different CILs as dual separation systems was investigated, resulting in a significant increase in the resolution. The use of 5 mM tetrabutylammonium-aspartic acid ([TBA][L-Asp]) in 50 mM formate buffer (pH 2.0) containing 4 mM sulfated-γ-CD were considered the best conditions in terms of resolution and migration times for ivabradine enantiomers. Nevertheless, as no inversion of the enantiomer migration order was observed when combining CILs and sulfated-γ-CD and a good enantiomeric resolution and efficiency were obtained using just sulfated-γ-CD as the sole chiral selector, the analytical characteristics of this method were evaluated, showing good recovery (98% and 103% for S- and R-ivabradine, respectively) and precision values (RSD 5% for instrumental repeatability,6% for method repeatability and7% for intermediate precision). The limits of detection (LODs) were 0.22 and 0.28 μg mL

The aim of this work was to develop a capillary electrophoresis-mass spectrometry (CE-ESI-QToF-MS) method to carry out the metabolic fingerprinting of green and roasted coffee samples (Arabica variety). To evaluate changes in the metabolic profiles of coffee occurring along the roasting process, green coffee beans were submitted to different roasting degrees. The effect of different parameters concerning the electrophoretic separation (background electrolyte, temperature, voltage, and injection time), the MS detection (temperature and flow of drying gas, sheath gas of jet stream temperature, and capillary, fragmentator, nozzle, skimmer, and octapole voltages) and the sheath liquid (composition and flow rate) was studied to achieve an adequate separation and to obtain the largest number of molecular features. The analyses were carried out in positive ESI mode allowing to detect highly polar cationic metabolites present in coffee beans. Non-supervised and supervised multivariate analyses were performed showing a good discrimination among the different coffee groups. Those features having a high variable importance in the projection values on supervised analyses were selected as significant metabolites for their identification. Thus, 13 compounds were proposed as potential markers of the coffee roasting process, being 7 of them tentatively identified and 2 of them unequivocally identified. Different families of compounds such as pyridines, pyrroles, betaines, or indoles could be pointed out as markers of the coffee roasting process. (C) 2019 Elsevier B.V. All rights reserved.

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).

Four amino acid chiral ionic liquids were evaluated in dual systems with hydroxypropyl-beta-cyclodextrin to investigate the enantioseparation by CE of a group of seven drugs as model compounds (duloxetine, verapamil, terbutaline, econazole, sulconazole, metoprolol, and nadolol). The use of two of these chiral ionic liquids (tetramethylammonium L-Lysine ([TMA][L-Lys]) and tetramethylammonium L-glutamic acid ([TMA][L-Glu]) as modifiers in CE is reported for the first time in this work whereas tetrabutylammonium L-lysine [TBA][L-Lys]) and tetrabutylammonium L-glutamic acid ([TBA][L-Glu]) were employed previously in CE although very scarcely. The effect of the nature and the concentration of each ionic liquid added to the separation buffer containing the neutral cyclodextrin on the enantiomeric resolution and the migration time obtained for each drug, was investigated. A synergistic effect was observed when combining each chiral ionic liquid with hydroxypropyl-beta-cyclodextrin in the case of the five compounds for which the cyclodextrin showed enantiomeric discrimination power when used as sole chiral selector (duloxetine, verapamil, terbutaline, econazole, sulconazole). Buffer concentration and pH, temperature and separation voltage were varied in order to optimize the enantiomeric separation of these five compounds using dual systems giving rise to resolutions ranging from 1.1 to 6.6. (C) 2019 Elsevier B.V. All rights reserved.

A novel analytical method based on hybrid trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has been developed to achieve fast enantiomeric separation of amino acids (AAs). Resolution of chiral AAs was achieved by forming diastereomers through derivatization with the chiral agent (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), avoiding the use of reference compounds. Electrospray ionization (ESI) in positive mode yielded sodiated FLEC-AAs ions of which the diastereomers could be separated by TIMS. The effect of other alkali metal ions (such as Li and K) on the enantioselectivity was studied, but chiral discrimination was only observed for Na. TIMS conditions, including voltage ramp, ramp time, and accumulation time were optimized for each AA, and collision cross sections (CCSs) were determined for all diastereomers. The migration order of the DL enantiomers was found to be dependent on the structure of the AA. The resulting TIMS resolution (K0/ΔK0) for the FLEC-AA diastereomers on average was 115, requiring a mobility (K0) difference of about 0.009 cm 2 /(V s) to achieve 50%-valley separation. From the 21 AAs studied, enantiomer separation was achieved for 17 AAs with mobility differences ranging from 0.009 for lysine up to 0.061 cm 2 /(V s) for asparagine. Moreover, the presented methodology provided mutual separation of various AAs, allowing chiral analysis of multiple AAs simultaneously which may be challenging with previous enantioselective IMS approaches. It appeared possible to fully resolve all studied DL-AAs using three distinct TIMS methods, resulting in a total MS run time of about 3 min (1 min per method) and a total analysis time (including derivatization) of less than 15 min. The method demonstrated capable to determine enantiomeric ratios down to 2.5% with detection limits for the D enantiomers in the nanomolar range. This new TIMS-based methodology opens up possibilities for easy and fast analysis of AA enantiomers.

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.

Macroalgae contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered peptides within their primary structures. In this work, an analytical methodology was developed for the separation and identification of peptides present in protein hydrolysates from three different edible macroalgae used for human consumption (Saccharina latissima (brown macroalga), Codium spp. (green macroalga), and Mastocarpus stellatus (red macroalga)). The extraction of aqueous and alkaline soluble proteins was carried out followed by their precipitation with HCl or acetone. The protein extracts obtained were submitted to enzymatic digestion with alcalase and subsequently analyzed by reversed-phase high-performance liquid chromatography-quadrupole-time-of flight mass spectrometry (RP-HPLC-QTOF/MS) and de novo sequencing tool to separate and identify different short chain peptides. Thirty-seven peptides were identified in the hydrolysed protein extracts from the three macroalgae, five of them being common in brown and red macroalgae. After checking against BIOPEP database, several sequenced peptides were found within longer peptides with potential antibacterial activity. Any of the identified peptides had previously been identified in macroalgae.

In this work, a non-targeted metabolomics approach based on the use of reversed-phase liquid chromatography coupled to a high-resolution mass spectrometer has been developed to provide the characterization of coffee beans roasted at three different levels (light, medium, and dark). In this way, it was possible to investigate how metabolites change during the roasting process in order to identify those than can be considered as relevant markers. Twenty-five percent methanol was selected as extracting solvent since it provided the highest number of molecular features. In addition, the effect of chromatographic and MS parameters was evaluated in order to obtain the most adequate separation and detection conditions. Data were analyzed using both non-supervised and supervised multivariate statistical methods to point out the most significant markers that allow group discrimination. A total of 24 and 33 compounds in positive and negative ionization modes, respectively, demonstrated to be relevant markers; most of them were from the hydroxycinnamic acids family. Graphical abstract ᅟ.

doi: 10.1080/10408347.2018.1439365, Capillary electrophoresis is one of the most widely employed analytical techniques to achieve enantiomeric separations. In spite of the fact that there are many chiral selectors commercially available to perform enantioseparations by CE, one of the most relevant topics in this field is the search for new selectors capable of providing high enantiomeric resolutions. Chiral ionic liquids have interesting characteristics conferring them a high potential in chiral separations although only some of them are commercially available.The aim of this article is to review all the works published on the use of chiral ionic liquids as chiral selectors in the development of enantioselective methodologies by CE, covering the period of time from 2006 (when the first research work on this topic was published) to 2017. The use of chiral ionic liquids as sole chiral selectors, as chiral selectors in dual systems or as chiral ligands will be considered. This review also provides detailed analytical information on the experimental conditions used to carry outenantioseparations in different fields as well as on the separation mechanism involved.

This chapter presents the fundamentals of capillary electrophoresis (CE) including separation principles, instrumentation, and different modes in which this technique can be used. The main applications of CE for ensuring the authenticity of foods and drinks are presented discussing the advantages and disadvantages of this methodology. Authenticity of foods and beverages based on DNA, proteins, and other compounds analyzed by CE is discussed., The present work was supported by the projects AGL2014-53609-P (Ministerio de Economía y Competitividad, Spain). G.A.-R. would like to acknowledge MINECO for a Juan de La Cierva-Formación postdoctoral grant (FJCI-2015-25504). M.C.P. also thanks MINECO for her “Ramón y Cajal” research contract (RYC-2013-12688).

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.

Robust, efficient, sensitive and cost-effective analytical methodologies need to be developed continuously to guarantee food safety, quality and traceability. The rapid development of metabolomics-based approaches using mass spectrometry (MS) has demonstrated their potential to face these challenges. This review presents a critical overview of the most recent applications of MS-based metabolomics approaches for food quality, safety and traceability assays, covering the most relevant works published from 2010 to 2013. We also provide information about the different steps needed to develop a MS-metabolomics approach (i.e. sample treatment, analytical platform, and data processing). © 2013 Elsevier Ltd., The authors want to thank Project AGL2011-29857-C03-01 (Ministerio de Educación y Ciencia) for economical support. M.C.P. thanks MICINN for her Juan de la Cierva research contract. M.H. would like to thank MICINN for a Ramón y Cajal research contract.

We demonstrate the possibilities of foodomics to emerge as a green discipline by applying the basic concepts of Green Chemistry to production of functional ingredients and development of analytical methodologies. We present possibilities to reduce the environmental impact of an analytical technique from sample preparation through separation and detection, together with different methodologies to quantify the greenness of a particular process. By no means this review tries to be exhaustive but to provide with some basic ideas about the different possibilities encountered in this field, thus making the reader be aware of how easy, or difficult, might be to make greener different aspects related to foodomics such as functional ingredients production and analytical processes. © 2013 Elsevier Ltd., Ther authors want to thank Projects AGL2011-29857-C03-01, CONSOLIDER INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Ministerio de Educación y Ciencia) and ALIBIRD, project S-505/AGR-0153 (Comunidad Autónoma de Madrid) for financial support. M.C.P. thanks MICINN for her Juan de la Cierva contract.

The improvement of sample-preparation and extraction techniques for determinations of natural bioactive compounds is very important. New concepts relate to not only enhancement of extraction efficiencies but also environmental impact. This evolution towards Green Analytical Chemistry is to new extraction and sample-preparation processes that should be faster, more reproducible and more environmentally friendly.Compressed fluid-based sample-preparation techniques (e.g., supercritical fluid extraction and pressurized liquid extraction) demonstrate good capabilities. In this review, we update knowledge on the techniques together with the main technical developments and the most notable recent applications for the extraction of bioactive compounds. © 2012 Elsevier Ltd., M.H. would like to thank MICIIN for a Ramón y Cajal research contract. M.C.P. thanks MINECO for her Juan de la Cierva research contract.The authors thank projects AGL2011-29857-C03-01, CONSOLIDER INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Ministerio de Educacion y Ciencia) and ALIBIRD, project S-505/AGR-0153, (Comunidad Autonoma de Madrid) for financial support.

In recent years, the green microalgae Neochloris oleoabundans have demonstrated to be an interesting natural source of carotenoids that could be used as potential food additive. In this work, different N. oleoabundans extracts obtained by pressurized liquid extraction (PLE) have been analyzed in depth to evaluate the influence of different culture conditions (effect of nitrogen, light intensity or carbon supplied) not only on the total carotenoid content but also on the carotenoid composition produced by these microalgae. Regardless of the cultivation conditions, lutein and carotenoid monoesters were the most abundant carotenoids representing more than 60% of the total content in all extracts. Afterwards, the effect of the different N. oleoabundans extracts and the dose-effect of the most potent algae extracts (namely, N9, PS and CO2 (−)) on the proliferation of human colon cancer cells lines (HT-29 and SW480) and a cell line established from a primary colon cancer cell culture (HGUE-C-1) were evaluated by an MTT assay whereas a stepwise multiple regression analysis was performed to get additional evidences on the relationship between carotenoid content and the antiproliferative activity. Results revealed that, as a general trend, those extracts with high total carotenoid content showed comparably antiproliferative activity being possible to establish a high correlation between the cell proliferation values and the carotenoid constituents. Monoesters showed the highest contribution to cell proliferation inhibition whereas lutein and violaxanthin showed negative correlation and diesters and zeaxanthin showed a positive significant contribution to cell proliferation., M.C.P. thanks the Ministry of Economy and Competitiveness (Spain) for her “Ramón y Cajal” research contract (RYC-2013-12688). This work was supported by the projects AGL2014-53609-P and AGL2015-67995-C3-1-R (Ministerio de Economía y Competitividad, Spain). A.V. thanks the Ministerio de Economía y Competitividad for his FPI pre-doctoral fellowship (BES-2012-057014).

Analysis of foods and food components using liquid chromatography (LC) is presented in this chapter, paying special attention to the analysis of carbohydrates, vitamins, amino acids, peptides, proteins, and lipids as well as other minor food compounds (e.g., polyphenols and carotenoids) and food additives used as preservatives, antioxidants, colorants, or sweeteners. The text includes fundamentals about their physiological roles together with sample preparation and LC conditions used for their analysis in foods.

A new and sophisticated saffron adulteration method with gardenia was recently discovered in the European saffron market. In this work, an analytical methodology using liquid chromatography (quadrupole-time of flight)-mass spectrometry has been developed for the detection of the adulteration of saffron samples with gardenia through the determination of geniposide as an adulteration marker. A fused-core C18 column was employed using an isocratic elution with water:acetonitrile (85:15 v/v) containing 0.1% formic acid. After optimization of the mass spectrometry conditions, the analytical characteristics related to the determination of geniposide in negative electrospray ionization mode were evaluated. Then, it was possible to detect up to 10 ng/mL geniposide after a dilution step of 50-fold of the saffron extract (LOD of 41.7 mu g of geniposide per gram of sample analysed (i.e up to 0.004%)). The developed LC-MS methodology was applied to the analysis of different authentic and suspicious saffron samples. (C) 2016 Elsevier Inc. All rights reserved.

The always more-demanding fields of food safety, quality and traceability are continuously fostering the development of robust, efficient, sensitive and cost-effective analytical methodologies. Mass spectrometry-based metabolomics is a key tool nowadays with great potential in many analytical fields and has been demonstrated to be capable of facing some important challenges related to these areas within the food science domain. The main aim of this review is to present a critical overview of the most recent applications of MS-based metabolomics approaches for food quality, safety and traceability assessment, covering the most relevant works published from 2014 to 2017. Information about the different steps needed to develop a MS-metabolomics approach, i.e. sample treatment, analytical platform, and data processing, is also provided and discussed., The authors would like to thank Projects AGL2014-53609-P (MINECO, Spain) and S2013/ABI-3028 (AVANSECAL-CM, from Comunidad Autónoma of Madrid, Spain, and the European funding from FEDER program) for financial support. M.C.P. thanks the Ministry of Economy and Competitiveness (Spain) for her “Ramón y Cajal” research contract (RYC-2013-12688). R.P.M. thanks the University of Alcalá for her pre-doctoral contract.

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.

Although subcritical water extraction (SWE) has already shown its great potential for the attainment of natural bioactive extracts, concerns still remain on possible unexpected reactions that can arise during the extraction process, usually taking place at high pressure and temperature. It is already well-known that different components might be formed during the SWE extraction protocol due e.g. to Maillard reaction, which can improve the bioactivity of the obtained extracts. On the other hand, the formation of other compounds derived from these reactions, such as 5-hydroxymethylfurfural (HMF), has raised some concerns, mainly related to its safety. In this work, the formation of HMF during subcritical water extraction, at different conditions, from olive leaves has been monitored by using liquid chromatography with mass spectrometry (LC-MS) and diode array detection (LC-DAD). The possible influence of this compound in the overall antioxidant and antiproliferative activities against colon cancer cells has been also studied. Results showed an increase of HMF formation when increasing the extraction temperature, being the maximum concentration achieved at 200 °C (3.17 μg. HMF/mg extract); nevertheless, the HMF contained in the olive leave extracts did not influence the antioxidant capacity or the antiproliferative activity of the natural extracts, thus demonstrating the safety of the SWE process. © 2012 Elsevier Ltd., This work has been financed by AGL2008-05108-C03-01 and AGL2011-29857-C03-01 (Ministerio de Educacion y Ciencia), CSD2007-00063 FUN-CFOOD (Programa CONSOLIDER-INGENIO 2010) and by ALIBIRD, S2009/AGR-1469 (Comunidad de Madrid) projects. M.H. would like to thank MICINN for a “Ramón y Cajal” research contract. M.C.P. thanks MICINN for her “Juan de la Cierva” contract.

The latest strategies and instrumental improvements for enhancing the detection sensitivity in chiral analysis by CE are reviewed in this work. Following the previous reviews by García-Ruiz et al. (Electrophoresis 2006, 27, 195-212) and Sánchez-Hernández et al. (Electrophoresis 2008, 29, 237-251; Electrophoresis 2010, 31, 28-43), this review includes those papers that were published during the period from June 2009 to May 2011. These works describe the use of offline and online sample treatment techniques, online sample preconcentration techniques based on electrophoretic principles, and alternative detection systems to UV-Vis to increase the detection sensitivity. The application of the above-mentioned strategies, either alone or combined, to improve the sensitivity in the enantiomeric analysis of a broad range of samples, such as pharmaceutical, biological, food and environmental samples, enables to decrease the limits of detection up to 10-12M. The use of microchips to achieve sensitive chiral separations is also discussed. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., The authors thank the Spanish Ministry of Science and Innovation (project CTQ2009-09022) and the Comunidad Autónoma of Madrid (Spain) and European funding from FEDER programme (project S2009/AGR-1464, ANALISYCII). They also thank the University of Alcalá and the Comunidad Autónoma of Madrid for project CCG10-UAH/AGR-5950. Laura Sánchez-Hernández thanks the Comunidad Autónoma of Madrid for her research contract. Maria Castro Puyana thanks the Spanish Ministry of Science and Innovation for her Juan de la Cierva contract (JCI-2009-05297).

Rosemary bioactivity is correlated with the presence of phenolic diterpenes (carnosic acid, carnosol) and phenolic acids (rosmarinic acid). In the present work, two processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF) were integrated to separate and enrich these families of compounds. The optimization of the SAF process was carried out using a response surface methodology (RSM) with three factors: (i) CO pressure, (ii) percentage of water in the PLE extract and (iii) PLE extract/SC-CO flow ratio. The selected responses were the relative amount of each compound (rosmarinic acid (RA) and carnosic acid + carnosol (CA + CS)), total phenolic content, antioxidant activity and antiproliferative activity against HT-29 colon cancer cells. The higher CA + CS content was obtained at 100 bar, 50% (v/v) of water in the feed solution and 0.025 feed/SC-CO mass flow ratio, achieving values of 478.1 mg/g extract and 17% of cell survival after 24 h of treatment., A.P.S.C. thanks to the Administrative Department of Science, Technology and Innovation COLCIENCIAS (Colombia) for her Ph.D. Scholarship. A.V. thanks the Ministerio de Economía y Competitividad for his FPI pre-doctoral fellowship. M.H. would like to thank MICINN for his “Ramón y Cajal” research contract. The authors would also like to thank Projects AGL2011-29857-C03-01, AGL2014-53609-P (MINECO, Spain) and ALIBIRD-CM, S2013/ABI-2728 (Comunidad de Madrid) for their financial support.

In addition to the 20 amino acids universally distributed as protein constituents in living organisms, there are other amino acids of non-protein origin that can be found in foods. The determination of these non-protein amino acids is interesting since they can be indicative of the quality and safety of foods. This work presents for the first time an updated and comprehensive review devoted to show the possibilities of capillary electrophoresis for the determination of non-protein amino acids in food samples. The results reported have been classified according to the chemical structure of the non-protein amino acid studied. Separation conditions as well as detection systems used have been detailed since most of these amino acidic compounds do not possess chromophore groups detectable by conventional UV-Vis detection, being in this case necessary a previous derivatization step. Finally, the application of microchip electrophoresis to the determination of non-protein amino acids in foodstuffs is also included in this review.

The enantioselective separation of a group of six weak base azole compounds was achieved in this work using EKC with three neutral beta-CDs as chiral selectors. The native beta-CD and two other beta-CD derivatives with different types and positions of the substituents on the CD rim ((2-hydroxy)propyl-beta-CD (HP-beta-CD) and heptakis-2,3,6-tri-O-methyl-beta-CD (TM-beta-CD)) were employed. Apparent binding constants for each pair compound-CD were determined in order to study analyte-CD interactions. The best enantiomeric resolutions for miconazole, econazole, and sulconazole were observed with HP-beta-CD whereas for the separation of the enantiomers of ketoconazole, terconazole, and bifonazole, TM-beta-CD was the best chiral selector. The enantioseparations obtained were discussed on the basis of the structure of the compounds taking into account that inclusion into the hydrophobic CD cavity occurred through the phenyl ring closer to the azole group. In addition, a change in the migration order for the enantiomers of two of the compounds studied (ketoconazole and terconazole) with the concentration of HP-beta-CD was observed for the first time.

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 Alcalá

In the current post-genomic era, Foodomics has been defined as a discipline that studies food and nutrition through the application of advanced omics approaches. Foodomics involves the use of genomics, transcriptomics, epigenetics, proteomics, peptidomics, and/or metabolomics to investigate food quality, safety, traceability and bioactivity. In this context, capillary electrophoresis-mass spectrometry (CE-MS) has been applied mainly in food proteomics, peptidomics and metabolomics. The aim of this review work is to present an overview of the most recent developments and applications of CE-MS as analytical platform for Foodomics, covering the relevant works published from 2008 to 2012. The review provides also information about the integration of several omics approaches in the new Foodomics field. © 2013 Elsevier B.V., This work was supported by AGL2011-29857-C03-01 (Ministerio de Economía y Competitividad, Spain), and CSD2007-00063 FUN-C-FOOD (Programa CONSOLIDER, Ministerio de Educacion y Ciencia, Spain) projects. C.I. thanks the Ministerio de Ciencia e Innovación for her FPI pre-doctoral fellowship. M.C.-P. thanks the Ministerio de Ciencia e Innovación for her Juan de la Cierva contract (JCI-2009-05297).

A novel experimental design was used to optimize the extraction of carotenoids from Neochloris oleoabundans using pressurized liquid extraction with food-grade solvents such as ethanol and limonene. Experimental factors, including the extraction temperature and the solvent composition, were optimized using a three-level factorial design. The response variables extraction yield and total amount of carotenoids were assessed. The statistical analysis of the results provided mathematical models to predict the behavior of the responses as a function of the factors involved in the process. The optimum conditions predicted by the model developed in this study were 112 °C as the extraction temperature and 100% ethanol as the extraction solvent. Chemical characterization of the extracts obtained was performed by means of high-performance liquid chromatography-tandem mass spectrometry. The results obtained demonstrated that, under certain growth conditions (photoautotrophically cultured in a medium supplemented with 0.3 g L(-1) KNO3), N. oleoabundans accumulated significant total amounts of the carotenoids (from 57.4 to 120.2 mg carotenoids per gram of extract depending on the extraction conditions), mainly lutein, cantaxanthin, zeaxanthin, and astaxanthin monoesters and diesters., This work was financed thanks to AGL2011-29857-C03-01 (Ministerio de Economía y Competitividad; MINECO) and ALIBIRD, S2009/AGR-1469 (Comunidad de Madrid) projects. M.C.P. thanks MINECO for her Juan de la Cierva research contract. M.H. thanks MINECO for his Ramón y Cajal research contract.

Instrumental methods based on gas chromatography coupled to mass spectrometry (GC-MS) have been developed and compared using two different MS ionisation modes, electron impact (EI) and electron capture negative ionisation (ECNI), for the fast, quantitative and simultaneous determination of polychlorinated biphenyls (PCBs) and their main metabolites (hydroxylated PCBs, OH-PCBs, and methyl sulfone PCBs, MeSO2-PCBs). Parameters affecting chromatographic separation and MS detection were evaluated in order to achieve the highest selectivity and sensitivity for both operation modes. The analytical characteristics of the developed methods were studied and compared in terms of linear range, limits of detection (LODs), limits of quantification (LOQs), and instrumental precision (repeatability and intermediate precision). Both ionisation methods showed similar precision, being relative standard deviations (RSD, %) lower than 9% and 14% for repeatability and intermediate precision, respectively. However, better LODs (from 0.01 to 0.14pg injected for the three families of congeners studied) were achieved using ECNI-MS as ionisation mode. The suitability of the developed method was demonstrated through their application to fish liver oil samples.

This chapter will examine the use of supercritical fluids as effective and selective solvents for the extraction of natural products. Their characteristics, advantages and disadvantages, and applications will be contextualized in maximizing their potential as an attractive alternative to reduce the use of organic solvents for the production of natural extracts.

A fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in food samples was developed for the first time using partial filling micellar electrokinectic chromatography-mass spectrometry (MEKC-MS). The use of MEKC was essential to achieve the separation of these neutral analytes, while the partial filling technique was necessary to avoid the contamination of the ion source with non-volatile micelles. MEKC separation and MS detection conditions were optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Filling 25% of the capillary with an MEKC solution containing 40 mM ammonium bicarbonate, 25 mM SDS, and 32.5% (v/v) acetonitrile, a baseline separation of the four azo-dyes was obtained in 10 min. Tandem MS was investigated in order to improve the sensitivity and selectivity of the analysis. Limits of detection (LOD) values 5, 8, 15, and 29 times better were obtained for Sudan III, I, II, and IV, respectively, using partial filling MEKC-MS/MS instead of partial filling MEKC-MS. Under optimized conditions, LOD from 0.05 to 0.2 μg/mL were obtained. The suitability of the developed method was demonstrated through the fast and sensitive determination of Sudan I, II, III, and IV in spiked chilli powder samples. This determination could not be achieved by MEKC-UV due to the existence of several interfering compounds from the matrix.

The potential of three capillary columns based on β-cyclodextrin (i.e., Chirasil-Dex, BGB-172, and BGB-176SE) has been studied for the simultaneous enantiomeric separation of polychlorinated biphenyls (PCBs) and methylsulfonyl metabolites of PCBs (MeSO(2)-PCBs) employing a heart-cut multidimensional gas chromatographic system (heart-cut MDGC). Among the columns studied, the BGB-176SE capillary column provided the best results, allowing the simultaneous enantioselective resolution of six MeSO(2)-PCBs and six chiral PCBs; the Chirasil-Dex column did not resolve any of the studied MeSO(2)-PCBs; and a poor resolution was obtained for three MeSO(2)-PCBs when the BGB-172 column was employed. The developed method was successfully applied to two fish oil and one cow liver samples commercially available, which showed different enantioselective pattern. PCBs 91 and 176 presented a clear enrichment of the second eluted atropisomer in codfish oil, whereas in fish oil sample, slight enrichment of the first eluted atropisomer of CB45 and the second eluted atropisomer of CB136 were observed.

The occurrence and incidence of different diseases such as cancer, cardiovascular diseases, obesity, and diabetes may be related to the consumption of high calorie calorie-containing diets combined with sedentary lifestyles. The concept of functional foods first appeared in Japan where it was considered to be a tool to promote health and well-being. In 1992, the Japanese government established a policy of “Foods of Specific Health Uses” (FOSHU). This concept was further developed in Europe within the “Functional Food Science in Europe” (FUFOSE) project supported by the European Commission (EC) and co-ordinated by the International Life Sciences Institute (ILSI). Several interesting points were observed at the end of this project (Bellisle, F., A.T. Diplock, G. Hornstra, B. Koletzko, M. Roberfroid, S. Salminen, et al. 1998. Functional food science in Europe. British Journal of Nutrition 80:1–193; Diplock, A.T., P.J. Agget, M. Ashwell, F. Bornet, E.B. Fern, M.B. Roberfroid. 1999. Scientific concepts of functional foods in Europe: Consensus document. British Journal of Nutrition 81:S1–S27), including a definition of a functional food as “a food which is demonstrated to positively affect one or more physiological functions, so that it is able to increase the well-being and/or to reduce the risk to suffer a disease” (Diplock, A.T., P.J. Agget, M. Ashwell, F. Bornet, E.B. Fern, M.B. Roberfroid. 1999. Scientific concepts of functional foods in Europe: Consensus document. British Journal of Nutrition 81:S1–S27). This definition implies that a functional food must maintain the shape of the food (thereby excluding pills and capsules) and that the functional food must impart a physiological effect following consumption that is above and beyond any observed nutritional effects.

The final publication is available at Springer via http://dx.doi.org/10.1002/elps.201100005, A capillary electrophoresis-tandem mass spectrometry methodology enabling the simultaneous determination of betaines (glycine betaine, trigonelline, proline betaine and total content of carnitines) in vegetable oils was developed. Betaines were derivatized with butanol previous to their baseline separation in 10 min using a 0.1 M formic acid buffer at pH 2.0. Ion trap conditions were optimized in order to maximize selectivity and sensitivity. Analytical characteristics of the proposed method were established by evaluating its selectivity, linearity, precision (RSDs ranged from 4.8% to 10.7% for corrected peak areas), accuracy by means of recovery studies (from 80% to 99%) and LODs and LOQs at 0.1 ppb level. The method was applied to the determination of the selected betaines in seed oils and extra virgin olive oils. MS2 experiments provided the fingerprint fragmentation for all betaines studied in seed oils. In extra virgin olive oils, carnitines were not detected being possible to propose them as a feasible novel marker for the detection of adulterations of olive oils. Application of the developed method to the analysis of different mixtures of extra virgin olive oil with seed oil (between 2-10 %) enabled the detection and quantitation of the total content of carnitines. The results obtained show the high potential of the developed method for the authentication and quality control of olive oils., Authors thank the Spanish Ministry of Science and Innovation (project CTQ2009- 09022) and the Comunidad Autónoma of Madrid (Spain) and european funding from FEDER programme (project S2009/AGR-1464, ANALISYC-II). They also thank the University of Alcalá and the Comunidad Autónoma of Madrid for project CCG10- UAH/AGR-5950. Laura Sánchez-Hernández thanks the Comunidad Autónoma of Madrid for her research contract.

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of l-MTX and its enantiomeric impurity d-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10−7 M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of l-MTX. Furthermore, l-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples. Figure A quenched phosphorescence method was developed and coupled to chiral EKC for the sensitive detection of the drug L-methotrexate and its impurity D-MTX; unspiked pharmaceutical formulation