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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.ᅟ [ABSTRACT FROM AUTHOR]

Plum ( Prunus domestica L.) and peach ( Prunus persica (L.) Batsch) seed proteins are a source of bioactive peptides. These seeds, though, are usual residues produced during canning and beverage preparation that, in most cases, are irreversibly lost. The recovery and identification of these proteins might be of importance in human nutrition. This work employs the combinatorial peptide ligand libraries (CPLLs) technology as a tool to reduce the proteins dynamic concentration range. The most suitable extraction and CPLL capture conditions have been obtained and applied for the comprehensive identification of seed proteins. The analysis of recovered species by nLC-MS/MS has allowed the identification of 141 and 97 unique gene products from plum and peach seeds, respectively. It was possible to identify 16 proteins belonging to the Prunus genus. Moreover, a high number of histones and seed storage proteins were identified. Additionally, 21 and 14 bioactive peptides previously identified were found within protein sequences in plum and peach seeds, respectively. Significance Plums and peaches seeds are cheap sources of proteins that are irretrievably lost after canning and beverage production. Although this kind of residues has been used in animal feed or production of biofuel, they are usually incinerated or sent to landfills, wasting their huge potential. In order to exploit this, it is important to comprehensively study proteins present in plum and peach seeds. Nevertheless, since proteomics analysis is in most cases handicapped by the presence of high-abundance proteins masking the detection of the low-abundance ones, it is important to overcome this challenge. In this sense, combinatorial peptide ligand libraries (CPLLs) have been used in this work to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, the better extracting conditions have been optimized and up to 141 and 97 unique gene products from plum and peach seeds have been found, respectively. Moreover, 21 and 14 peptides previously identified as bioactive peptides were ascertained within protein sequences in plum and peach seeds, respectively. For that reason, this research takes the first step in the recovery of these valuable proteins and in the extraction of bioactive peptides, which could be successfully adopted in human nutrition. [ABSTRACT FROM AUTHOR]

Oxygen deficiency in cells, tissues, and organs can not only prevent the proper development of biological functions but it can also lead to several diseases and disorders. In this sense, the kidney deserves special attention since hypoxia can be considered an important factor in the pathophysiology of both acute kidney injury and chronic kidney disease. To provide better knowledge to unveil the molecular mechanisms involved, new studies are necessary. In this sense, this work aims to study, for the first time, an in vitro model of hypoxia-induced metabolic alterations in human proximal tubular HK-2 cells because renal proximal tubules are particularly susceptible to hypoxia. Different groups of cells, cultivated under control and hypoxia conditions at 0.5, 5, 24, and 48 h, were investigated using untargeted metabolomic approaches based on reversed-phase liquid chromatography–mass spectrometry. Both intracellular and extracellular fluids were studied to obtain a large metabolite coverage. On the other hand, multivariate and univariate analyses were carried out to find the differences among the cell groups and to select the most relevant variables. The molecular features identified as affected metabolites were mainly amino acids and Amadori compounds. Insights about their biological relevance are also provided. [ABSTRACT FROM AUTHOR]

An untargeted metabolomics strategy using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) was developed in this work enabling the study of the coffee roasting process. Green coffee beans and coffee beans submitted to three different roasting degrees (light, medium, and strong) were analyzed. Chromatographic separation was carried out using water containing 10 mM ammonium formate with 0.2 % formic acid (mobile phase A) and acetonitrile containing 10 mM ammonium formate with 0.2 % formic acid (mobile phase B). A total of 93 molecular features were considered from which 31 were chosen as the most statistically significant using variable in the projection values. 13 metabolites were tentatively identified as potential biomarkers of the coffee roasting process using this metabolomic platform. Results obtained in this work were complementary to those achieved using orthogonal techniques such as reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and capillary electrophoresis-mass spectrometry (CE-MS) since only one metabolite was found to be common between HILIC-MS and RPLC-MS platforms (caffeoylshikimic acid isomer) and other between HILIC-MS and CE-MS platforms (choline). On the basis of these results, an untargeted metabolomics multiplatform is proposed in this work based on the integration of the three orthogonal techniques as a powerful tool to expand the coverage of the roasted coffee metabolome. [ABSTRACT FROM AUTHOR]

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. • Peptide composition of coffee silverskin protein hydrolysates was investigated. • A LC-(QTOF)MS method was developed for peptide separation and identification. • Protein hydrolysates showed antioxidant and hypocholesterolemic activities. • The degree of coffee roasting had little influence on peptide composition. • Coffee silverskin is proposed as a source of peptides with potential bioactivities. [ABSTRACT FROM AUTHOR]

Apoptotic bodies play an important role in the cellular communication as a consequence of the great variety of biomolecules they harbor. There is evidence that 1st generation apoptotic bodies from HK-2 cells induced by cisplatin or UV light trigger apoptosis in naïve HK-2 cells whereas 2nd generation apoptotic bodies activate cell proliferation showing an opposite effect. Thus, the development of new analytical strategies to quantify the changes in the involved metabolites is imperative to shed light on the biological mechanisms which trigger apoptosis and cell proliferation. A LC-(Q-Orbitrap)MS method has been developed to quantify the metabolites unequivocally identified in the apoptotic body fluid from HK-2 cells in our previous works based on untargeted metabolomics. Thus, two different columns and gradients were tested and the HILIC column was selected taking into account the retention times and chromatographic separation. Also, different normal collision energies were tested for each metabolite and the parallel reaction monitoring was chosen to carry out the quantitative analysis. Once the method was optimized, it was evaluated in terms of linearity, limits of detection and quantification, matrix effects, accuracy, and precision, for each metabolite. Limits of detection ranged from 0.02 to 1.4 ng mL−1. A total of 9 relevant metabolites proposed as potential biomarkers to reveal metabolic differences among apoptotic bodies from HK-2 cells were quantified and some insights about the biological relevance were discussed. The first targeted metabolomics methodology enabling the quantification of relevant metabolites in apoptotic bodies from HK-2 cells was developed using LC-(Q-Orbitrap)MS. Pyridoxine, kynurenine, and creatine concentrations were determined in apoptotic bodies from HK-2 cells treated with cisplatin and UV light. Phenylacetylglycine, hippuric acid, butyrylcarnitine, acetylcarnitine, carnitine, and phenylalanine were determined in 1st and 2nd generation apoptotic bodies from HK-2 cells treated with cisplatin. Concentrations determined were useful to establish their biological role in the metabolism. [Display omitted] • LC-(Q-Orbitrap)MS method developed to analyze apoptotic bodies from HK-2 cells. • Metabolites proposed as biomarkers in these apoptotic bodies were quantified. • 1st generation apoptotic bodies induced by cisplatin and UV light were compared. • 1st and 2nd generation apoptotic bodies induced by cisplatin were compared. • Significant changes in metabolite concentrations in both comparative studies. [ABSTRACT FROM AUTHOR]

Abstract: Different types of extraction protocols are described for identifying proteins in seed and pulp of olive (Olea europea), by employing both conventional extraction methods and capture with ProteoMiner as well as with in house-made combinatorial peptide ligand libraries (HM-CPLLs) at pH 7.4 and at pH 2.2. Thanks to the use of CPLLs, able to dramatically amplify the signal of low-abundance species, a quite large number of compounds has been indeed identified: 61 in the seed (vs. only four reported in current literature) and 231 in the pulp (vs. 56 described so far), the deepest investigation up to the present of the olive proteome. In the seed, it highlights the presence of seed storage proteins, oleosins and histones. In the pulp, the allergenic thaumatin-like protein (Ole e 13) was confirmed, among the other 231, as the most abundant protein in the olive pulp. The present research has also been undertaken with the aim of identifying proteins in olive oil and ascertaining the relative contribution of seed and pulp proteins in their presence, if any, in oils. [Copyright &y& Elsevier]

• Synthesis and characterization of new L -carnitine-based ionic liquids. • Synthesis and characterization of salts based on L -carnitine. • Evaluation of the effect of the compounds synthesized as additives in chiral EKC. • γ-CD-based dual system for the enantioseparation of cysteine and homocysteine. • Effect of anionic counterions and substituents of L -carnitine on the separation. 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 (NTf 2 −), 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, high-performance liquid chromatography-mass spectrometry, and elemental analysis. Subsequently, they were used as additives to establish a γ-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 (μ eff) and the effective electrophoretic selectivity (α eff). Practically, all the dual systems evaluated exhibited substantially improved enantioseparations for the two amino acids compared with the single γ-CD system. [ABSTRACT FROM AUTHOR]

• Development of a LC–MS platform to study diabetic nephropathy in HK-2 cells. • First metabolomic work to study diabetic nephropathy in HK-2 cells. • Combination of RPLC and HILIC analysis on intra- and extracellular fluids. • Affected metabolic pathways include amino acidic and polyol, among others. 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 separation 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. [ABSTRACT FROM AUTHOR]

A liquid chromatography-(quadrupole-time of flight)-mass spectrometry methodology was developed to assess the authenticity of saffron through the analysis of a group of kaempferol derivatives recently proposed as novel authenticity markers as a result of a metabolomic study of saffron (kaempferol 3-O-glucoside, kaempferol 3-O-sophoroside, kaempferol 3,7-O-diglucoside, kaempferol 3,7,4′-O-triglucoside, kaempferol 3-O-sophoroside-7-O-glucoside). Geniposide was also studied as an adulteration marker of saffron with gardenia. The optimized chromatographic conditions enabling the simultaneous separation of glycosylated kaempferols and geniposide consisted of the use of a C18 column and an elution gradient with acetonitrile and water as mobile phases (both with formic acid at 0.1%). A strategy was proposed to evaluate the minimum quantifiable adulteration percentage which was established at a 0.2% regardless of the adulterant employed. The analysis of nineteen commercial samples showed the method to be specific and suitable for saffron quality control. [ABSTRACT FROM AUTHOR]

Alcohol is the most widely consumed legal drug, whereas cocaine is the illicit psychostimulant most commonly used in Europe. The combined use of alcohol and cocaine is frequent among drug-abuse consumers and leads to further exacerbation of health consequences compared to individual consumption. The pharmacokinetic and metabolic interactions leading to an increase in their combined toxicity still remains poorly understood. Here, the first metabolomics study of combined cocaine and ethanol chronic exposure effects is reported. A Liquid Chromatography strategy based on sample derivatization with 9-fluorenylmethyloxycarbonyl chloride and using a C18 column coupled to high resolution Mass Spectrometry (time of flight analyzer) was employed to analyze plasma from rats exposed intravenously to these drugs in a 52-min analysis. Using a combination of non-supervised and supervised multivariate analysis the metabolic differences between our experimental groups were explored and unraveled. A comparative analysis of the individual models and their variable importance in the projection values have shown that every experiment intervention includes a subset of specific metabolites. Eleven of these metabolites were annotated, where eight were unequivocally identified using standards and three were tentatively identified by matching the MS/MS spectra to libraries. The results demonstrated that the affected metabolic pathways were mainly those related to the metabolism of different amino acids. [ABSTRACT FROM AUTHOR]

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 μg 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. [ABSTRACT FROM AUTHOR]

This work is focused on the study of the metabolomic changes that occur in cocoa powder throughout the alkalization process by using an untargeted ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-QTOF-MS) metabolomics approach. With this aim, cocoa powder samples submitted to different alkalization degrees (light, medium, and strong) were analyzed. Metabolite extraction was performed using 75% MeOH in water since it provided the highest number of molecular features. After data processing, non-supervised and supervised methods were applied to carry out the statistical data analysis. Thus, the most significant metabolites that allow establishing differences among the cocoa powder samples submitted to the alkalization processes were pointed out. Thus, 43 and 30 metabolites in positive and negative ionization modes, respectively, demonstrated to be relevant. Among them, 9 compounds were unequivocally identified and 22 tentatively identified. Most of them were amino acids, alkaloids, organic acids or polyphenols, among others. • First UHPLC-MS metabolomics strategy to find markers of cocoa alkalization process. • 43 and 30 different markers were found relevant in ESI+ and ESI-, respectively. • 9 compounds were unequivocally identified and 22 tentatively identified. • Different amino acids, alkaloids, organic acids, or polyphenols were identified. [ABSTRACT FROM AUTHOR]

Progress in liquid chromatography and mass spectrometry technologies offers a great opportunity for the determination of bioactive peptides. Nevertheless, in many cases, the direct application of this technology does not enable the detection of the investigated peptides due to serious signal suppression. This is the case of lunasin, a cancer preventive, anti-inflammatory, and cholesterol-reducing peptide originally isolated from soybean and later found in some cereals. Most methods applied for the quantitation of this peptide were immunological and based on the detection of just a fragment of the lunasin sequence. At this regard, there is a peptide commercially available with a sequence similar to lunasin but differing in just one amino acid that has been wrongly used for the quantitation of lunasin. The use of high resolution mass spectrometry has enabled to be aware of this issue and of the need for new methods enabling the reliable identification and determination of lunasin. However, when different approaches were evaluated in this work for the reduction of the interferences originating signal suppression, such as matrix dilution, previous lunasin purification by reversed-phase or ion-exchange solid-phase extraction, and use of different chromatographic columns, no one resulted successful in the case of soybean. Just a one-dimensional separation of the soybean extract by isoelectrofocusing followed by a second dimension separation by reversed-phase liquid chromatography enabled a significant reduction of matrix interferences and the detection of lunasin in soybean products by high resolution mass spectrometry with a time of flight (TOF) analyzer. After method optimization, selectivity, linearity, accuracy, precision, and limits of detection and quantitation were evaluated, being possible to quantitate as low as 25 ng/mL (1.5 μg lunasin/g protein). Concentration of lunasin in the analyzed soybean flour and textured soybean ranged from 14.0 to 22.5 mg lunasin/g protein. [ABSTRACT FROM AUTHOR]

• Determination of six glycopeptide antibiotics in biological samples using glycosyl molecularly imprinted mesoporous microspheres. • A boronic acid functional monomer by click chemistry reaction to prepare glycosyl imprinted mesoporous microspheres. • The size exclusion effect of the mesoporous structure prevents interfering large molecules from entering the recognition cavities. Glycopeptide antibiotics are critical weapons against serious Gram-positive resistant bacteria, and therefore the development of analytical methods for their determination is essential. In this work, with the aim of extending the scope of molecularly imprinted mesoporous materials to the recognition of large molecules such as proteins and peptides, we selected the glycosyl moiety of glycopeptide antibiotics as a template and synthesised a boronic acid functional monomer by click chemistry reaction to prepare glycosyl imprinted mesoporous microspheres. On the basis of boronate affinity, the template and the functional monomer formed a self-assembly structure that was incorporated into the silica framework during polymerisation. The removal of the glycosyl moiety created cavities with boronic acid groups covalently anchored to the pore walls of the glycosyl imprinted mesoporous microspheres. The resultant microspheres showed regular spherical shape, narrow size distribution and porous structure and exhibited high adsorption capability and fast adsorption kinetics. The size exclusion effect of the mesoporous structure prevents large molecules from entering the cavities, while the glycosyl imprinted cavities provide selectivity for glycopeptide antibiotics. The glycosyl imprinted mesoporous microspheres were employed to separate six glycopeptide antibiotics in serum samples, which were then determined using ultra-high performance liquid chromatography tandem mass spectrometry. The proposed method exhibited satisfactory linearity in the range of 0.1 to 20.0 μg/L, demonstrating great potential for the determination of glycopeptide antibiotics in serum samples. [ABSTRACT FROM AUTHOR]