Capillary electromigration methods for food analysis and Foodomics: Advances and applications in the period March 2021 to March 2023.

This work presents a revision of the main applications of capillary electromigration (CE) methods in food analysis and Foodomics. Papers that were published during the period March 2021 to March 2023 are included. The work shows the multiple CE methods that have been developed and applied to analyze different types of molecules in foods and beverages. Namely, CE methods have been applied to analyze amino acids, biogenic amines, heterocyclic amines, peptides, proteins, phenols, polyphenols, pigments, lipids, carbohydrates, vitamins, DNAs, contaminants, toxins, pesticides, additives, residues, small organic and inorganic compounds, and other minor compounds. In addition, new CE procedures to perform chiral separation and for evaluating the effects of food processing as well as the last developments of microchip CE and new applications in Foodomics will be also discussed. The new procedures of CE to investigate food quality and safety, nutritional value, storage, and bioactivity are also included in the present review work.

Rapid indirect separation of glutamine enantiomers by micellar electrokinetic chromatography. Analysis of dietary supplements.

Glutamine is the most abundant free proteinogenic α-amino acid. It is naturally produced in the organism and acts as a precursor for the synthesis of different biologically important molecules (such as proteins or nucleotides). However, under stressful conditions, the organism is unable to produce it in enough amounts to function properly. Thus, glutamine (Gln)-based supplements have become increasingly popular over the last decade. Since legal regulations establish that amino acid-based dietary supplements must contain only the L-enantiomer and not the racemate, adequate chiral methodologies are required to achieve their quality control. In this work, an analytical methodology based on the use of micellar electrokinetic chromatography is proposed for the rapid enantiomeric determination of DL-Gln in dietary supplements. Using (+)-1-(9-fluorenyl)-ethyl chloroformate as a derivatizing agent and ammonium perfluorooctanoate as separation medium, the Gln diastereoisomers formed under optimal conditions were separated in 8 min with a resolution of 2.8. The analytical characteristics of the method were evaluated in terms of linearity, precision, accuracy, and limits of detection/quantitation, and they were found appropriate for the analysis of L-Gln-based dietary supplements.

Enantiomeric analysis of drugs in water samples by using liquid-liquid microextraction and nano-liquid chromatography.

The nano-LC technique is increasingly used for both fast studies on enantiomeric analysis and test beds of novel stationary phases due to the small volumes involved and the short conditioning and analysis times. In this study, the enantioseparation of 10 drugs from different families was carried out by nano-LC, utilizing silica with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) column. The effect on chiral separation caused by the addition of different salts to the mobile phase was evaluated. To simultaneously separate as many enantiomers as possible, the effect of buffer concentration in the mobile phase was studied, and, to increase the sensitivity, a liquid-liquid microextraction based on the use of isoamyl acetate as sustainable extraction solvent was applied to pre-concentrate four chiral drugs from tap and environmental waters, achieving satisfactory recoveries (>70%).

Exploring the Metabolic Differences between Cisplatin- and UV Light-Induced Apoptotic Bodies in HK-2 Cells by an Untargeted Metabolomics Approach.

Among the extracellular vesicles, apoptotic bodies (ABs) are only formed during the apoptosis and perform a relevant role in the pathogenesis of different diseases. Recently, it has been demonstrated that ABs from human renal proximal tubular HK-2 cells, either induced by cisplatin or by UV light, can lead to further apoptotic death in naïve HK-2 cells. Thus, the aim of this work was to carry out a non-targeted metabolomic approach to study if the apoptotic stimulus (cisplatin or UV light) affects in a different way the metabolites involved in the propagation of apoptosis. Both ABs and their extracellular fluid were analyzed using a reverse-phase liquid chromatography-mass spectrometry setup. Principal components analysis showed a tight clustering of each experimental group and partial least square discriminant analysis was used to assess the metabolic differences existing between these groups. Considering the variable importance in the projection values, molecular features were selected and some of them could be identified either unequivocally or tentatively. The resulting pathways indicated that there are significant, stimulus-specific differences in metabolites abundancies that may propagate apoptosis to healthy proximal tubular cells; thus, we hypothesize that the share in apoptosis of these metabolites might vary depending on the apoptotic stimulus.

Exploratory Metabolomic Analysis Based on Reversed-Phase Liquid Chromatography-Mass Spectrometry to Study an In Vitro Model of Hypoxia-Induced Metabolic Alterations in HK-2 Cells.

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.

Untargeted HILIC-MS-Based Metabolomics Approach to Evaluate Coffee Roasting Process: Contributing to an Integrated Metabolomics Multiplatform.

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.

A Non-Targeted Capillary Electrophoresis-Mass Spectrometry Strategy to Study Metabolic Differences in an In vitro Model of High-Glucose Induced Changes in Human Proximal Tubular HK-2 Cells.

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.

Chiral Discrimination of DL-Amino Acids by Trapped Ion Mobility Spectrometry after Derivatization with (+)-1-(9-Fluorenyl)ethyl Chloroformate.

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 cm2/(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 cm2/(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.

Nuclear magnetic resonance to study the interactions acting in the enantiomeric separation of homocysteine by capillary electrophoresis with a dual system of γ-cyclodextrin and the chiral ionic liquid EtCholNTf2.

The enantiomeric separation of 9-fluorenylmethoxycarbonyl chloride (FMOC)-homocysteine (Hcy) by CE was investigated using γ-CD and the chiral ionic liquid (R)-(1-hydroxybutan-2-yl)(trimethyl)azanium-bis(trifluoromethanesulfon)imidate (also called (R)-N,N,N-trimethyl-2-aminobutanol-bis(trifluoromethane-sulfon)imidate) (EtCholNTf2 ) as chiral selectors. Using 2 mM γ-CD and 5 mM EtCholNTf2 in 50 mM borate buffer (pH 9), FMOC-Hcy enantiomers were separated with a resolution value of 3.8. A reversal in the enantiomer migration order in comparison with the single use of γ-CD in the separation buffer was obtained. Then, NMR experiments were carried out to elucidate the interactions taking place in the enantiomeric separation of FMOC-Hcy. NMR analyses highlighted the formation of an inclusion complex since the hydrophobic group of FMOC-Hcy was inserted into the γ-CD cavity. Moreover, interactions between EtCholNTf2 and γ-CD were also observed, suggesting that the chiral ionic liquid would also enter the cavity of the γ-CD.

A micellar electrokinetic chromatography approach using diastereomeric derivatization and a volatile surfactant for the enantioselective separation of selenomethionine.

A MEKC methodology with UV detection was developed for the enantioselective separation of selenomethionine (SeMet). The use of (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral derivatization reagent to form SeMet diastereomers enabled their subsequent separation using ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase. The effect of APFO concentration and pH, temperature, injection volume, and derivatization conditions (time and FLEC/SeMet ratio) were evaluated in order to select the best separation conditions. A chiral resolution of 4.4 for DL-SeMet was achieved in less than 6 min using 100 mM APFO at pH 8.5 as electrophoretic buffer. Satisfactory results were obtained in terms of linearity, precision (RSD from 3.4 to 5.1% for migration times and from 1.8 to 4.6% for corrected peak areas), accuracy, and LODs (3.1 × 10-6  M and 3.7 × 10-6  M for d and l enantiomers, respectively). The method was successfully applied to the determination of l-SeMet in food supplements.

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

Natural, synthetic or semisynthetic antibiotics are highly used to prevent or treat diseases in humans and animals, and to promote animal growth. This fact makes that antibiotics residues or their transformation products may be present in food or in the environment after human or animal excretion. For this reason, it is imperative to develop reliable and sensitive analytical methodologies for their analysis. The main aim of this work is to present and discuss the most recent applications of capillary electromigration methods for the analysis of antibiotics, including the developments and applications of their use as chiral selectors in CE. The literature published from June 2015 to June 2017 is included following the previous review by Domínguez-Vega et al. (Electrophoresis, 2016, 37, 189-211). Information about the use of different detection systems, off-line and on-line strategies to improve sensitivity, and microchip devices for the analysis of antibiotics is provided and properly discussed.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics.

This review work presents and discusses the main applications of capillary electromigration methods in food analysis and Foodomics. Papers that were published during the period February 2015-February 2017 are included following the previous review by Acunha et al. (Electrophoresis 2016, 37, 111-141). The paper shows the large variety of food related molecules that have been analyzed by CE including amino acids, biogenic amines, carbohydrates, chiral compounds, contaminants, DNAs, food additives, heterocyclic amines, lipids, peptides, pesticides, phenols, pigments, polyphenols, proteins, residues, toxins, vitamins, small organic and inorganic compounds, as well as other minor compounds. This work describes the last results on food quality and safety, nutritional value, storage, bioactivity, as well as uses of CE for monitoring food interactions and food processing including recent microchips developments and new applications of CE in Foodomics.

A non-targeted metabolomic approach based on reversed-phase liquid chromatography-mass spectrometry to evaluate coffee roasting process.

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.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics.

In this work, the analysis of foods and food components using capillary electromigration methods is reviewed. The present work presents and discusses the main CE applications performed in Food Science and Technology including the new field of Foodomics, reviewing recent results on food quality and safety, nutritional value, storage, bioactivity, as well as applications of CE for monitoring food interactions and food processing. The CE analysis of a large variety of food-related molecules with different chemical properties, including amino acids, peptides, proteins, phenolic compounds, carbohydrates, DNA fragments, vitamins, toxins, pesticides, additives, and other minor compounds is described. The use of microchips, CE-MS, and chiral-CE in food analysis is also discussed as well as other current and foreseen trends in this area of research. Following the previous review by Castro-Puyana et al. (Electrophoresis, 2012, 33, 147­167), the current review covers the papers that were published from February 2011 to February 2013.

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.

Recovering bioactive compounds from olive oil filter cake by advanced extraction techniques.

The potential of by-products generated during extra-virgin olive oil (EVOO) filtration as a natural source of phenolic compounds (with demonstrated bioactivity) has been evaluated using pressurized liquid extraction (PLE) and considering mixtures of two GRAS (generally recognized as safe) solvents (ethanol and water) at temperatures ranging from 40 to 175 °C. The extracts were characterized by high-performance liquid chromatography (HPLC) coupled to diode array detection (DAD) and electrospray time-of-flight mass spectrometry (HPLC-DAD-ESI-TOF/MS) to determine the phenolic-composition of the filter cake. The best isolation procedure to extract the phenolic fraction from the filter cake was accomplished using ethanol and water (50:50, v/v) at 120 °C. The main phenolic compounds identified in the samples were characterized as phenolic alcohols or derivatives (hydroxytyrosol and its oxidation product), secoiridoids (decarboxymethylated and hydroxylated forms of oleuropein and ligstroside aglycones), flavones (luteolin and apigenin) and elenolic acid derivatives. The PLE extraction process can be applied to produce enriched extracts with applications as bioactive food ingredients, as well as nutraceuticals.

Rapid enantiomeric separation of indacaterol by electrokinetic chromatography.

The first chiral methodology enabling the separation of indacaterol enantiomers was developed in this work by cyclodextrin-electrokinetic chromatography. Indacaterol (IND) is a chiral drug marketed as a pure enantiomer. Then, the separation and quantification of each enantiomer is of great importance for the quality control of pharmaceutical formulations. After selecting the most suitable chiral selector and background electrolyte, two Box-Behnken designs were achieved to optimize the electrophoretic conditions using two different approaches to shorten analysis times: i) decreasing the capillary length, or ii) performing a short-end injection. Indacaterol enantiomers were separated in less than 5 min with a resolution value of 3.6 under the optimal separation conditions: 0.7% (m/v) carboxymethyl-α-cyclodextrin in 50 mM sodium formate buffer (pH 4.0) and using a short-end injection. Then, the analytical characteristics of the method were evaluated and LODs of 0.05 mg/L for S-IND and 0.04 mg/L for R-IND were achieved. Also, the method allowed the detection of a 0.1% enantiomeric impurity (S-IND) in the R-IND-based pharmaceutical formulations. The developed method was applied to the analysis of two pharmaceutical formulations. Percentages of 97 ± 3% and 103 ± 6% of R-IND with respect to the labeled amounts were found.

Optimization of clean extraction methods to isolate carotenoids from the microalga Neochloris oleoabundans and subsequent chemical characterization using liquid chromatography tandem mass spectrometry.

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.

Untargeted HPLC-MS-based metabolomics approach to reveal cocoa powder adulterations.

Cocoa powder is a highly consumed product all over the world which could be substituted by cheaper raw materials resulting in food fraud. In this work, a non-targeted metabolomics approach based on the use of reversed-phase liquid chromatography coupled to high-resolution mass spectrometry was developed to carry out the characterization of cocoa powder samples adulterated, at two different levels, with carob flour, soy flour, and chicory. The sample preparation protocol and the chromatographic parameters were optimized to extract and detect the highest number of molecular features. Both non-supervised and supervised statistical methods were employed to analyze the most significant variables that gave rise to group discrimination. From the 21 and 37 significant variables analyzed in positive and negative ionization modes, respectively, a total of 20 were tentatively identified. Different families of compounds including flavonoids, fatty acids, terpenoids, lysophospholipids, and a galactolipid could be pointed out as cocoa adulteration markers.

Quantitative multiple fragment monitoring with enhanced in-source fragmentation/annotation mass spectrometry.

Analytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold standard in clinical chemistry. However, tandem mass spectrometers come at high capital expenditure and maintenance costs. We recently showed that it is possible to generate very similar results using a much simpler single mass spectrometry detector by performing enhanced in-source fragmentation/annotation (EISA) combined with correlated ion monitoring. Here we provide a step-by-step protocol for optimizing the analytical conditions for EISA, so anyone properly trained in liquid chromatography-mass spectrometry can follow and apply this technique for any given analyte. We exemplify the approach by using 2-hydroxyglutarate (2-HG) which is a clinically relevant metabolite whose D-enantiomer is considered an 'oncometabolite', characteristic of cancers associated with mutated isocitrate dehydrogenases 1 or 2 (IDH1/2). We include procedures for determining quantitative robustness, and show results of these relating to the analysis of DL-2-hydroxyglutarate in cells, as well as in serum samples from patients with acute myeloid leukemia that contain the IDH1/2 mutation. This EISA-mass spectrometry protocol is a broadly applicable and low-cost approach for the quantification of small molecules that has been developed to work well for both single-quadrupole and time-of-flight mass analyzers.