De Novo Access to BODIPY C-Glycosides as Linker-Free Nonsymmetrical BODIPY-Carbohydrate Conjugates.

Recent years have witnessed an increasing interest in the synthesis and study of BODIPY-glycoconjugates. Most of the described synthetic methods toward these derivatives involve postfunctional modifications of the BODIPY core followed by the covalent attachment of the fluorophore and the carbohydrate through a "connector". Conversely, few de novo synthetic approaches to linker-free carbohydrate-BODIPY hybrids have been described. We have developed a reliable modular, de novo, synthetic strategy to linker-free BODIPY-sugar derivatives using the condensation of pyrrole C-glycosides with a pyrrole-carbaldehyde derivative mediated by POCl3. This methodology allows labeling of carbohydrate biomolecules with fluorescent-enough BODIPYs within the biological window, stable in aqueous media, and able to display singlet oxygen generation.

What Are We Eating? Surveying the Presence of Toxic Molecules in the Food Supply Chain Using Chromatographic Approaches.

Consumers in developed and Western European countries are becoming more aware of the impact of food on their health, and they demand clear, transparent, and reliable information from the food industry about the products they consume. They recognise that food safety risks are often due to the unexpected presence of contaminants throughout the food supply chain. Among these, mycotoxins produced by food-infecting fungi, endogenous toxins from certain plants and organisms, pesticides, and other drugs used excessively during farming and food production, which lead to their contamination and accumulation in foodstuffs, are the main causes of concern. In this context, the goals of this review are to provide a comprehensive overview of the presence of toxic molecules reported in foodstuffs since 2020 through the Rapid Alert System for Food and Feed (RASFF) portal and use chromatography to address this challenge. Overall, natural toxins, environmental pollutants, and food-processing contaminants are the most frequently reported toxic molecules, and liquid chromatography and gas chromatography are the most reliable approaches for their control. However, faster, simpler, and more powerful analytical procedures are necessary to cope with the growing pressures on the food chain supply.

Chiral Monolithic Silica-Based HPLC Columns for Enantiomeric Separation and Determination: Functionalization of Chiral Selector and Recognition of Selector-Selectand Interaction.

This review draws attention to the use of chiral monolithic silica HPLC columns for the enantiomeric separation and determination of chiral compounds. Properties and advantages of monolithic silica HPLC columns are also highlighted in comparison to conventional particle-packed, fused-core, and sub-2-µm HPLC columns. Nano-LC capillary monolithic silica columns as well as polymeric-based and hybrid-based monolithic columns are also demonstrated to show good enantioresolution abilities. Methods for introducing the chiral selector into the monolithic silica column in the form of mobile phase additive, by encapsulation and surface coating, or by covalent functionalization are described. The application of molecular modeling methods to elucidate the selector-selectand interaction is discussed. An application for enantiomeric impurity determination is also considered.

New Advanced Materials and Sorbent-Based Microextraction Techniques as Strategies in Sample Preparation to Improve the Determination of Natural Toxins in Food Samples.

Natural toxins are chemical substances that are not toxic to the organisms that produce them, but which can be a potential risk to human health when ingested through food. Thus, it is of high interest to develop advanced analytical methodologies to control the occurrence of these compounds in food products. However, the analysis of food samples is a challenging task because of the high complexity of these matrices, which hinders the extraction and detection of the analytes. Therefore, sample preparation is a crucial step in food analysis to achieve adequate isolation and/or preconcentration of analytes and provide suitable clean-up of matrix interferences prior to instrumental analysis. Current trends in sample preparation involve moving towards "greener" approaches by scaling down analytical operations, miniaturizing the instruments and integrating new advanced materials as sorbents. The combination of these new materials with sorbent-based microextraction technologies enables the development of high-throughput sample preparation methods, which improve conventional extraction and clean-up procedures. This review gives an overview of the most relevant analytical strategies employed for sorbent-based microextraction of natural toxins of exogenous origin from food, as well as the improvements achieved in food sample preparation by the integration of new advanced materials as sorbents in these microextraction techniques, giving some relevant examples from the last ten years. Challenges and expected future trends are also discussed.

Bi-functionalized mesostructured silicas as reversed-phase/strong anion-exchange sorbents. Application to extraction of polyphenols prior to their quantitation by UHPLC with ion-trap mass spectrometry detection.

Hybrid mesostructured silicas with wormhole-like pore structure were synthesized and bi-functionalized with n-octyl (C8) and quaternary ammonium (NR4+) groups to obtain new sorbent materials for dispersive solid-phase extraction (dSPE) of polyphenols. Due to their nature of being both a reversed-phase and a strong anion-exchanger, the materials display mixed-mode retention mechanism. During the synthesis, the functionalization conditions were varied to obtain materials with different functionalization degree. The resulting materials (denoted as HMS-RPC8-SAX-1, HMS-RPC8-SAX-2 and HMS-RPC8-SAX-3) show high surface area, wormhole-like framework and controlled pore size. They were evaluated for multicomponent extraction of 22 polyphenols, including phenolic acids, flavonoids and stilbenes, from spiked juice samples. The sample extracts were analyzed by ultra-high performance liquid chromatography coupled to ion-trap tandem mass spectrometry. The adsorption capability, the amount of sorbent, the eluent and the elution volume were optimized. Best performance was achieved by using HMS-RPC8-SAX-2, which is the material with the highest fraction of NR4+ groups. This material has a large extraction capability and provides high recovery values of the target analytes (70-101%) as a result of its hydrophobic and anion-exchange interactions. The detection limits for polyphenols in juice range from 1 to 560 ng mL-1. Graphical abstract Schematic presentation of dispersive solid-phase extraction of polyphenols from juice samples using a novel sorbent based on the bi-functionalization of mesostructured silica with n-octyl and quaternary ammonium groups, followed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) analysis. Due to the sorbent nature of being both a reversed-phase and strong anion-exchanger, the material displays mixed mode retention mechanism that improves its extraction capability.

Miniaturized Analytical Strategy Based on µ-SPEed for Monitoring the Occurrence of Pyrrolizidine and Tropane Alkaloids in Honey.

Currently, the analysis of trace-level contaminants in food must be addressed following green analytical chemistry principles and with a commitment to the sustainable development goals. Accordingly, a sustainable and ecofriendly microextraction procedure based on µ-SPEed followed by ultrahigh liquid chromatography coupled to ion-trap tandem mass spectrometry analysis was developed to determine the occurrence of pyrrolizidine and tropane alkaloids in honey samples. The µ-SPEed procedure took approximately 3 min per sample, using only 100 µL of organic solvent and 300 µL of diluted sample. The method was properly validated (overall recoveries 72-100% and precision RSD values ≤15%), and its greenness was scored at 0.61 out of 1. The method was applied to different honey samples, showing overall contamination levels from 32 to 177 µg/kg of these alkaloids. Atropine was found in all the samples, whereas retrorsine N-oxide, lasiocarpine, echimidine, and echimidine N-oxide were the main pyrrolizidine alkaloids in the samples analyzed.

Quick and Green Microextraction of Pyrrolizidine Alkaloids from Infusions of Mallow, Calendula, and Hibiscus Flowers Using Ultrahigh-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry Analysis.

A sustainable microextraction of pyrrolizidine alkaloids (PAs) from edible flower infusions using the innovative µSPEed technique is proposed. Different sorbents and extraction conditions were tested, achieving the highest extraction efficiency with an octadecylsilane sorbent (4 mg). The extraction procedure just took 1 min per sample, and only 300 µL of methanol and 300 µL of the sample were used per extraction. Ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry was used for analysis. The method was properly validated, providing suitable linearity, selectivity, sensitivity (quantification limits 0.3-1 µg/L), overall recoveries (79-97%), and precision (≤17% relative standard deviation). Its application to the analysis of different infusions of mallow, calendula, and hibiscus flowers revealed similar total PA values (23-41 µg/L) and contamination profile among the mallow and hibiscus samples, with predominance of senecionine-type and heliotrine-type PAs, respectively. Conversely, calendula samples showed more variations (23-113 µg/L), highlighting the occurrence of intermedine N-oxide and europine N-oxide on them.

The Potential of Microextraction Techniques for the Analysis of Bioactive Compounds in Food.

For a long time, the importance of sample preparation and extraction in the analytical performance of the most diverse methodologies have been neglected. Cumbersome techniques, involving high sample and solvent volumes have been gradually miniaturized from solid-phase and liquid-liquid extractions formats and microextractions approaches are becoming the standard in different fields of research. In this context, this review is devoted to the analysis of bioactive compounds in foods using different microextraction approaches reported in the literature since 2015. But microextraction also represents an opportunity to mitigate the environmental impact of organic solvents usage, as well as lab equipment. For this reason, in the recent literature, phenolics and alkaloids extraction from fruits, medicinal herbs, juices, and coffee using different miniaturized formats of solid-phase extraction and liquid-liquid microextraction are the most popular applications. However, more ambitious analytical limits are continuously being reported and emergent sorbents based on carbon nanotubes and magnetic nanoparticles will certainly contribute to this trend. Additionally, ionic liquids and deep eutectic solvents constitute already the most recent forefront of innovation, substituting organic solvents and further improving the current microextraction approaches.

Miniaturized and modified QuEChERS method with mesostructured silica as clean-up sorbent for pyrrolizidine alkaloids determination in aromatic herbs.

This work proposes the miniaturization and modification of the QuEChERS strategy using different large pore mesostructured silicas, non-modified and modified with amino groups (NH2), as dispersive clean-up sorbents for multi-component extraction of 21 pyrrolizidine alkaloids from different aromatic herbs, combined with ultra-high performance liquid chromatography coupled to tandem mass spectrometry analysis. The procedure was miniaturized by reducing the amounts of sample (0.2 g), solvents (2 mL), clean-up sorbents (25 mg sorbent + 150 mg MgSO4) and partitioning salts (0.65 g) employed. Best results were achieved using mesostructured silicas (LP-MS-NH2) than conventional PSA. The method was validated (overall recoveries 73-105%) and applied to the analysis of 17 samples. All the samples were contaminated with PAs (average concentration 262 µg/Kg). Thyme and basil samples were the most contaminated, whereas rosemary was the least. Lasiocarpine, senecivernine N-oxide and europine N-oxide were the main PAs that contributed to their contamination.

A Miniaturized QuEChERS Method Combined with Ultrahigh Liquid Chromatography Coupled to Tandem Mass Spectrometry for the Analysis of Pyrrolizidine Alkaloids in Oregano Samples.

Recent and unexpected food alerts about relatively high amounts of pyrrolizidine alkaloids in oregano samples have stressed the need to develop analytical strategies to ensure food safety in this type of foodstuff. Accordingly, this work presents the development of a miniaturized strategy based on the QuEChERS (quick, easy, cheap, effective, rugged and safe) method combined with ultrahigh liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the determination of 21 pyrrolizidine alkaloids suggested by the European Food Safety Authority to be monitored in food. The analytical method was properly validated, with overall average recoveries from 77 to 96% and relative standard deviations <13% (n = 9). The method proved to be a sustainable analytical strategy which meets green analytical chemistry principles as it showed good performance by using small amounts of sample (0.2 g), organic solvents (1000 µL), clean-up sorbents (175 mg) and partitioning salts (0.65 g). Its feasibility was verified through the analysis of 23 oregano samples. Of the samples analyzed, 100% were contaminated, with an average concentration of 1254 µg/kg. Lasiocarpine, lasiocarpine N-oxide, europine, europine N-oxide, senecivernine, senecionine, echimidine N-oxide, lycopsamine N-oxide and intermedine N-oxide were the alkaloids which significantly contributed to the contamination of the samples.

Modeling-based optimization of the simultaneous enantiomeric separation of multicomponent mixtures of phenoxy acid herbicides using dual cyclodextrin systems by Capillary Electrophoresis.

In this work, the Dubsky's model proposed for Capillary Electrophoresis (CE) enantioseparation systems with a mixture of chiral selectors was applied to the rapid optimization of the simultaneous enantiomeric separation of a multicomponent mixture of six phenoxy acid herbicides using a dual system of two cyclodextrins (CDs), (2-hydroxypropyl)-ß-CD (HP-ß-CD) and heptakis(2,3,6-tri-O-methyl)-ß-CD (TM-ß-CD). Simply by carrying out a small number of individual experiments separately with each CD, the Dubsky's model enabled to foresee the results that could be obtained for any possible combination of concentrations and relative proportion of both CDs in the mixture. Results obtained in this work demonstrated that the model was successful by improving the previous results experimentally obtained by the trial and error method for the simultaneous enantiomeric separation of the six phenoxy acid herbicides studied in this work. In fact, the separation was improved in terms of enantiomeric resolutions obtained (from 1.2 to 4.2 for concentrations of CDs of 4 mM HP-ß-CD and 16 mM TM-ß-CD) and by considerably reducing the time to optimize the separation conditions enabling to find, in a faster and efficient way, the most adequate proportion of both CDs and the concentration of each CD in the mixture to obtain baseline separation of the twelve enantiomers. Additionally, the apparent complexation constants between enantiomers and each CD were calculated. This is the first time that the above-mentioned model was applied to a multicomponent mixture of chiral compounds.

Enantiomeric Determination of Drugs in Pharmaceutical Formulations and Biological Samples by Electrokinetic Chromatography.

Chirality is a relevant issue in the pharmaceutical field due to the different biological activity that enantiomers of a chiral drug can show. In fact, the desired biological or pharmaceutical activity might be present in only one of the enantiomers, while the other enantiomer(s) may have different biological activity, be inactive or even toxic. This has motivated in recent years the development of drugs marketed as pure enantiomers to avoid exposing the organism to the action of enantiomers that may not be active or even harmful to health. Thus, it is of high interest to develop enantioselective analytical methodologies to control the presence of enantiomeric impurities and to understand the enantioselective metabolism of chiral drugs. This review gives an overview about the analytical strategies developed by electrokinetic chromatography (EKC) from 2010 to June 2019 for the enantiomeric determination of drugs in both pharmaceutical formulations and biological samples. The types of chiral selectors used, the migration order of enantiomers, their resolution, the detection technique employed and the sensitivity achieved are revised and compared. Also, applications to assess the enantiomeric purity control of pharmaceutical formulations and to determine chiral drugs in biological samples to study their metabolism are included. Advantages and limitations of the chiral methods developed by EKC are also discussed.

Enantiomeric analysis of pyrethroids and organophosphorus insecticides.

The use of pesticides has increased sharply in the last decades, not only in agriculture, but also in industry, public health, and other areas. Pyrethroids and organophosphorus insecticides are among the most employed pesticides. These chemicals usually contain asymmetric chiral atoms; thus, they are characterized by stereoisomerism. Although most of these chiral pesticides are produced, used, and released as racemic mixtures, the different enantiomers of these compounds can present different insecticidal activity, different toxicity against vertebrates and invertebrates, and also different persistence in the environment. In fact, in some cases, only one enantiomer is active, while the other can be less active or even toxic to non-target organisms. Therefore, the development of enantioselective analytical methodologies enabling their determination presents a high interest. Different separation techniques, including high performance liquid chromatography, gas chromatography, supercritical fluid chromatography, and capillary electrophoresis, have been employed to achieve the chiral analysis of pyrethroids and organophosphorus insecticides. This review presents the characteristics of the stereoselective analytical methodologies developed with this aim from 2010 to April 2019 and their applications to the analysis of real samples as well as for toxicity and biodegradation studies.

Enantiomeric separation of ivabradine by cyclodextrin-electrokinetic chromatography. Effect of amino acid chiral ionic liquids.

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 and < 7% for intermediate precision). The limits of detection (LODs) were 0.22 and 0.28 µg mL-1 for S- and R-ivabradine, respectively, and the method enabled to detect a 0.1% of the enantiomeric impurity, allowing to accomplish the requirements of the International Conference on Harmonisation (ICH) guidelines. Finally, the method was applied to the analysis of a pharmaceutical formulation of ivabradine. The content of R-ivabradine was below the LOD and the amount of S-ivabradine was in agreement to the labeled content.

Comparison of high-throughput microextraction techniques, MEPS and µ-SPEed, for the determination of polyphenols in baby food by ultrahigh pressure liquid chromatography.

In this study, two different high-throughput microextraction techniques, microextraction by packed sorbents (MEPS) and micro solid phase extraction (µ-SPEed®), were evaluated and compared, regarding the performance criteria, for the isolation of polyphenols from baby foods prior to their determination by ultrahigh pressure liquid chromatography (UHPLC). To achieve the best performance, influential parameters affecting extraction efficiency (including type of sorbent, number of extraction cycles, pH, elution solvent and elution volume) were systematically studied and optimized. To enable an effective comparison, selectivity, linear dynamic range, method detection (LODs) and quantification limits (LOQs), accuracy, precision and extraction yields, were determined and discussed for both techniques. Both methods provided the analytical selectivity required for the analysis of polyphenols in baby foods. However, µ-SPEed® sample treatment in combination with UHPLC-PDA has demonstrated to be more sensitive, selective and efficient than MEPS. Appropriate linearity in solvent and matrix-based calibrations, very low LODs and LOQs, ranging between 1.37 and 13.57 µg kg-1 and 4.57 - 45.23 µg kg-1, respectively, suitable recoveries (from 67 to 97%) and precision (RSD values < 5%) were achieved for the selected analytes by µ-SPEed®/UHPLC-PDA. Finally, the validated methodologies were applied to different commercial baby foods. Gallic acid, chlorogenic acid, epicatechin, ferulic acid, rutin, naringenin and myricetin are the most dominant polyphenols present in the studied baby food samples. The proposed methodology revealed a promising approach to evaluate the nutritional quality of this kind of products.

Dispersive Solid-Phase Extraction of Polyphenols from Juice and Smoothie Samples Using Hybrid Mesostructured Silica Followed by Ultra-high-Performance Liquid Chromatography-Ion-Trap Tandem Mass Spectrometry.

A wormhole-like mesostructured silica was synthesized and modified with octadecylsilane (C18) groups. The resulting hybrid material (HMS-C18) was characterized and evaluated as sorbent for simultaneous extraction of 20 polyphenols from mixed fruit-vegetable juices and smoothies by dispersive solid-phase extraction (dSPE). The samples were first subjected to solvent extraction followed by dSPE procedure. The extraction step was optimized and combined with a reversed-phase ultra-high-performance liquid chromatography method coupled to ion-trap tandem mass spectrometry (UHPLC-IT-MS/MS), which was also optimized. HMS-C18 showed high potential to extract and purify the target analytes, being more effective than commercial C18 amorphous silica. The proposed method was validated for both samples, obtaining average recoveries from 57% to 99% with relative standard deviations lower than 9%. Its applicability in the analysis of commercial mixed fruit-vegetable juices and smoothies revealed mainly contents of rutin, 4-hydroxybenzoic acid, chlorogenic acid, epicatechin, caffeic acid, and naringin in the samples analyzed.

Evaluation of bi-functionalized mesoporous silicas as reversed phase/cation-exchange mixed-mode sorbents for multi-residue solid phase extraction of veterinary drug residues in meat samples.

A SBA-15 type mesoporous silica was synthesized and bi-functionalized with octadecylsilane (C18) or octylsilane (C8), and sulfonic acid (SO3-) groups in order to obtain materials with reversed-phase/strong cation-exchange mixed-mode retention mechanism. The resulting hybrid materials (SBA-15-C18-SO3- and SBA-15-C8-SO3-) were comprehensively characterized. They showed high surface area, high pore volume and controlled porous size. Elemental analysis of the materials revealed differences in the amount of C18 and C8. SBA-15-C18-SO3- contained 0.19mmol/g of C18, while SBA-15-C8-SO3- presented 0.54mmol/g of C8. The SO3- groups anchored to the silica surface of the pore walls were 0.20 and 0.09mmol/g, respectively. The bi-functionalized materials were evaluated as SPE sorbents for the multi-residue extraction of 26 veterinary drug residues in meat samples using ultra-high-performance liquid chromatography coupled to mass spectrometry detector (UHPLC-MS/MS). Different sorbent amounts (100 and 200mg) and organic solvents were tested to optimize the extraction procedure. Both silicas showed big extraction potential and were successful in the extraction of the target analytes. The mixed-mode retention mechanism was confirmed by comparing both silicas with SBA-15 mesoporous silica mono-functionalized with C18 and C8. Best results were achieved with 200mg of SBA-15-C18-SO3- obtaining recoveries higher than 70% for the majority of analytes.

Application of a hybrid ordered mesoporous silica as sorbent for solid-phase multi-residue extraction of veterinary drugs in meat by ultra-high-performance liquid chromatography coupled to ion-trap tandem mass spectrometry.

A quick, sensitive and selective analytical reversed-phase multi-residue method using ultra-high performance liquid chromatography coupled to an ion-trap mass spectrometry detector (UHPLC-IT-MS/MS) operating in both positive and negative ion mode was developed for the simultaneous determination of 23 veterinary drug residues (ß-blockers, ß-agonists and Non-Steroidal Anti-inflammatory Drugs (NSAIDs)) in meat samples. The sample treatment involved a liquid-solid extraction followed by a solid-phase extraction (SPE) procedure. SBA-15 type mesoporous silica was synthetized and modified with octadecylsilane, and the resulting hybrid material (denoted as SBA-15-C18) was applied and evaluated as SPE sorbent in the purification of samples. The materials were comprehensively characterized, and they showed a high surface area, high pore volume and a homogeneous distribution of the pores. Chromatographic conditions and extraction procedure were optimized, and the method was validated according to the Commission Decision 2002/657/EC. The method detection limits (MDLs) and the method quantification limits (MQLs) were determined for all the analytes in meat samples and found to range between 0.01-18.75µg/kg and 0.02-62.50µg/kg, respectively. Recoveries for 15 of the target analytes ranged from 71 to 98%. In addition, for comparative purpose SBA-15-C18 was evaluated towards commercial C18 amorphous silica. Results revealed that SBA-15-C18 was clearly more successful in the multi-residue extraction of the 23 mentioned analytes with higher recovery values. The method was successfully tested to analyze prepacked preparations of mince bovine meat. Traces of propranolol, ketoprofen and diclofenac were detected in some samples.