A liquid chromatography-mass spectrometry method for the enantioselective multiresidue determination of nine chiral agrochemicals in urine using an enrichment procedure based on graphitized carbon black.

Many agrochemicals are chiral molecules, and most of them are marketed as racemates or diastereomeric mixtures. Stereoisomers that are not the active enantiomer have little or no pesticidal activity and can exert serious toxic effects towards non-target organisms. Thus, investigating the possible exposure to different isomers of chiral pesticides is an urgent need. The present work was aimed at developing a new enantioselective high-performance liquid chromatography-mass spectrometry method for the simultaneous determination of nine chiral pesticides in urine. Two solid-phase extraction (SPE) procedures, based on different carbon-based sorbents (graphitized carbon black (GCB) and buckypaper (BP)), were developed and compared. By using GCB, all analytes were recovered with yields ranging from 60 to 97%, while BP allowed recoveries greater than 54% for all pesticides except those with acid characteristics. Baseline separation was achieved for the enantiomers of all target agrochemicals on a Lux Cellulose-2 column within 24 min under reversed-phase mode. The developed method was then validated according to the FDA guidelines for bioanalytical methods. Besides recovery, the other evaluated parameters were precision (7-15%), limits of detection (0.26-2.21 µg/L), lower limits of quantitation (0.43-3.68 µg/L), linear dynamic range, and sensitivity. Finally, the validated method was applied to verify the occurrence of the pesticide enantiomers in urine samples from occupationally exposed workers.

Miniaturized matrix solid-phase dispersion assisted by deep eutectic solvent for acrylamide determination in bread samples.

Acrylamide determination is important to state its quantity in baked food preventing any potential carcinogenic effects. Matrix solid-phase dispersion (MSPD) extraction is an extraction procedure based on a homogenization phase between a solid sample and a solid dispersing material to break sample increasing analyte extraction yield, often used for acrylamide determination. The addition of a green deep eutectic solvent (DES) during the MSPD homogenization phase improves the analyte extraction, giving the possibility to reduce the amount of organic solvent used. In this work, a miniaturized MSPD extraction assisted by a DES was developed to determine acrylamide in bread, using high-performance liquid chromatography coupled with mass spectrometry detection. The optimized procedure provides 1:1 (w/w) matrix-to-dispersing material ratio, 2 mL of methanol as extraction solvent, and 50 µL of choline chloride-glycerol DES added during the homogenization phase. Method validation ensured good results with minimum recoveries of 90%, high precision with a maximum intra-day error of 4%, and inter-day error of 6%. Limit of detection and limit of quantification resulted to be 16 µg/kg and 35 µg/kg, respectively. This miniaturized extraction procedure represents a good alternative to those reported in the literature, guaranteeing great performance and respecting green chemistry principles.

Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes.

AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. CONCLUSIONS/INTERPRETATION: Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.

Chemical Composition of Hazelnut Skin Food Waste and Protective Role against Advanced Glycation End-Products (AGEs) Damage in THP-1-Derived Macrophages.

Glycation and the accumulation of advanced glycation end-products (AGEs) are known to occur during aging, diabetes and neurodegenerative diseases. Increased glucose or methylglyoxal (MGO) levels in the blood of diabetic patients result in increased AGEs. A diet rich in bioactive food compounds, like polyphenols, has a protective effect. The aim of this work is to evaluate the capacity of hazelnut skin polyphenolic extract to protect THP-1-macrophages from damage induced by AGEs. The main polyphenolic subclass was identified and quantified by means of HPLC/MS and the Folin-Ciocalteu method. AGEs derived from incubation of bovine serum albumin (BSA) and MGO were characterized by fluorescence. Cell viability measurement was performed to evaluate the cytotoxic effect of the polyphenolic extract in macrophages. Reactive oxygen species' (ROS) production was assessed by the H2-DCF-DA assay, the inflammatory response by real-time PCR for gene expression, and the ELISA assay for protein quantification. We have shown that the polyphenolic extract protected cell viability from damage induced by AGEs. After treatment with AGEs, macrophages expressed high levels of pro-inflammatory cytokines and ROS, whereas in co-treatment with polyphenol extract there was a reduction in either case. Our study suggests that hazelnut skin polyphenol-rich extracts have positive effects and could be further investigated for nutraceutical applications.

Development and Box-Behnken design optimization of a green extraction method natural deep eutectic solvent-based for phenolic compounds from barley malt rootlets.

In recent years, attention has been turned finding new sources of phenolic compounds, antioxidant molecules, main by-products from the agri-food chain like barley malt rootlets (BMRs). Traditionally, phenolic compounds are extracted from food matrices using different procedures, for example, solid-liquid, liquid-liquid, or solid-phase extraction techniques employing organic solvents. With the advent of green chemistry, attention has been paid to the search for green, nontoxic, inexpensive, and nonflammable solvents and the natural deep eutectic solvents (NADESs) respect these characteristics. The aim of this project was to develop and optimize an environmentally friendly, inexpensive, and rapid extraction method for phenolic compounds from BMRs using natural DESs as extractive solvents. Several natural DESs were tested as extractive solvents and, among them, the best results in terms of total phenolic content were obtained using a choline chloride-malic acid (1:2 molar ratio)-based mixture. Box-Behnken experimental design guaranteed the extraction of 9.51 ± 0.83 gallic acid equivalent/g of BMRs, under the following optimal extraction conditions: 1:21 solid-to-liquid ratio, 80°C as extraction temperature, 43 min as the time of extraction, and 29% as a percentage of added water in the NADESs. Phenolic acids and flavonoids were detected in the BMRs extract through HPLC-PDA/MS analysis.

Potentiality of miniaturized techniques for the analysis of drugs of abuse.

Capillary electromigration (CE) and liquid chromatographic techniques (CLC/nano-LC) are miniaturized techniques offering distinct advantages over conventional ones in the field of separation science. Among these, high efficiency, high chromatographic resolution, and use of minute volumes of both mobile phase and sample volumes are the most important. CE and CLC/nano-LC have been applied to the analysis of many compounds including peptides, proteins, drugs, enantiomers, ions, etc. Over the years, the methods described here have also been used for the analysis of compounds of clinical, forensic, and toxicological interest. In this review article, the main features of the mentioned techniques are summarized. Their potentiality for the analysis of drugs of abuse are discussed. Some selected applications in this field in the period of 2015-present are also reported.

Chiral Nano-Liquid Chromatography and Dispersive Liquid-Liquid Microextraction Applied to the Analysis of Antifungal Drugs in Milk.

A novel chromatographic application in chiral separation by using the nano-LC technique is here reported. The chiral recognition of 12 antifungal drugs was obtained through a 75 µm I.D. fused-silica capillary, which was packed with a CSP-cellulose 3,5-dichlorophenylcarbamate (CDCPC), by means of a lab-made slurry packing procedure. The mobile phase composition and the experimental conditions were optimized in order to find the optimum chiral separation for some selected racemic mixtures of imidazole and triazole derivatives. Some important parameters, such as retention faction, enantioresolution, peak efficiency, and peak shape, were investigated as a function of the mobile phase (pH, water content, type and concentration of both the buffer and the organic modifier, and solvent dilution composition). Within one run lasting 25 min, at a flow rate of approximately 400 nL min-1, eight couples of enantiomers were baseline-resolved and four of them were separated in less than 25 min. The method was then applied to milk samples, which were pretreated using a classical dispersive liquid-liquid microextraction technique preceded by protein precipitation. Finally, the DLLME-nano-LC-UV method was validated in a matrix following the main FDA guidelines for bioanalytical methods.

Choline Chloride-Lactic Acid-Based NADES As an Extraction Medium in a Response Surface Methodology-Optimized Method for the Extraction of Phenolic Compounds from Hazelnut Skin.

Deep eutectic solvents (DESs) are promising green solvents for the extraction of compounds from food byproducts. Hazelnut (Corylus avellana L.) is one of the most commonly cultivated tree nuts worldwide. The skin represents one of the major byproducts of the hazelnut industry and accounts for 2.5% of the total hazelnut kernel weight. It is a rich source of phenolic compounds like flavan-3-ols, flavonols, dihydrochalcones, and phenolic acids. In this work, fifteen DESs based on choline chloride and betaine, with different compositions, were studied in order to test their phenolic compounds extraction efficiency through the determination of their total concentration via Folin-Ciocalteu assay. A qualitative analysis of extracted phenolic compounds was assessed by HPLC with UV and MS detection. Using the DES with the best extraction efficiency, a new ultrasound-assisted solid liquid extraction (UA-SLE) method was optimized though the response surface methodology (RSM), taking into account some extraction parameters. Efficient recovery of extracted phenolic compounds was achieved using a 35% water solution of choline chloride and lactic acid (molar ratio 1:2) as an extraction solvent, working at 80 °C and with a solid-to-solvent ratio of 1:25 gmL-1. The optimized conditions made it possible to recover 39% more phenolic compounds compared to a classic organic solvent.

Application of a Low Transition Temperature Mixture for the Dispersive Liquid-Liquid Microextraction of Illicit Drugs from Urine Samples.

The use of psychoactive substances is a serious problem in today's society and reliable methods of analysis are necessary to confirm their occurrence in biological matrices. In this work, a green sample preparation technique prior to HPLC-MS analysis was successfully applied to the extraction of 14 illicit drugs from urine samples. The isolation procedure was a dispersive liquid-liquid microextraction based on the use of a low transition temperature mixture (LTTM), composed of choline chloride and sesamol in a molar ratio 1:3 as the extracting solvent. This mixture was classified as LTTM after a thorough investigation carried out by FTIR and DSC, which recorded a glass transition temperature at -71 °C. The extraction procedure was optimized and validated according to the main Food and Drug Administration (FDA) guidelines for bioanalytical methods, obtaining good figures of merit for all parameters: the estimated lower limit of quantitation (LLOQ) values were between 0.01 µg L-1 (bk-MMBDB) and 0.37 µg L-1 (PMA); recoveries, evaluated at very low spike levels (in the ng-µg L-1 range), spanned from 55% (MBDB) to 100% (bk-MMBDB and MDPV); finally, both within-run and between-run precisions were lower than 20% (LLOQ) and 15% (10xLLOQ).

Application of deep eutectic solvents for the extraction of phenolic compounds from extra-virgin olive oil.

A HPLC-DAD/ESI-MS method has been developed and validated for the analysis of the most representative phenolic compounds in extra-virgin olive oil (EVOO) samples using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. We examined ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea. Five phenolic compounds, belonging to the classes of secoiridoids and phenolic alcohols, were selected for the evaluation of extraction efficiency. A betaine-based DES with glycerol (molar ratio 1:2) was found to be the most effective for extracting phenolic compounds as compared to a conventional solvent. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Thirty percent of water added to DES and sample to solvent ratio 1:1 (w/v) were selected as the best conditions. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range. Recovery values obtained spiking seed oil sample aliquots with standard compounds at 5 and 100 µg/g concentration were in the range between 75.2% and 98.7%.

Enantioseparation of tryptophan and its unnatural derivatives by nano-LC on CSP-teicoplanin silica based.

This work deals with the potentiality of nano liquid chromatography (Nano-LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory-assembled nano-LC-UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.

Enantiomers separation by capillary electrochromatography using polysaccharide-based stationary phases.

The separation of chiral compounds is an interesting and important topic of research because these compounds are involved in some biological processes, fundamentally in human health. Among the various application fields where enantiomers are remarkable, drug analysis has to be considered. Most of the drugs contain enantiomers and very often one of the two isomers could be pharmacologically more active or even dangerous. Therefore, the separation of these compounds is very important. Among the different analytical techniques usually employed, capillary electrochromatography has demonstrated great capability in enantiomers resolution. The great potential of this electromigration technique stands mainly in its high efficiency due to the use of an electrosmotic flow (flat flow profile) and on the high selectivity because of the use of a stationary phase. Chiral separation can be obtained utilizing several chiral stationary phases including a polysaccharide derivative. The aim of this review paper is to summarize the main features of capillary electrochromatography and polysaccharide derivatives of chiral stationary phase. It also report examples of practical applications utilizing this approach.

Analysis of Enantiomers in Products of Food Interest.

The separation of enantiomers has been started in the past and continues to be a topic of great interest in various fields of research, mainly because these compounds could be involved in biological processes such as, for example, those related to human health. Great attention has been devoted to studies for the analysis of enantiomers present in food products in order to assess authenticity and safety. The separation of these compounds can be carried out utilizing analytical techniques such as gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, and other methods. The separation is performed mainly employing chromatographic columns containing particles modified with chiral selectors (CS). Among the CS used, modified polysaccharides, glycopeptide antibiotics, and cyclodextrins are currently applied.

Optimization of pressurized liquid extraction by response surface methodology of Goji berry (Lycium barbarum L.) phenolic bioactive compounds.

Pressurized liquid extraction (PLE) has been used for the first time in this work to extract phenolic compounds from Goji berries according to a multilevel factorial design using response surface methodology. The global yield (% w/dw, weight/dry-weight), total phenolic content (TPC), total flavonoid (TF) and antioxidant activity (determined via ABTS assay, expressed as TEAC value) were used as response variables to study the effects of temperature (50-180°C) and green solvent composition (mixtures of ethanol/water). Phenolic compounds characterization was performed by high performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS). The optimum PLE conditions predicted by the model were as follows: 180°C and 86% ethanol in water with a good desirability value of 0.815. The predicted conditions were confirmed experimentally and once the experimental design was validated for commercial fruit samples, the PLE extraction of phenolic compounds from three different varieties of fruit samples (Selvatico mongolo, Bigol, and Polonia) was performed. Nine phenolic compounds were tentatively identified in these extracts, including phenolic acids and their derivatives, and flavonols. The optimized PLE conditions were compared to a conventional solid-liquid extraction, demonstrating that PLE is a useful alternative to extract phenolic compounds from Goji berry.

Effect of solvent on the extraction of phenolic compounds and antioxidant capacity of hazelnut kernel.

Hazelnut kernel phenolic compounds were recovered applying two different extraction approaches, namely ultrasound-assisted solid/liquid extraction (UA-SLE) and solid-phase extraction (SPE). Different solvents were tested evaluating total phenolic compounds and total flavonoids contents together to antioxidant activity. The optimum extraction conditions, in terms of the highest value of total phenolic compounds extracted together to other parameters like simplicity and cost were selected for method validation and individual phenolic compounds analysis. The UA-SLE protocol performed using 0.1 g of defatted sample and 15 mL of extraction solvent (1 mL methanol/1 mL water/8 mL methanol 0.1% formic acid/5 mL acetonitrile) was selected. The analysis of hazelnut kernel individual phenolic compounds was obtained by HPLC coupled with DAD and MS detections. Quantitative analysis was performed using a mixture of six phenolic compounds belonging to phenolic classes' representative of hazelnut. Then, the method was fully validated and the resulting RSD% values for retention time repeatability were below 1%. A good linearity was obtained giving R2 no lower than 0.997.The accuracy of the extraction method was also assessed. Finally, the method was applied to the analysis of phenolic compounds in three different hazelnut kernel varieties observing a similar qualitative profile with differences in the quantity of detected compounds.

Analysis of phenolic compounds in different parts of pomegranate (Punica granatum) fruit by HPLC-PDA-ESI/MS and evaluation of their antioxidant activity: application to different Italian varieties.

The analysis of pomegranate phenolic compounds belonging to different classes in different fruit parts was performed by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detection. Two different separation methods were optimized for the analysis of anthocyanins and hydrolyzable tannins along with phenolic acids and flavonoids. Two C18 columns, core-shell and fully porous particle stationary phases, were used. The parameters for separation of phenolic compounds were optimized considering chromatographic resolution and analysis time. Thirty-five phenolic compounds were found, and 28 of them were tentatively identified as belonging to four different phenolic compound classes; namely, anthocyanins, phenolic acids, hydrolyzable tannins, and flavonoids. Quantitative analysis was performed with a mixture of nine phenolic compounds belonging to phenolic compound classes representative of pomegranate. The method was then fully validated in terms of retention time precision, expressed as the relative standard deviation, limit of detection, limit of quantification, and linearity range. Phenolic compounds were analyzed directly in pomegranate juice, and after solvent extraction with a mixture of water and methanol with a small percentage of acid in peel and pulp samples. The accuracy of the extraction method was also assessed, and satisfactory values were obtained. Finally, the method was used to study identified analytes in pomegranate juice, peel, and pulp of six different Italian varieties and one international variety. Differences in phenolic compound profiles among the different pomegranate parts were observed. Pomegranate peel samples showed a high concentration of phenolic compounds, ellagitannins being the most abundant ones, with respect to pulp and juice samples for each variety. With the same samples, total phenols and antioxidant activity were evaluated through colorimetric assays, and the results were correlated among them.

Extraction, Analysis, and Antioxidant Activity Evaluation of Phenolic Compounds in Different Italian Extra-Virgin Olive Oils.

The analysis of phenolic compounds in extra virgin olive oils was carried out by high-performance liquid chromatography utilizing photodiode array and mass spectrometry detectors. The chromatographic profile of thirty samples from four Italian Regions highlighted the presence of secoiridoids, phenolic alcohols, flavonoids, and phenolic acid classes. A similar qualitative profile was observed with some differences in peak area and fifteen compounds were tentatively identified. Quantitative analysis was performed by UV detection considering eight standard phenolic compounds. The chromatographic method, after optimization, was validated studying some parameters, e.g., intra-day and inter-day retention time precision, limit of detection, limit of quantification, and linearity. Recovery of the method was performed achieving good results (10 and 50 g·g-1 with recovery of 72.9⁻92.1% (w/w) and 79.1⁻102.8% (w/w), respectively). In all samples secoiridoids were the main compounds ranging from 85 to more than 99% (w/w) of the total concentration of detected phenolic compounds while phenolic acids accounted for the lowest percentage (0.1⁻0.6%, w/w). Finally, total concentration of phenolic compounds and antioxidant activity were determined with different chemical assays. A good and significant correlation among total phenolic compound concentration and antioxidant activity was observed. A significant different phenolic compound concentration and antioxidant activity was determined between samples from Puglia and Sicily. This was studied performing statistical analysis by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test.

Lifelong imbalanced LA/ALA intake impairs emotional and cognitive behavior via changes in brain endocannabinoid system.

Imbalanced dietary n-3 and n-6 PUFA content has been associated with a number of neurological conditions. Endocannabinoids are n-6 PUFA derivatives, whose brain concentrations are sensitive to modifications of fatty acid composition of the diet and play a central role in the regulation of mood and cognition. As such, the endocannabinoid system appears to be an ideal candidate for mediating the effects of dietary fatty acids on mood and cognition. Lifelong administration of isocaloric α-linolenic acid (ALA)-deficient and -enriched diets induced short-term memory deficits, whereas only dietary ALA enrichment altered emotional reactivity in adult male rats compared with animals fed a standard diet that was balanced in ALA/linoleic acid (LA) ratio. In the prefrontal cortex, both diets reduced 2-AG levels and increased MAG lipase expression, whereas only the enriched diet reduced AEA levels, simultaneously increasing FAAH expression. In the hippocampus, an ALA-enriched diet decreased AEA content and NAPE-PLD expression, and reduced 2-AG content while increasing MAG lipase expression. These findings highlight the importance of a diet balanced in fatty acid content for normal brain functions and to support a link between dietary ALA, the brain endocannabinoid system, and behavior, which indicates that dietary ALA intake is a sufficient condition for altering the endocannabinoid system in brain regions modulating mood and cognition.

Antioxidant activity evaluation and HPLC-photodiode array/MS polyphenols analysis of pomegranate juice from selected italian cultivars: A comparative study.

Chemical composition of pomegranate juice can vary due to cultivar, area of cultivation, ripening, climate, and other variables. This study investigates the polyphenolic composition and antioxidant activity of juices obtained from six old Italian pomegranate cultivars. Fruit accessions physicochemical characteristics were determined. Total polyphenols content (TPC), anthocyanin content (TAC) and proanthocyanidin content (TPAC) were measured in the juice samples. Phenolic bioactive molecules were analyzed by HPLC-photodiode array (PDA)/ESI-MS in all the pomegranate juices. In total, seven nonanthocyanidinic and six anthocyanidinic compounds were identified. The six anthocyanins were found in all juices although at different amounts. These results were correlated with antioxidant activity measured by three different chemical assays: 2,2 diphenyl-1-picrylhydrazyl (DPPH(•) ) scavenging activity assay, Trolox equivalent antioxidant capacity (TEAC) method and ferric reducing-antioxidant power (FRAP) assay. Pomegranate juices obtained by six different varieties show variable polyphenolic content and antioxidant activity. The antioxidant capacity methods used have shown variable sensitivity, supporting the hypothesis that different methods for the assessment of antioxidant capacity of food compounds are indeed necessary, due to complexity of sample composition and assay chemical mechanism and sensitivity. Juices from Italian pomegranate show good levels of polyphenols content and antioxidant activity making them potential candidates for employment in the food industry.

Determination of key flavonoid aglycones by means of nano-LC for the analysis of dietary supplements and food matrices.

A method for the analysis of flavonoids (myricetin, quercetin, naringenin, hesperitin, and kaempferol), with interesting bioactivity, has been developed and validated utilizing nano-LC technique. In order to find optimal conditions, capillary columns (75 µm id × 10 cm) packed with different types of stationary phases, Kinetex® C18 core-shell (2.6 µm particle size), Hydride-based RP-C18 (sub-2 µm particle size), and LiChrospher® 100 RP-18 endcapped (5 µm particle size) were evaluated. The method was validated using Hydride-based RP-C18 stationary phase, with sub-2 µm particle size. A good chromatographic performance, expressed in terms of repeatability (RSD, in the range 1.63-4.68% for peak area), column-to-column reproducibility (RSD not higher than 8.01% for peak area), good linearity and sensitivity was obtained. In particular limit of detection values between 0.07 and 0.31 µg/mL were achieved with on column focusing technique. The method was applied to the determination of studied flavonoids in dietary supplements as well as in food matrices. The amount of quercetin found in the first analyzed dietary supplement, was in agreement to the labeled content. In the other samples, where the content of flavonoids was not labeled, most of the studied flavonoids were determined in amounts somewhere comparable to those reported in literature.