Development, Characterization and In Vitro Gastrointestinal Release of PLGA Nanoparticles Loaded with Full-Spectrum Cannabis Extracts.

Cannabinoids, such as ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD), are effective bioactive compounds that improve the quality of life of patients with certain chronic conditions. The copolymer poly(lactic-co-glycolic acid) (PLGA) has been used to encapsulate such compounds separately, providing pharmaceutical grade edible products with unique features. In this work, a variety of PLGA based nanoformulations that maintain the natural cannabinoid profile found in the plant (known as full-spectrum) are proposed and evaluated. Three different cannabis sources were used, representing the three most relevant cannabis chemotypes. PLGA nanocapsules loaded with different amounts of cannabinoids were prepared by nanoemulsion, and were then functionalized with three of the most common coating polymers: pectin, alginate and chitosan. In order to evaluate the suitability of the proposed formulations, all the synthesized nanocapsules were characterized, and their cannabinoid content, size, zeta-potential, morphology and in vitro bioaccessibility was determined. Regardless of the employed cannabis source, its load and the functionalization, high cannabinoid content PLGA nanocapsules with suitable particle size and zeta-potential were obtained. Study of nanocapsules' morphology and in vitro release assays in gastro-intestinal media suggested that high cannabis source load may compromise the structure of nanocapsules and their release properties, and hence, the use of lower content of cannabis source is recommended.

Multiresidue analytical method for the determination of 41 multiclass organic pollutants in mussel and fish tissues and biofluids by liquid chromatography coupled to tandem mass spectrometry.

In this work, the full optimisation and validation procedure to analyse a wide set of emerging organic contaminants in biotissues (mussel and fish muscle, liver, gills and brain) and biofluids (fish plasma and bile) is described. The target families include artificial sweeteners, industrial products, hormones, pharmaceutical and personal care products, pesticides and phytoestrogens. Different clean-up strategies (hydrophilic-lipophilic-balanced (HLB) solid-phase extraction, Florisil solid-phase extraction and liquid-liquid extraction followed by HLB solid-phase extraction and microextraction based on polyethersulfone polymer) were evaluated for the clean-up of focused ultrasonic solid-liquid extraction (FUSLE) extracts before the analysis by liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The methods afforded satisfactory apparent recovery values (71-126%) using isotopically labelled analytes and matrix-matched calibration approach, regardless of the matrix. Method detection limits in the range of 4-48 ng/g and 0.3-111 ng/L were obtained for biotissues and biofluids, respectively. The developed method was applied to determine the uptake and tissue distribution in juvenile gilt-head bream (Sparus aurata) during 7 days in seawater, and unexpectedly, perfluoro-1-butanesulfonate tended to accumulate in liver and, to a lesser extent, in muscle and gills. Furthermore, real mussel samples collected in the Basque coast were also analysed and the presence of the highly consumed valsartan (7 ng/g) and telmisartan (6.8 ng/g) compounds in bivalves is reported for the first time here. Graphical abstract ᅟ.

Determination of fluoroquinolones in fish tissues, biological fluids, and environmental waters by liquid chromatography tandem mass spectrometry.

This work describes the optimization, validation, and application in real samples of accurate and precise analytical methods to determine ten fluoroquinolones (FQs) (norfloxacin, enoxacin, pefloxacin, ofloxacin, levofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin, and sparfloxacin) in different environmental matrices, such as water (estuarine, seawater, and wastewater treatment plant effluent), fish tissues (muscle and liver), and fish biofluids (plasma and bile). The analysis step performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was fully optimized to improve the separation and detection steps. The extraction of analytes from fish tissues was accomplished using focused ultrasound solid-liquid extraction using methanol/acetic acid (95:5 v/v) as extractant. The preconcentration and clean-up steps were optimized in terms of extraction efficiency and cleanliness and the best strategy for each matrix was selected: (i) Oasis HLB for seawater and muscle, (ii) liquid-liquid extraction combined with Oasis HLB for the lipid-rich liver, (iii) the combination of Evolute-WAX and Oasis HLB for estuarine water and wastewater treatment plant effluent, and (iv) molecular imprinted polymers for biofluids. The methods afforded satisfactory apparent recoveries (80-126%) and repeatability (RSD < 15%), except for sparfloxacin, which showed a lack of correction with the available isotopically labeled surrogates ([2H8]-ciprofloxacin and [2H5]-enrofloxacin). Ciprofloxacin, norfloxacin, and ofloxacin were detected in both water and fish liver samples from the Biscay Coast at concentrations up to 278 ng/L and 4 ng/g, respectively. To the best of our knowledge, this work is one of the few analyzing up to ten FQs and in so many fish tissues and biofluids. Graphical abstract Determination of fluoroquinolones in different environmental matrices, such as water (estuarine, seawater, and wastewater treatment plant effluent), fish tissues (muscle and liver), and fish biofluids (plasma and bile).

Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes.

The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.

Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatography-mass spectrometry.

High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been successfully applied to cannabis plant extracts in order to identify cannabinoid compounds after their quantitative isolation by means of supercritical fluid extraction (SFE). MS conditions were optimized by means of a central composite design (CCD) approach, and the analysis method was fully validated. Six major cannabinoids [tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabigerol (CBG), and cannabinol (CBN)] were quantified (RSD < 10%), and seven more cannabinoids were identified and verified by means of a liquid chromatograph coupled to a quadrupole-time-of-flight (Q-ToF) detector. Finally, based on the distribution of the analyzed cannabinoids in 30 Cannabis sativa L. plant varieties and the principal component analysis (PCA) of the resulting data, a clear difference was observed between outdoor and indoor grown plants, which was attributed to a higher concentration of THC, CBN, and CBD in outdoor grown plants.

Chitosan-Coated Alginate Microcapsules of a Full-Spectrum Cannabis Extract: Characterization, Long-Term Stability and In Vitro Bioaccessibility.

Cannabinoids present in Cannabis sativa are increasingly used in medicine due to their therapeutic potential. Moreover, the synergistic interaction between different cannabinoids and other plant constituents has led to the development of full-spectrum formulations for therapeutic treatments. In this work, the microencapsulation of a full-spectrum extract via vibration microencapsulation nozzle technique using chitosan-coated alginate is proposed to obtain an edible pharmaceutical-grade product. The suitability of microcapsules was assessed by their physicochemical characterization, long-term stability in three different storage conditions and in vitro gastrointestinal release. The synthetized microcapsules contained mainly ∆9-tetrahydrocannabinol (THC)-type and cannabinol (CBN)-type cannabinoids and had a mean size of 460 ± 260 µm and a mean sphericity of 0.5 ± 0.3. The stability assays revealed that capsules should be stored only at 4 °C in darkness to maintain their cannabinoid profile. In addition, based on the in vitro experiments, a fast intestinal release of cannabinoids ensures a medium-high bioaccessibility (57-77%) of therapeutically relevant compounds. The full characterization of microcapsules indicates that they could be used for the design of further full-spectrum cannabis oral formulations.

Quantification of Endocannabinoids in Human Plasma.

The determination of the concentration of endocannabinoids and related compounds in human plasma has become a matter of interest due to their implication in physiological processes and, thus, their possible relation with physiological conditions or illnesses. The analysis of these compounds though has to be carefully designed as they are found in very low concentrations, and some of them degrade easily once blood is collected. In this chapter, a simple method based on a liquid-liquid extraction and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) is described to determine the concentration of eight of the most relevant endocannabinoids in plasma.

Review: Metabolomics as a prediction tool for plants performance under environmental stress.

Metabolomics as a diagnosis tool for plant performance has shown good features for breeding and crop improvement. Additionally, due to limitations in land area and the increasing climate changes, breeding projects focusing on abiotic stress tolerance are becoming essential. Nowadays no universal method is available to identify predictive metabolic markers. As a result, research aims must dictate the best method or combination of methods. To this end, we will introduce the key aspects to consider regarding growth scenarios and sampling strategies and discuss major analytical and data treatment approaches that are available to find metabolic markers of plant performance.

Focused ultrasound-assisted acceleration of enzymatic hydrolysis of alkylphenols and 17ß-oestradiol glucuronide in fish bile.

According to the European Water Framework Directive (WFD), alkylphenols, such as octylphenols and nonylphenols, and 17ß-oestradiol are considered as priority or emerging pollutants, respectively, mainly due to their possible properties as endocrine-disrupting compounds (EDCs). EDCs are accumulated in liver, fat, kidney and bile in the glucuronide form. In order to determine the concentration of these compounds in bile, an enzymatic hydrolysis step is necessary. This step is usually long (~16 h), and in this sense, ultrasound probes were studied as a possible alternative energy source to accelerate this process. Enzymatic hydrolysis was reduced to 20 min using an ultrasound probe at one cycle and 10% of amplitude. For validation of analytical procedure, nonylphenol glucuronide (4NP-G), 4-tert-octylphenol glucuronide (4tOP-G) and 4-n-octylphenol glucuronide (4nOP-G) were synthesised while 17ß-oestradiol glucuronide (E2-G) was commercially available. Bile from thick-lip grey mullets (Chelon labrosus) was spiked with known amounts of 4NP-G, 4tOP-G, 4nOP-G and E2-G and submitted to the optimised procedure. Good recoveries (77-122%), precision in the 5% to 12% range and limits of detection, ranging from the low nanogramme per gramme level for 4tOP, 4nOP and E2 to the low microgramme per gramme level for nonylphenols, were obtained. The optimised method was applied for the determination of alkylphenol in the bile of thick-lip grey mullets fish bile from the Urdaibai estuary (UNESCO reserve of the Biosphere, Bay of Biscay), and high concentrations (2.3-14.2 µg/g), such as those obtained in polluted areas, were measured. E2 was determined in the bile of thick-lip grey mullets, intraperitoneally injected with E2.

Ultrasonic-assisted derivatization of estrogenic compounds in a cup horn booster and determination by GC-MS.

Cup horn boosters are miniaturized ultrasound baths that maximize efficiency and precision. The optimization of an ultrasonic-assisted derivatization step by means of a cup horn booster and the determination of estrone, 17beta-estradiol, estriol, 17alpha-ethynyl estradiol and mestranol was developed by GC-MS. Different derivatization reagents and solvents were studied for maximizing the di-derivatization of 17alpha-ethynyl estradiol under ultrasound energy. Only N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane in pyridine gave satisfactory results and this mixture was further used in the optimization of the ultrasound assisted derivatization. The experiment designs included sonication time (1-10 min), sonication power (20-80%), sonication cycles (1-9), derivatization reagent volume (25-125 microL) and solvent volume (25-125 microL). Once the optimum conditions were fixed, the effect of organic matter and the frequency of the water bath change were studied. Finally, the validation of the analytical method was carried out using spiked natural and synthetic waters. Recoveries (natural (138-70%) and synthetic (112-89%)), the LODs (0.35-1.66 ng/L), and LOQs (1.16-5.52 ng/L) and the precision (0.2-5.3%) of the method were studied. This is the first work in the literature where a cup horn booster is used with the aim of minimizing derivatization time during the determination of estrogenic compounds.

Distribution of organic microcontaminants, butyltins, and metals in mussels from the Estuary of Bilbao.

Mussels are used as bioindicators of chemical pollution in coastal and estuarine waters. We measured the concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), butyltins, and metals (Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, V, and Zn) in mussel tissues collected from the lower Bilbao estuary (Arriluze, north of Spain) every 2 months from November 2002 to March 2004. The concentration (microg g(-1) dry weight) of PAHs, PCBs, and PEs ranged from 5.1 to 18.3, from 0.04 to 0.2, and from 1.5 to 27.6, respectively. Temporal pattern variations, including maximum and minimum values, were determined for metals and BTs from their concentration profiles during a period of 1 year. The main feature of organic microcontaminants was relatively high concentration values, reflecting the overall industrial and harbour activities of the site. Moreover, the ratios of methylated species and certain other diagnostic ratios suggested a petrogenic origin for PAHs. Finally, the relations among the concentrations found in mussel tissues and the levels of several cell biomarkers were established by a partial least squares model.

Optimisation of the headspace-solid phase microextraction for organomercury and organotin compound determination in sediment and biota.

Headspace solid-phase microextraction was optimised for the simultaneous preconcentration of methylmercury (MeHg+), monobutyltin, dibutyltin, tributyltin, monophenyltin (MPhT), diphenyltin (DPhT), and triphenyltin (TPhT) from sediments and biota. Extraction time (3-24 min), extraction temperature (20-90 degrees C), desorption time (1-10.4 min), desorption temperature (152-260 degrees C), and sample volume (5-22 mL) were simultaneously optimised, while variables such as fibre type (30 microm polydimethylsiloxane, PDMS), pH (acetic acid/sodium acetate, HOAc/NaOAc, 2 mol/L, pH approximately 4.8), the concentration of the derivatisation agent (sodium tetraethylborate, NaBEt4, 0.1% m/v), and the ionic strength (fixed by the buffer solution) were kept constant. The variables were optimised according to the experiments proposed by the MultiSimplex program and the responses were considered in order to establish the optimum conditions. The repeatability (relative standard deviation, RSD, 5-20.6%) and limits of detection (LODs, 0.05-0.97 ng/g) of the overall method were also estimated. The lowest precisions were obtained for DPhT and TPhT. The optimised preconcentration method was applied to the determination of MeHg+), butyl- and phenyltins in certified reference materials (IAEA-405 MeHg+) in estuarine sediment, BCR-646 butyl- and phenyltins in marine sediment, BCR-463 MeHg+ in tuna fish, DOLT-2 MeHg+ in dogfish liver, and BCR-477 butyltins in mussel tissue) by GC with microwave-induced plasma/atomic-emission detection.

Evaluation of polar organic chemical integrative and hollow fibre samplers for the determination of a wide variety of organic polar compounds in seawater.

The calibration of two passive samplers for the determination of 20 emerging organic compounds in seawater is described in this work: i) a new version of polar organic chemical integrative sampler (POCIS) containing 100 mg of mixed-mode anion exchanger (Strata X-AW) and 100 mg of polymeric HLB (Plexa) sorbent materials and using a highly porous Nylon membrane (30-µm pore size) and ii) polyethersulfone (PES) hollow fibre. Among the studied contaminants, herbicides, hormones, life style products (stimulants and artificial sweeteners), industrial chemicals (corrosion inhibitor and fluorinated compounds), personal care products and several pharmaceuticals were included. In the case of POCIS, both the sorbents and the Nylon membranes were extracted and analysed independently. The calibration set up consisted on a continuous-flow tank that was fed with a continuous flow of seawater (2 L/h) and a stock mixture of contaminants (20 mL/h), assuring a nominal concentration of ~ 600 ng/L (each analyte) in the tank. The uptake was linear in POCIS sorbent and Nylon membranes but exponential for PES hollow fibres. Furthermore, the highest sampling rates (Rs) values were obtained in POCIS sorbent (between 2.7 for acetaminophen and 491 mL/day for perfluoro-n-octanoic acid, PFOA) followed by Nylon membranes (between 3.6 for OBT and 50 mL/day for telmisartan) and the lowest were those from PES fibres (between 1.7 for bezafibrate and 157 mL/day for butylparaben). Additionally, five deuterated compounds ([2H5]-atrazine, [2H3]-amitriptyline, [2H7]-irbesartan, [2H3]-ketoprofen and [2H9]-progesterone) were studied as candidates for performance reference compounds (PRCs) in both POCIS and PES, and though [2H5]-atrazine, [2H9]-progesterone and [2H3]-amitriptyline showed acceptable results in the case of POCIS, only [2H5]-atrazine provided a good validation. In the case of PES fibres, the PRC corrections did not provide acceptable results due to a low dissipation of the PRCs. Finally, POCIS were deployed in two sites of the low part of the estuary of Bilbao (northern Spain) from where water samples were also taken and analysed. As a result, in addition to the overall good agreement between the passive and active samplings, passive samplers allowed the determination of several compounds that were below the detection limits in the active sampling.

Non-targeted metabolomics reveals alterations in liver and plasma of gilt-head bream exposed to oxybenzone.

The extensive use of the organic UV filter oxybenzone has led to its ubiquitous occurrence in the aquatic environment, causing an ecotoxicological risk to biota. Although some studies reported adverse effects, such as reproductive toxicity, further research needs to be done in order to assess its molecular effects and mechanism of action. Therefore, in the present work, we investigated metabolic perturbations in juvenile gilt-head bream (Sparus aurata) exposed over 14 days via the water to oxybenzone (50 mg/L). The non-targeted analysis of brain, liver and plasma extracts was performed by means of UHPLC-qOrbitrap MS in positive and negative modes with both C18 and HILIC separation. Although there was no mortality or alterations in general physiological parameters during the experiment, and the metabolic profile of brain was not affected, the results of this study showed that oxybenzone could perturb both liver and plasma metabolome. The pathway enrichment suggested that different pathways in lipid metabolism (fatty acid elongation, α-linolenic acid metabolism, biosynthesis of unsaturated fatty acids and fatty acid metabolism) were significantly altered, as well as metabolites involved in phenylalanine and tyrosine metabolism. Overall, these changes are signs of possible oxidative stress and energy metabolism modification. Therefore, this research indicates that oxybenzone has adverse effects beyond the commonly studied hormonal activity, and demonstrates the sensitivity of metabolomics to assess molecular-level effects of emerging contaminants.

Chemical fingerprinting of petroleum biomarkers in biota samples using retention-time locking chromatography and multivariate analysis.

This work was conducted to study a new separation and evaluation approach for the chemical fingerprinting of petroleum biomarkers in biota samples. The final aim of this work was to study the correlation between the observed effects in the shore habitats (mussels and limpets) and one pollution source: the oil spill of the Prestige tanker. The method combined a clean-up step of the biota extracts (mussels and limpets), the retention-time locking of the gas chromatographic set up, and the multivariate data analysis of the chromatograms. For clean-up, solid-phase extraction and gel permeation chromatography were compared, and 5g Florisil cartridges assured the lack of interfering compounds in the last extracts. In order to assure reproducible retention times and to avoid the realignment of the chromatograms, the retention-time locking feature of our gas chromatography-mass spectrometry (GC-MS) set up was used. Finally, in the case of multivariate analysis, the GC-MS chromatograms were treated, essentially by derivatization and by normalization, and all the chromatograms at m/z 191 (terpenes), m/z 217-218 (steranes and diasteranes) and m/z 231 (triaromatic steranes) were treated by means of principal component analysis. Furthermore, slightly different four oil samples from the Prestige oil spill were analyzed following the Nordtest method, and the GC-MS chromatograms were considered as the reference chemical fingerprints of the sources. In this sense, the correlation between the studied samples, including sediments and biota samples, and the source candidate was completed by means of a supervised pattern recognition method. As a result, the method proposed in this work was useful to identify the Prestige oil spill as the source of many of the analyzed samples.

Fast method for routine simultaneous analysis of methylmercury and butyltins in seafood.

A simple, fast, and accurate method for the simultaneous determination of methylmercury (MeHg(+)), monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) in seafood is proposed. The method makes use of relatively cheap instrumentation and allows simultaneous analysis of those four species in a routine basis. The sample is treated with methanolic potassium hydroxide in an ultrasound bath, derivatised with sodium tetraethylborate (NaBEt(4)), preconcentrated into n-hexane and analysed by gas chromatography with atomic emission detection (GC-MIP/AES). The soft extraction conditions provided by ultrasound energy prevent chemical decomposition of the analytes and allow fast and efficient recovery of the species considered. Both the extraction and the derivatisation/preconcentration steps were optimised. Detection limits of 34, 3, 6 and 8 ng g(-1) (dry mass) were obtained for MeHg(+), MBT, DBT and TBT, respectively, using the best experimental conditions found. The uncertainty of the analysis ranged from 11% (MeHg(+)) to 15% (MBT). The accuracy of the method was checked by the analysis of several certified reference materials, e.g., BCR 477 (mussel tissue, MBT, DBT and TBT), DOLT-2 (dogfish liver, MeHg(+)), BCR 463 (tuna fish, MeHg(+)) and NIST 2976 (mussel tissue, MeHg(+)) with satisfactory results. Several oyster samples collected in the estuary of the Oka River (Urdaibai, Unesco Reserve of the Biosphere, Basque Country) during four sampling campaigns in 2003-2004 were processed following the proposed procedure. Concentrations ranging from 65 to 149 ng g(-1) (MeHg(+)),

Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC-MS/MS and enzyme-alkaline hydrolysis.

A high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of Δ9-tetrahydrocannabinol (THC), its two metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), and four additional cannabinoids (cannabidiol (CBD), cannabigerol (CBG), tetrahydrocannabivarin (THCV), and cannabinol (CBN)) in 1 mL of human urine and plasma was developed and validated. The hydrolysis process was studied to ensure complete hydrolysis of glucuronide conjugates and the extraction of a total amount of analytes. Initially, urine and plasma blank samples were spiked with THC-COOH-glucuronide and THC-glucuronide, and four different pretreatment methods were compared: hydrolysis-free method, enzymatic hydrolysis with Escherichia Coli ß-glucuronidase, alkaline hydrolysis with 10 M NaOH, and enzyme-alkaline tandem hydrolysis. The last approach assured the maximum efficiencies (close to 100%) for both urine and plasma matrices. Regarding the figures of merit, the limits of detection were below 1 ng/mL for all analytes, the accuracy ranged from 84% to 115%, and both within-day and between-day precision were lower than 12%. Finally, the method was successfully applied to real urine and plasma samples from cannabis users. Copyright © 2016 John Wiley & Sons, Ltd.

Targeting the endocannabinoid system: future therapeutic strategies.

The endocannabinoid system (ECS) is involved in many physiological regulation pathways in the human body, which makes this system the target of many drugs and therapies. In this review, we highlight the latest studies regarding the role of the ECS and the drugs that target it, with a particular focus on the basis for the discovery of new cannabinoid-based drugs. In addition, we propose some key steps, such as the creation of a cannabinoid-receptor interaction matrix (CRIM) and the use of metabolomics, toward the development of improved and more specific drugs for each relevant disease.

Microencapsulation and storage stability of polyphenols from Vitis vinifera grape wastes.

Wine production wastes are an interesting source of natural polyphenols. In this work, wine wastes extracts were encapsulated through vibration nozzle microencapsulation using sodium alginate as polymer and calcium chloride as hardening reagent. An experimental design approach was used to obtain calcium-alginate microbeads with high polyphenol content and good morphological features. In this way, the effect of pressure, frequency, voltage and the distance to the gelling bath were optimized for two nozzles of 150 and 300 µm. Long-term stability of the microbeads was studied for 6 months taking into account different storage conditions: temperatures (4 °C and room temperature), in darkness and in presence of light, and the addition of chitosan to the gelling bath. Encapsulated polyphenols were found to be much more stable compared to free polyphenols regardless the encapsulation procedure and storage conditions. Moreover, slightly lower degradation rates were obtained when chitosan was added to the gelling bath.

Optimization of supercritical fluid consecutive extractions of fatty acids and polyphenols from Vitis vinifera grape wastes.

In this study, supercritical fluid extraction has been successfully applied to a sequential fractionation of fatty acids and polyphenols from wine wastes (2 different vitis vinifera grapes). To this aim, in a 1st step just fatty acids were extracted and in a 2nd one the polyphenols. The variables that affected to the extraction efficiency were separately optimized in both steps following an experimental design approach. The effect of extraction temperature flow, pressure, and time were thoroughly evaluated for the extraction of fatty acids, whereas the addition of methanol was also considered in the case of the polyphenols extraction. A quantitative extraction with high efficiency was achieved at a very short time and low temperatures. Concerning quantification, fatty acids were determined by means of gas chromatography coupled to mass spectrometry after a derivatization step, whereas the polyphenols were analyzed by means of high performance liquid chromatography coupled to tandem mass spectrometry and the Folin-Ciocalteu method.