Ultrasound-assisted Solid-liquid Extraction Research Articles

Screening of Bioactive Compounds in Lettuce: Multivariate Optimization of an Ultrasound-Assisted Solid–Liquid Extraction Procedure

Screening of Bioactive Compounds in Lettuce: Multivariate Optimization of an Ultrasound-Assisted Solid–Liquid Extraction Procedure

Determination of the Nucleic Acid Constituents in Edible Mushrooms by High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD)

Nucleosides and nucleobases in edible mushrooms collected from the Tekirdağ region of Turkey were determined by high performance liquid chromatography (HPLC). Separation of compounds was accomplished by gradient elution on a C18 column. A mixture of methanol (MeOH):50 mM sodium phosphate buffer (1:99, v/v) at pH 3.70 and methanol:50 mM sodium phosphate buffer (20:80) at pH 6.20 was used as the solvent. Two methods were used to treat the lyophilized mushrooms: ultrasound-assisted solid-liquid extraction (US-SLE) and pressurized (accelerated) solid-liquid extraction (PSLE). Deionized water was used as the extraction solvent. The extraction temperature and time were optimized. Fourteen nucleosides and nucleobases were simultaneously monitored at 254 nm within 25 min. Ultrasound-assisted solid-liquid was demonstrated to be more effective, perhaps because the pressurized liquid extraction system is a closed system and the matrix-solvent interaction may be insufficient. Adenosine, guanosine and uridine were the most abundant compounds in all studied mushrooms.

Development of a green ultrasound-assisted procedure for the extraction of phenolic compounds from avocado peel with deep eutectic solvents

Avocado (Persea americana Mill.) is a tropical fruit grown in different areas of the world. Avocado pulp is of interest to food companies while seed and peel are wasted. In 2021, about 25 thousand tons of avocado peel were produced as waste. In accordance with circular economy and green chemistry principles, avocado peel could be considered a source of bioactive molecules, specifically phenolic compounds, to be recovered using green methods. Deep eutectic solvents (DESs) are green solvents whose extractive efficiency for phenolic compounds from food and waste products of the agri-food chain has already been demonstrated. In this work, an ultrasound-assisted solid-liquid extraction, using DESs, was developed to recover phenolic compounds from avocado (Hass variety) peel. For this purpose, twelve DESs were tested as extraction solvents considering the provided total phenolic content. Then, in order to obtain the largest amount of phenolic compounds, the most relevant factors affecting solid-liquid extraction were evaluated. Matrix-to-solvent ratio of 1:30 (w/v), 25°C and 15 min as temperature and time of extraction, respectively, were found as optimal conditions to guarantee an extracted amount of phenolic compounds of 8.29 ± 0.07 g GAE/100g of dry avocado peel. Through high-performance liquid chromatography coupled with photodiode array and mass spectrometry detection, some flavonoids and phenolic acids were identified in the avocado peel extract.

Determination of phytocannabinoids in cannabis samples by ultrasound-assisted solid-liquid extraction and high-performance liquid chromatography with diode array detector analysis

The characterisation of cannabis plants, especially the determination of specific phytocannabinoids, has gained enormous importance in the last decade, mainly due to the recent changes in cannabis control in several countries or states. This is particularly relevant for the forensic, medical or recreative industry to have a rapid, inexpensive, and reliable methodology to identify and quantify phytocannabinoids. Furthermore, spiking cannabis products with Δ8-tetrahydrocannabinol (THC) is a contemporary trend that demands improving or replacing current methods to include this cannabinoid. The current study presents an ultrasound-assisted solid-liquid extraction followed by high-performance liquid chromatography with diode array detection (HPLC-DAD) methodology to identify and quantify Δ9-THC, Δ8-THC, cannabidiol, cannabinol, Δ9-tetrahydrocannabinolic acid and cannabidiolic acid in cannabis products. The herbal samples were extracted with ethanol:acetonitrile (50:50, v:v) by ultrasonication using only 50 mg of sample. The plant oils were diluted in ethanol. The optimised procedure allowed ≈100% extraction efficiency of the target cannabinoids. The validation assays showed that the method is linear (R2 > 0.997), selective, sensitive, precise and accurate, with suitable limits of detection (0.125–0.250 µg mL−1) and quantification (0.500 µg mL−1). The method was successfully applied to cannabis samples, demonstrating its suitability for routine analyses. This contribution follows the current demand for fast and straightforward analysis services of this plant and its derivatives, using small amounts of sample. The present study compares very favourably against other works, particularly in regards to the extraction efficiency, speed of the overall procedure, method sensitivity, and ability to monitor Δ8-THC spiked samples using a novel solvent mixture.

Incorporation of Moringa oleifera Leaf Extract in Yoghurts to Mitigate Children's Malnutrition in Developing Countries.

Moringa oleifera, which is rich in bioactive compounds, has numerous biological activities and is a powerful source of antioxidants and nutrients. Therefore, M. oleifera can be incorporated into food to mitigate children's malnutrition. In this work, the bioactive compounds were extracted from M. oleifera leaf powder by ultrasound-assisted solid-liquid extraction. The antioxidant and antimicrobial activities and the phenolic composition of the extract were evaluated. The extract presented a total phenolic content of 54.5 ± 16.8 mg gallic acid equivalents/g and IC50 values of 133.4 ± 12.3 mg/L for DPPH and 60.0 ± 9.9 mg/L for ABTS. Catechin, chlorogenic acid, and epicatechin were the main phenolics identified by HPLC-DAD. The obtained extract and M. oleifera leaf powder were incorporated into yoghurts and their physicochemical and biological properties were studied. The incorporation of M. oleifera did not impair the yoghurts' stability over eight weeks when compared to both negative and positive controls. The extract presented higher stability regarding syneresis but lower stability regarding TPC compared to the powder. Also, the fortified yoghurts presented higher antioxidant properties than the negative control. These findings highlight the potential use of M. oleifera powder and extract as natural additives to produce fortified foods that can be used in the mitigation of malnutrition.

Effects of Extraction Methods on Phenolic Content, Antioxidant and Antiplatelet Activities of Tomato Pomace Extracts

Aqueous and ethanolic extracts of tomato pomace were examined with the aim of optimizing the extraction process of compounds with cardioprotective activity. Once the results of the ORAC response variables, total polyphenols, °Brix, and antiplatelet activity of the extracts were obtained, a multivariate statistical analysis was performed using the Statgraphics Centurion XIX software. This analysis showed that the most relevant positive effects in the inhibition of platelet aggregation were 83 ± 2% when using the agonist TRAP-6, when the working conditions were the type of tomato pomace conditioning (drum-drying process at 115 °C), phase ratio (1/8), type of solvent (ethanol 20%), and type of extraction (ultrasound-assisted solid-liquid extraction). The extracts with the best results were microencapsulated and characterized by HPLC. The presence of chlorogenic acid (0.729 mg/mg of dry sample) was found, a compound that has a potential cardioprotective effect documented in various studies, in addition to rutin (2.747 mg/mg of dry sample) and quercetin (0.255 mg/mg of dry sample). These results show that the extraction efficiency of compounds with cardioprotective activity depends largely on the polarity of the solvent, thus playing an important role in the antioxidant capacity of the extracts of tomato pomace.

Combining Ultrafiltration and Nanofiltration to Obtain a Concentrated Extract of Purified Polyphenols from Wet Olive Pomace

Despite the environmental concerns raised every year by the generation of high volumes of wet olive pomace, it contains valuable phenolic compounds that are essential for the valorization of this by-product. In this work, an integrated process to recover phenolic compounds from wet olive pomace is proposed. It consists of ultrasound-assisted solid-liquid extraction, followed by ultrafiltration and nanofiltration. Several commercial membranes were studied at different operational conditions. The ultrafiltration stage allowed the purification of biophenols, which were obtained in the permeate stream. Regarding organic matter, satisfactory rejection values were obtained with both commercial UH030 and UP005 membranes (Microdyn Nadir), but the latter provided more efficient purification and higher values of permeate flux, above 18 L·h-1·m-2 at 2.5 bar and 1.5 m·s-1. Later, this permeate stream was concentrated by means of a nanofiltration process, obtaining polyphenol rejection values that surpassed 85% with the commercial NF270 membrane (DuPont), then achieving the concentration of the previously purified polyphenols.

Characterization and Biological Activity of Fiber-Type Cannabis sativa L. Aerial Parts at Different Growth Stages.

Currently, there is a renewed interest in cannabis-related products in different fields because of the rich phytocomplex of this plant, together with its fiber and agricultural features. In this context, the current study aims to chemically characterize different samples of fiber-type Cannabis sativa L. grown in Italy as a potential health promoting source. An ultrasound-assisted solid-liquid extraction (UA-SLE) method was first developed and optimized to obtain a fingerprinting of the investigated phytocomplex. Analyses were carried out through an ultra high performance liquid chromatography equipped with a photodiode array detector in series with triple quadrupole system with an electrospray ionization (ESI) interface (UHPLC-UV-ESI-MS/MS) and showed that the phytocomplex mainly includes flavonoids and non-psychotomimetic cannabinoids. The method was then applied to characterize and compare 24 samples of fiber-type Cannabis sativa L. aerial parts (mainly stems and leaves), which differed for the growth stages (from mid-vegetative to early flowering), growth land plots, and methods of drying (forced-draft oven or freeze-drying). The quali-quantitative analysis showed that a freeze-drying method seems to better preserve the chemical composition of the samples, while the location of the land plot and the growth stage of the plant (which did not comprise inflorescences) had minor influences on the chemical pattern. These results were also supported by spectrophotometric in-vitro assays (scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2′-azinobis-3-ethyl-benzthiazoline-6-sulphonate (ABTS+•) radicals and inhibitory activity against tyrosinase and elastase enzymes) to investigate the potential biological activity of these samples and the contribution of non-psychotomimetic cannabinoids.

Ultrasound-assisted Solid-liquid Extraction Devices - Harnessing Active Ingredients from Plant materials – Overview and Analysis

Background: In the growing environmental concern use of natural products, efficient processes and devices are necessary. Solid-Liquid extraction of active Ingredients from Plant materials is one of the important unit operations in Chemical Engineering and need to be enhanced. Objectives: Since, these active ingredients are firmly bound to the plant cell wall membrane, which pose mass-transfer resistance and need to get detached through the use of suitable process intensification tools such as ultrasound and suitable devices. Therefore, detailed analysis and review is essential on development made in this area through Publications and Patents. Hence, the present paper illustrates the development of ultrasound assisted device for solid-liquid extraction are presented in this paper. Methods: Advantages such as % Yield, Reduction in extraction time, use of ambient conditions, better process control, avoidance or minimizing multi stage extraction could be achieved due to the use of ultrasound in extraction as compared to conventional processes. Conclusions: Use of ultrasound to provide significant improvements in the extraction of Vegetable tannins, Natural dyes for application in Leather processing has been demonstrated and reported earlier. These enhancement could be possible through various effects of ultrasound such as better flow of solvents through micro-jet formation, mass transfer enhancement due to rupture of plant cell wall membranes through acoustic cavitation, better leaching due to micro-mixing and acoustic streaming effects. This approach would minimize material wastage; thereby, leading to eco-conservation of plant materials, which is very much essential for better environment. Hence, various methods and design for application of ultrasound assisted solid-liquid extractor device are necessary.

Determination of veterinary drugs in microalgae biomass from photobioreactors fed with piggery wastewater

Concentration data of veterinary drugs in microalgae biomass collected from photobioreactors fed with piggery wastewaters are presented for the first time in this work. To this aim, a QuEChERS methodology and an ultrasound-assisted solid-liquid extraction have been assessed as sample preparation procedures with the purpose of determining 20 veterinary drugs, mainly antibiotics of different physico-chemical properties in addition to dexamethasone, fenbendazole and progesterone. Some critical operation parameters of the QuEChERS procedure were optimized by an experimental design but tetracycline, oxytetracycline, doxycycline, marbofloxacin and ciprofloxacin were not detected by the QuEChERS sample preparation. The use of a longer and thorough approach, a solid-liquid extraction with water/methanol in presence of primary secondary amine as a clean-up agent followed by solid-phase extraction on Oasis HLB cartridges, is recommended to monitor all intended analytes. The determination in extracts is carried out by ultra-high performance liquid chromatography–tandem mass spectrometry in selected reaction monitoring mode. Limits of detection about 0.2–42 ng per g of lyophilized microalgae sample, and repeatabilities about 6–46% (n = 5, RSDs) are reached. The solid-liquid extraction method was applied to microalgae biomass samples collected from a photobioreactor. Nine drugs were detected in the samples at relatively low concentration and a proportional relationship between the found concentrations and the octanol/water partition coefficients of the drugs has been outlined. Moreover, a linear ratio between the concentrations measured in biomass and effluent has been observed for most of the drugs.

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.

N-compounds speciation analysis in environmental samples using ultrasound-assisted solid-liquid extraction and non-chromatographic techniques.

A fast, efficient, and non-chromatographic method was presented in this study for nitrite, nitrate, and p-nitrophenol (N-compounds) extraction and speciation analysis of environmental samples. By applying ultrasound-assisted solid-liquid extraction (USLE), analytes were efficiently extracted from water, soil, or sediment collected in areas of environmental disaster. These analytes were selectively converted to NO(g) through UV photolysis (NO3-), H2O2/UV photocatalysis (PNP), and direct conversion (NO2-). Following conversion, NO(g) was separated from the liquid phase and determined by high-resolution continuum source molecular absorption spectrometry (HR-CS MAS). The LODs obtained were 0.097 ± 0.004mgL-1 for nitrite, 0.119 ± 0.004mgL-1 for nitrate, and 0.090 ± 0.006mgL-1 for p-nitrophenol. On applying this speciation method to environmental samples, concentrations were found to be up to 0.99 ± 0.03mgL-1 (NO2-), 49.80 ± 2.5mgL-1 (NO3-), and 0.10 ± 0.02mgL-1 (PNP). Finally, addition/recovery study of real water, soil, and sediment samples showed 101 ± 2% recovery for NO2-, 100 ± 1% for NO3-, and 96 ± 5% for PNP.

Levels of volatile methylsiloxanes in urban wastewater sludges at various steps of treatment

Volatile methylsiloxanes are wastewater organic pollutants originating from industrial and personal care products. They accumulate in sewage sludge and hamper biogas production through the formation of silicon dioxide on the surfaces of gas engines and turbines, yet there is few knowledge on their fate in sludge treatment systems. Here we studied the occurrence and removal of seven volatile methylsiloxanes along the solid treatment line of an urban wastewater treatment plant. We analyzed 75 sludge samples from seven locations using ultrasound-assisted solid–liquid extraction followed by gas chromatography-mass spectrometry. Results show that volatile methylsiloxanes occur in all samples with a predominance of cyclic homologues, particularly decamethylcyclopentasiloxane. Total siloxane levels reach 6087 ng/g dry weight in the secondary sludge, average at 3575 ng/g in digested sludge, and 5703 ng/g in the dewatered sludge, the latter being likely due to the addition of a polyelectrolyte. Overall, these levels call for measures to reduce methylsiloxane levels before the biogas production step.

Analysis of highly polar marine biotoxins in seawater by hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry

The monitoring of marine biotoxins (MBTs) in seawater is presented as an alternative strategy to determine their presence and the possible implications in the ecosystem. For this, an analytical method based on hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry (HILIC-HRMS) has been developed to identify and quantify some hydrophilic MBTs in seawater: saxitoxin (STX), decarbamoyl-saxitoxin (dcSTX), neosaxitoxin (NeoSTX), gonaytoxin-2,3 (GTX-2,3) and tetrodotoxin (TTX), which are responsible of gastrointestinal and central nervous system distress in humans when are consumed via seafood. Particulate and filtrate portion were analyzed separately in order to characterize the extracellular toxins dissolved in the water and those present in the particulate. Ultrasound assisted solid-liquid extraction with methanol was used for the isolation of the MBTs from particulate and solid phase extraction using silica cartridges for the filtrate. Extraction procedure was the most critical step during the analytical method due to the high polarity of the toxins and the absolute recoveries obtained ranged from 15 to 47 % in the filtrate and 26 to 71 % in the particulate portions. Limits of detection of the method ranged from 0.5 to 5 µg/L in the filtrate portion and from 3.1 to 62 µg/L in the particulate portion.•Saxitoxins and tetrodotoxins have been analysed by using HILIC-HRMS.•UAE with methanol and SPE with silica cartridges have been employed for the extractions of the polar MBTs from seawater.

A multi-residue method for determining twenty-four endocrine disrupting chemicals in vegetables and fruits using ultrasound-assisted solid–liquid extraction and continuous solid-phase extraction

In this work, we developed an analytical approach using an ultrasound-assisted extraction (UAE) followed by continuous solid-phase extraction (SPE) and gas chromatography–mass spectrometry (GC–MS) detection in order to determine simultaneously 24 endocrine disrupting chemicals such as alkylphenols, organophosphorus pesticides, parabens, phenylphenols, triclosan and bisphenol A in vegetable and fruit samples. Different variables influencing UAE and SPE performance were optimized in order to maximize removal of the sample matrix and preconcentration of the analytes. The optimized extraction and GC–MS quantitation conditions provided acceptable sensitivity, selectivity, accuracy and precision. Limits of detection spanned the range 0.6–25 ng kg−1, recoveries were near-quantitative and relative standard deviations ranged from 4.5 to 7.6%. The proposed method was used to analyse 11 vegetable samples and 7 fruit samples purchased at various Spanish and Moroccan supermarkets. Most samples contained more than three of the analytes, at levels between 5.8 and 580 ng kg−1.

Application of response surface methodology for optimization of ultrasound‐assisted solid‐liquid extraction of phenolic compounds from Cenostigma macrophyllum

AbstractThis study determines the optimal conditions for the ultrasound‐assisted solid‐liquid extraction (UA‐SLE) of the total phenolic compounds (TPC) of Cenostigma macrophyllum leaves. The experiments were carried out according to three‐level, four‐variable Box‐Behnken design combined with the response surface methodology (RSM). Extraction (sonication) time, liquid‐solid ratio, methanol concentration, and ultrasonic power were investigated as independent variables to obtain the optimal extraction conditions for TPC. Data were fitted to a second‐order polynomial model, and the model fitness was evaluated by the analysis of variance, which indicated the model (P < 0.0001), presenting precision and reliability with the experiments performed (R2 = 0.9947, coefficient of variance [CV] = 4.29, SD = 1.67). Individual and interactive effects of the independent variables on TPC extraction were interpreted using the proposed mathematical model. The results showed that for the maximum TPC extraction, the optimal conditions included 32 min extraction time, 29.73 mL·g−1 liquid‐solid ratio, 47.72% methanol concentration, and 145.71 W ultrasonic power. Under these optimal conditions, the predicted values were in agreement with the experimental values, thus validating the RSM model. When the optimal conditions were compared with those of conventional extraction techniques, the results showed that UA‐SLE presented significantly better results for TPC extraction (71.2% to 138.3% more efficient) and for antioxidant activity (115.0% to 179.2% more efficient). High‐performance liquid chromatography analysis of the extracts revealed that UA‐SLE did not interfere with the extract chemical composition, thus confirming that it was more effective than conventional techniques.

Focused ultrasound-based extraction for target analysis and suspect screening of organic xenobiotics in fish muscle

The development of multitarget and/or suspect screening methods for the analysis of xenobiotics in fish samples is compulsory due to the lack of works in the literature where a deep evaluation of the variables affecting extraction and clean-up steps is performed. The aim of the present work was to optimize and validate a multitarget (180 compounds) method for the analysis of priority and emerging xenobiotics in fish muscle using focused ultrasound-assisted solid-liquid extraction. From the different extraction solvents studied, a single extraction in cold acetonitrile rendered the best consensus results in terms of absolute recoveries and the number of target compounds extracted. Matrix effect was minimized using commercially available Captiva ND-Lipid filters, which provided clean extracts and satisfactory repeatability compared to other approaches. Absolute recoveries were corrected using matrix-matched calibration and apparent recoveries in the 43%-105%, 73%–131% and 78%–128% ranges were obtained at low (20 ng g−1), medium (100 ng g−1), and high (200 ng g−1) spiking levels, respectively. A 60% of the xenobiotics showed limits of identification lower than 20 ng g−1. The developed method was successfully applied to the quantification and suspect screening of samples bought in a local market (hake, gilt-head bream, sea bass and prawn) and fished (thicklip grey mullet) at the Urdaibai estuary (north of Spain). Food additives, antiparasitic drugs and PFOS were quantified at ng g−1 level. Moreover, the targeted method was extended to the suspect screening, revealing the presence of plastic related products (caprolactam, phthalates, polyethylenglycols), pharmaceutical products (albendazole, mebendazole, valpromide) and pesticides or insect repellents (icaridin, myristyl sulfate, nootkatone). Therefore, FUSLE in cold acetonitrile combined with Captiva ND-Lipid filters and liquid chromatography tandem high-resolution mass spectrometry (LC-q-Orbitrap) were successfully applied to both multitarget quantitative analysis and suspect screening of approx. 17,800 compounds.

Determination of transformation products of per- and polyfluoroalkyl substances at trace levels in agricultural plants

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous in the environment. However, only a limited number of predominantly persistent perfluoroalkyl acids (PFAAs) have been analyzed in edible plants so far. We present a generic trace analytical method that allows for quantification of 16 intermediate fluorotelomer alcohol (FTOH)- or perfluoroalkane sulfonamidoethanol (FASE)-based transformation products as well as 18 PFAAs in plants. Additionally, 36 suspected intermediate PFAS transformation products were qualitatively analyzed. The ultrasound-assisted solid-liquid extraction of wheat and maize grain, maize leaves, Jerusalem artichoke and ryegrass (1-5 g plant sample intake) was followed by a clean-up with dispersive solid-phase extraction using graphitized carbon adsorbent (5-10 mg per sample) and chemical analysis by reversed phase liquid chromatography-tandem mass spectrometry. The method was based on matrix matched and extracted calibration and displayed good precision with relative standard deviations in triplicate analyses typically below 15% for all quantified analytes and matrices. An average deviation of 12% between quantified concentrations obtained by matrix matched and extracted calibration and a method based on isotopically labelled internal standards underlines the good trueness of the method. The method quantification limits for the majority of analytes in all plant samples were in the low ng/kg concentration range on a dry weight basis. Plant matrices were analyzed from crops grown on agricultural fields that have been contaminated with PFASs. FTOH- and/or FASE-based intermediate transformation products were detected in all samples with N-ethyl perfluorooctane sulfonamidoacetic acid (EtFOSAA) and perfluorooctane sulfonamide (FOSA) as the prevailing compounds in concentrations up to several hundred ng/kg in maize leaves. The 9:3 Acid (a transformation product of 10:2 FTOH) was tentatively identified. In accordance with these findings, the final degradation products perfluorooctane sulfonic acid (PFOS) and perfluorodecanoic acid (PFDA) were frequently detected. For perfluoroalkyl carboxylic acids (PFCAs), according to earlier findings, short chain homologues generally displayed the highest levels (up to 98 µg/kg for perfluorobutanoic acid (PFBA) in maize leaves). However, maize grain was an exception showing the highest concentrations for long chain PFCAs, whereas PFBA was not detected. The uptake of high levels of PFASs into plants is of concern since these may be used as animal feed or represent a direct exposure medium for humans.

Determination of free tyrosine in equestrian supplements by LC–MS/MS and comparison of its quantity with total free amino acids content in view of doping control

The reports on the probable beneficial impact of tyrosine (TYR) supplementation on performance enhancement have contributed to the growth of interest in TYR in equestrian sports field and related industries, such as the manufacture of dietary and nutritional supplements. In this study, the first attempt to the assessment of horses exposure to TYR during nutritional supplementation was demonstrated by quantification of unbound TYR and a total free amino acids (AAs), and subsequent determination of the ratio of TYR to a total free AAs content in equestrian supplement samples. Within the scope of this study, a rapid and sensitive LC–MS/MS-based method for the determination of unbound TYR and a simple spectrophotometric protocol for the quantification of a total free AAs have been developed and validated according to the international guidelines for bioanalytical methods. For sample preparation, an ultrasound-assisted solid-liquid extraction (USLE; for solid samples) and ultrasound-assisted liquid-liquid extraction (ULLE; for liquid samples) have been optimized. The comprehensive approach on the simultaneous assessment of matrix effect, recovery, process efficiency, accuracy, precision, curve factor and internal standard association demonstrated satisfactory validation parameters for LC–MS/MS-based procedure, such as global matrix effect in the range of 89.9–91.9%, good linearity (R2 > 0.9929), limit of detection (0.6 ng/mL) and recovery within 89.2–108.0%. For spectrophotometric method, limit of detection was 1100 ng/mL, recovery ranged from 103.2 to 108.8%, both, intra- and inter-day accuracy was within 89.5–108.7%, and both, intra- and inter-assay precision was below 8.5%. In view of the satisfactory validation parameters obtained for both methods, the procedures can be utilized for routine analysis of dietary and nutritional supplements.

Pumpkin Peel Flour Extracts Obtained by an Ultrasound-Assisted System as a Rich Source of Bioactive Compounds with Antioxidant Properties

This study evaluated an Ultrasound-Assisted solid-liquid Extraction (UAE) aided by Response Surface Methodology (RSM) to obtain bioactive-rich extracts from pumpkin ( Cucurbita moschata ) dried peel. Total Phenolic Compounds (TPC), Total Flavonoids Content (TFC), as well as the in vitro Antioxidant Activity (AA), were assessed. The physicochemical analysis revealed that Pumpkin Peel Flour (PPF) has an excellent nutritional profile, comprised by lipids (11.80%), proteins (6.02%) and total fibre (1.96%), in addition to valuable quantity of minerals such as P, Mg, Ca, Fe, Na, Zn and Mn. A Box-Behnken design was applied to analyse the effect of the concentration of solvent, the extraction time and the solid-liquid ratio as independent variables and the TPC, TFC and AA in response. PPF is a rich source of TPC (145.51-479.05 mg GAE/L) and TFC (44.08-89.68 mg CTE/L), which contribute to a high antioxidant activity (653.90-1698.20 &mumol TE/L). Using the RSM, a simultaneous optimisation was performed by the desirability function and the optimum condition to maximise the obtaining of target compounds was with 80% ethanol in the proportion of 10 mL/g (solvent vs raw material) during 45 min. External validation was subsequently conducted on the proposed optimal point and all results were within ±95% of the prediction intervals proposed by the models. Thus, the UAE aided by RSM was shown to be an adequate approach to model the recovery of bioactive compounds with antioxidant properties from a by-product of the pumpkin industry.