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- A powerful methodological approach combining headspace solid phase microextraction, mass spectrometry and multivariate analysis for profiling the volatile metabolomic pattern of beer starting raw materialsPublication . Gonçalves, João L.; Figueira, José A.; Rodrigues, Fátima P.; Ornelas, Laura P.; Branco, Ricardo N.; Silva, Catarina L.; Câmara, José S.The volatile metabolomic patterns from different raw materials commonly used in beer production, namely barley, corn and hop-derived products - such as hop pellets, hop essential oil from Saaz variety and tetra-hydro isomerized hop extract (tetra hop), were established using a suitable analytical procedure based on dynamic headspace solid-phase microextraction (HS-SPME) followed by thermal desorption gas chromatography-quadrupole mass spectrometry detection (GC-qMS). Some SPME extraction parameters were optimized. The best results, in terms of maximum signal recorded and number of isolated metabolites, were obtained with a 50/30 μm DVB/CAR/PDMS coating fiber at 40 °C for 30 min. A set of 152 volatile metabolites comprising ketones (27), sesquiterpenes (26), monoterpenes (19), aliphatic esters (19), higher alcohols (15), aldehydes (11), furan compounds (11), aliphatic fatty acids (9), aliphatic hydrocarbons (8), sulphur compounds (5) and nitrogen compounds (2) were positively identified. Each raw material showed a specific volatile metabolomic profile. Monoterpenes in hop essential oil and corn, sesquiterpenes in hop pellets, ketones in tetra hop and aldehydes and sulphur compounds in barley were the predominant chemical families in the targeted beer raw materials. β-Myrcene was the most dominant volatile metabolite in hop essential oil, hop pellets and corn samples while, in barley, the predominant volatile metabolites were dimethyl sulphide and 3-methylbutanal and, in tetra hop, 6-methyl-2-pentanone and 4-methyl-2-pentanone. Principal component analysis (PCA) showed natural sample grouping among beer raw materials.
- Beer volatile fingerprinting at different brewing stepsPublication . Alves, Vera; Gonçalves, João; Figueira, José A.; Ornelas, Laura P.; Branco, Ricardo N.; Câmara, José S.; Pereira, Jorge A. M.Volatile fingerprints of a lager beer were carried out throughout five brewing steps to characterize the changes encompassing this process. Overall, 60 volatile organic metabolites (VOMs) were identified by headspace solid phase microextraction followed by gas chromatography mass spectrometry (HS-SPME/GC–MS). Specific profiles were observed at different brewing steps - aldehydes and furans dominate in wort, whereas the aliphatic esters and alcohols predominate in the following steps. Such variations can be assigned to specific VOMs, as 3-me thylbutanal (wort), ethyl alcohol and ethyl octanoate (fermentation, maturation and filtration), or ethyl alcohol and isoamyl acetate (final product). These VOMs can influence the beer final flavour. Ethyl alcohol contributes to its strong and pungent smell and taste, while isoamyl acetate adds intense ‘fruity’ and ‘banana’ odours. These beer volatile fingerprints constitute a valuable tool to obtain insights on the impact of each brewing step on the final product, being also very useful for certification purposes.
- Microextraction using packed sorbent as an effective and high-throughput sample extraction technique: recent applications and future trendsPublication . Pereira, J.; Gonçalves, J.; Alves, V.; Câmara, J. S.In the last decade, several developments, particularly in the liquid chromatography and hyphenated techniques fields, have allowed researchers to reach analytical limits that are becoming very close to the single molecule level. Sample preparation, however, despite being a key step in the analytical methodology, did not track these developments and very few approaches able to cope with these stringent analytical requirements were developed. One such approach is microextraction by packed sorbent (MEPS), a sophisticated and miniaturized form of solid phase extraction that has reduced to the microliter scale every step of the sample extraction methodology including sample volume and solvent usage. Simultaneously, the amount of extraction phase used was also reduced and, more importantly, the sorbent is reusable dozens of times which significantly lowers the cost of analysis versus other solid-phase extraction approaches. In this review, we will update the state-of-the-art of the MEPS technique, focusing on the trends and applications reported since 2010 and future perspectives and developments that in our view will further improve the high-throughput potential and applications of this sample preparation methodology.
- Development of a novel microextraction by packed sorbent-based approach followed by ultrahigh pressure liquid chromatography as a powerful technique for quantification phenolic constituents of biological interest in winesPublication . Gonçalves, João; Mendes, Berta Rodrigues; Silva, Catarina L.; Câmara, José S.A novel analytical approach, based on a miniaturized extraction technique, the microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the quantitative determination of sixteen biologically active phenolic constituents of wine. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (linearity, sensitivity, selectivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters on the MEPS performance such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). The wine bioactive phenolics were eluted by 250 μL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method gave satisfactory results in terms of linearity with r2-values > 0.9986 within the established concentration range. The LOD varied from 85 ng mL−1 (ferulic acid) to 0.32 μg mL−1 ((+)-catechin), whereas the LOQ values from 0.028 μg mL−1 (ferulic acid) to 1.08 μg mL−1 ((+)-catechin). Typical recoveries ranged between 81.1 and 99.6% for red wines and between 77.1 and 99.3% for white wines, with relative standard deviations (RSD) no larger than 10%. The extraction yields of the MEPSC8/UHPLC–PDA methodology were found between 78.1 (syringic acid) and 99.6% (o-coumaric acid) for red wines and between 76.2 and 99.1% for white wines. The inter-day precision, expressed as the relative standard deviation (RSD%), varied between 0.2% (p-coumaric and o-coumaric acids) and 7.5% (gentisic acid) while the intra-day precision between 0.2% (o-coumaric and cinnamic acids) and 4.7% (gallic acid and (−)-epicatechin). On the basis of analytical validation, it is shown that the MEPSC8/UHPLC–PDA methodology proves to be an improved, reliable, and ultra-fast approach for wine bioactive phenolics analysis, because of its capability for determining simultaneously in a single chromatographic run several bioactive metabolites with high sensitivity, selectivity and resolving power within only 10 min. Preliminary studies have been carried out on 34 real whole wine samples, in order to assess the performance of the described procedure. The new approach offers decreased sample preparation and analysis time, and moreover is cheaper, more environmentally friendly and easier to perform as compared to traditional methodologies.
- Chemical analysis and in vitro cytotoxicity assessment of synthetic cathinones found in seized productsPublication . Gonçalves, João Luís Jesus; Câmara, José Sousa; Teixeira, Helena Maria de Sousa Ferreira eThe abuse of synthetic cathinones (SCat) represents a serious public health problem, and despite the legislative efforts to control the availability and use of these substances, new derivatives continue to appear in the market. Monitoring these substances is a challenging task faced by forensic and toxicological laboratories, due to the closely related molecular structures that lead to similar analytical characteristics. Moreover, with the emergence of new substances, information about their potential toxicity is unknown and the effects can be dangerous and unpredictable. The first purpose of this work was to chemically characterize fourteen seized products suspected to contained SCat, using several complementary analytical techniques, namely infrared spectroscopy (IR), gas chromatography coupled to mass spectrometry (GC-MS), ultrahigh performance liquid chromatography with photodiode array detector (UHPLC-PDA) and nuclear magnetic resonance (NMR). It was possible to identify nine SCat, namely methylone, methedrone, N-ethylcathinone, buphedrone, pentedrone, 3-fluoromethcathinone (3-FMC), 4- fluoromethcatinone (4-FMC), α-pyrrolidinohexanophenone (α-PHP) and 4-methyl-α pyrrolidinohexanophenone (MPHP). Subsequently, a new analytical methodology, based on the microextraction technique μSPEed® followed by UHPLC-PDA analysis, was developed for the determination of SCat in urine samples. Under the optimized conditions, the method showed satisfactory results in terms of linearity, with determination coefficients (R2 ) > 0.998, within the studied concentration ranges, and low detection limits (LOD) ranging from 36.8 to 95.3 ng mL−1. Once validated, the method was applied to real samples and α-PHP was detected in several samples. The cytotoxic potential of SCat, individually and in the samples, was evaluated in HepG2 cells. The elucidation of the cellular mechanisms underlying the observed hepatotoxicity, particularly those involved in oxidative stress was also evaluated. MPHP and α-PHP were the most cytotoxic compounds, and all SCat were able to disrupt the balance between reactive oxygen and nitrogen species (ROS and RNS) generation and the scavenging capacity of the antioxidant system.
- Characterization of phenolic compounds and antioxidant activity of ethanolic extracts from flowers of Andryala glandulosa ssp. varia (Lowe ex DC.) R.Fern., an endemic species of Macaronesia regionPublication . Gouveia, Sandra; Gonçalves, João; Castilho, Paula C.Andryala glandulosa spp. varia (Lowe ex DC.) R.Fern. (Asteraceae), is a small shrub that grows in moun tains of Madeira Island, Fuerteventura and Lanzarote from Canary Islands. The flowerheads are used traditionally for the treatment of edemas and in homemade dermo-cosmetic preparations. In this paper the chemical composition of the extracts of this plant, used in folk medicine, and their antioxidant capacity were established; the presence of potentially harmful lactones, so commonly associated with related species used for the same purposes was also evaluated. A reversed-phase high performance liquid chromatography method (RP-HPLC) coupled with diode-array detection (DAD) and electrospray ionization mass spectrometry (ESI/MSn) was used for the characterization of phenolic com pounds in ethanol extracts of flowers from A. glandulosa spp. varia collected in Madeira Island. Total phenolic content (TPC) and total flavonoid content (TFC) were established and three assays (DPPH, ABTS and FRAP) were used to measure the antioxidant capacity of the dichloromethane and ethanol extracts. The dichloromethane extract of A. glandulosa contain long linear chain hydrocarbons and esters. In the alcoholic extracts, a total of 16 compounds were characterized based on their UV, mass spectra and HPLC retention time. Quinic acid and luteolin derivatives were found to be the main compounds. Quantification of caffeoylquinic acids (CQA) detected was performed by HPLC-DAD and 5-O-CQA and 3,5-O-diCQA were the major compounds (with values of 22.40 ± 0.21 and 59.69 ± 1.07 mg/100 g dried plant, respectively). Only the ethanol extract was active, revealing a high radical scavenging capacity and a moderate reducing potential. The potent antioxidant alcoholic extracts are composed mainly of hydroxycinnamic acid derivatives and flavonoids. The presence of sesquiterpene lactones was not detected. Since lactones are very common among related plants, like arnicas, and known to cause dermatitis and other unwanted effects, this can be an explanation for the preference for Andryala over other more easily available alternatives
- New method for determination of (E)resveratrol in wine based on microextraction using packed sorbent and ultra-performance liquid chromatographyPublication . Gonçalves, João; Câmara, José S.An ultra-fast and improved analytical methodology based on microextraction by packed sorbent (MEPS) combined with ultra-performance LC (UPLC) was developed and validated for determination of (E)-resveratrol in wines. Important factors affecting the performance of MEPS such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles, and sample volume were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (50–250mL) in one extraction cycle (extract–discard) and in a short time period (about 3 min for the entire sample preparation step). (E)-Resveratrol was eluted by 1 250mL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a highstrength silica HSS T3 analytical column (100 mm 2.1 mm, 1.8mm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, extraction yield, accuracy, and inter/intra-day precision, using a Madeira wine sample (ET) spiked with (E)-resveratrol at concentration levels ranging from 5 to 60mg/mL. Validation experiments revealed very good recovery rate of 9575.8% RSD, good linearity with r2 values 40.999 within the established concentration range, excellent repeatability (0.52%), and reproducibility (1.67%) values (expressed as RSD), thus demonstrating the robustness and accuracy of the MEPSC8/UPLC-photodiode array (PDA) method. The LOD of the method was 0.21mg/mL, whereas the LOQ was 0.68mg/mL. The validated methodology was applied to 30 commercial wines (24 red wines and six white wines) from different grape varieties, vintages, and regions. On the basis of the analytical validation, the MEPSC8/UPLC-PDA methodology shows to be an improved, sensitive, and ultra-fast approach for determination of (E)-resveratrol in wines with high resolving power within 6 min.
- Re-exploring the high-throughput potential of microextraction techniques, SPME and MEPS, as powerful strategies for medical diagnostic purposes. Innovative approaches, recent applications and future trendsPublication . Pereira, Jorge; Silva, Catarina Luís; Perestrelo, Rosa; Gonçalves, João; Alves, Vera; Câmara, José S.The human population continues to grow exponentially in the fast developing and most populated countries, whereas in Western Europe it is getting older and older each year. This inevitably raises the demand for better and more efficient medical services without increasing the economic burden in the same proportion. To meet these requirements, improvement of medical diagnosis is certainly a key aspect to consider. Therefore, we need powerful analytical methodologies able to go deeper and further in the characterization of human metabolism and identification of disease biomarkers and endogenous molecules in body fluids and tissues. The ultimate goal is to have a reliable and early medical diagnosis, mitigating the disease complications as much as possible. Microextraction techniques (METs) represent a key step in these analytical methodologies by providing samples in the suitable volumes and purification levels necessary for the characterization of the target analytes. In this aspect, solid-phase microextraction (SPME) and, more recently, microextraction by packed sorbent (MEPS), are powerful sample preparation techniques, characterized by their reduced time of analysis, low solvent consumption, and broad application. Moreover, as miniaturized techniques, they can be easily automatized to have a high-throughput performance in the clinical environment. In this review, we explore some of the most interesting MEPS and SPME applications, focusing on recent trends and applications to medical diagnostic, particularly the in vivo and near real time applications.
- Food bioactive compounds and emerging techniques for their extraction: Polyphenols as a case studyPublication . Câmara, José S.; Albuquerque, Bianca R.; Aguiar, Joselin; Corrêa, Rúbia C. G.; Gonçalves, João L.; Granato, Daniel; Pereira, Jorge A. M.; Barros, Lillian; Ferreira, Isabel C. F. R.Experimental studies have provided convincing evidence that food bioactive compounds (FBCs) have a positive biological impact on human health, exerting protective effects against non communicable diseases (NCD) including cancer and cardiovascular (CVDs), metabolic, and neu rodegenerative disorders (NDDs). These benefits have been associated with the presence of sec ondary metabolites, namely polyphenols, glucosinolates, carotenoids, terpenoids, alkaloids, saponins, vitamins, and fibres, among others, derived from their antioxidant, antiatherogenic, anti-inflammatory, antimicrobial, antithrombotic, cardioprotective, and vasodilator properties. Polyphenols as one of the most abundant classes of bioactive compounds present in plant-based foods emerge as a promising approach for the development of efficacious preventive agents against NCDs with reduced side effects. The aim of this review is to present comprehensive and deep insights into the potential of polyphenols, from their chemical structure classification and biosynthesis to preventive effects on NCDs, namely cancer, CVDs, and NDDS. The challenge of polyphenols bioavailability and bioacces sibility will be explored in addition to useful industrial and environmental applications. Advanced and emerging extraction techniques will be highlighted and the high-resolution analytical techniques used for FBCs characterization, identification, and quantification will be considered.
- Development of MEPS–UHPLC/PDA methodology for the quantification of clozapine, risperidone and their major active metabolites in human urinePublication . Gonçalves, João L.; Alves, Vera L.; Conceição, Carlota J.F.; Teixeira, Helena M.; Câmara, José S.A new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the simultaneous determination of clozapine and risperidone, and their active metabolites, in urine samples. Important parameters affecting MEPS performance, including type of sorbent material (C2, C8, C18, SIL, M1, R-AX, R-CX, PGC, PEP, HLB-DVB and H-DVB), elution solvent system, number of extraction cycles, sample volume, elution volume, and sample pH, were evaluated. The optimal experimental conditions involved the loading of 500 μl of sample through a C18 sorbent in a five extraction cycles and in a short time period (5 min for the entire sample preparation step). The obtained extract was analyzed by UHPLC, using a binary mobile phase, composed of aqueous 0.1% formic acid and methanol in the gradient elution mode (10 min total analysis). Good results were obtained under the optimized conditions, in terms of linearity with correlation coefficients (r2) values higher than 0.9986. Low detection limits, ranging between 0.052 and 0.055 μg ml− 1 were achieved, whereas the lowest quantification limit was 0.1 μg ml− 1, corresponding to the lowest concentration of the standard curve. Satisfactory results were found with accuracy values ranging from 83% to 116%, and with precision results from 2.65% to 18%. Recoveries ranged between 76% and 96%, which was in agreement with results reported by other studies.
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