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- Ultrasound-assisted liquid-liquid extraction followed by ultrahigh pressure liquid chromatography for the quantification of major carotenoids in tomatoPublication . Figueira, José A.; Pereira, Jorge A.M.; Porto-Figueira, Priscilla; Câmara, José S.Lycopene and β-carotene, the main carotenoids present in different tomatoes varieties (gordal, cherry, roma and campari) of Solanum lycopersicum L. (Solanaceae), were investigated using ultrasound-assisted liquid-liquid extraction (LLUSAE) followed by ultra-high pressure liquid chromatography with PDA detection (UHPLC-PDA) analysis. Experimental parameters influencing the LLUSAE were optimized using an univariate design, resulting in a 30-min ACN/MeOH extraction by sonication of a lyophilized sample, followed by PSA/C18/MgSO4 clean-up and fast centrifugation before UPLC analysis. Using this greener methodology, high recoveries (above 97%), good linearity (r2 > 0.98) and improved sensitivity, with limits of detection and quantification of 24.0 and 80.0 ng/mL for lycopene and 3.0 and 9.9 ng/mL for β-carotene, respectively, were obtained. This sensitivity is about five times better than previously reported in literature, making LLUSAE/UHPLC-PDA a promising strategy for lycopene and β-carotene quantification in tomato and eventually in other matrices. The carotenoids studied, lycopene and β-carotene, were found at highest concentrations in the gordal tomato variety, followed by cherry, roma and campari (727.1, 342.2, 267.2 and 218.2 μg/g and 80.4, 44.0, 45.7 and 44.0 μg/g for lycopene and β-carotene, respectively). Additionally, an exponential increase of both carotenoids occurs during ripening and mainly in the skin and locular cavity of the gordal variety. These results provide further evidences of the potential of tomatoes as an interesting source of lycopene and β-carotene.
- Antitumor efficacy of doxorubicin-loaded laponite/alginate hybrid hydrogelsPublication . Gonçalves, Mara; Figueira, Priscilla; Maciel, Dina; Rodrigues, João; Shi, Xiangyang; Tomás, Helena; Li, YulinDegradable hybrid hydrogels with improved stability are prepared by incorporating nanodisks of biocompatible laponite (LP) in alginate (AG) hydrogels using Ca2+ as a crosslinker. The Dox‐loaded hybrid hydrogels give a controlled Dox release at physiological environment in a sustained manner. Under conditions that mimic the tumor environment, both the sustainability in the Dox release (up to 17 d) and the release efficiency from LP/AG‐Dox hydrogels are improved. The in situ degradation of these hybrid hydrogels gives rise to nanohybrids that might serve as vehicles for carrying Dox through the cell membrane and diminish the effect of Dox ion‐trapping in the acidic extracellular environment of the tumor and/or in the endo‐lysosomal cell compartments.
- Exploring the potentialities of an improved ultrasound-assisted quick, easy, cheap, effective, rugged, and safe-based extraction technique combined with ultrahigh pressure liquid chromatography-fluorescence detection for determination of Zearalenone in cerealsPublication . Porto-Figueira, Priscilla; Camacho, Irene; Câmara, José S.Zearalenone (ZEA), a secondary metabolite from Fusarium graminearum fungi that invade crops and grow during blooming in moist cool field conditions, is a nonsteroidal oestrogen, biologically very potent, although hardly toxic. Different analytical methods, among which a quick, easy, cheap, effective, rugged, and safe (QuEChERS) strategy, combined with or liquid chromatography, have been proposed for ZEA determination in foods. However, this extraction procedure has the disadvantage of using large amount of organic solvents, partitioning salts and sample volume, which limits the technique application on the fields where the sample amount is critical. Therefore in this work an improved sample preparation step based on the original QuEChERS (O-QuEChERS) approach, the μ-QuEChERS, combined with ultrasound-assisted extraction (USAE) and ultrahigh pressure liquid chromatography (UHPLC) analysis with fluorescence detection (FLR) is proposed for the sensitive and high-throughput quantification of ZEA in cereals. The proposed analytical strategy, μ-QuEChERSUSAE, uses up to 32 times lower amounts of partitioning salts, lower sample amount and lower extraction solvents, in comparison with O-QuEChERS technique. The performance of the analytical approach was assessed by studying the selectivity, specificity, limits of detection and quantification, linear dynamic range, matrix effect and precision. Good linearity (r(2)>0.99) was achieved for ZEA, and limits of detection (LOD=3.4μgkg(-1)) and limits of quantification (LOQ=4.7μgkg(-1)) were found below the tolerance levels set by European Commission. Good recoveries were obtained with different spiked concentrations, ranged from 80.2% to 109.7%, with relative standard deviations (RSD) lower than 5.0%. The comparison of the analytical performance of the proposed μ-QuEChERSUSAE with O-QuEChERS showed the powerful ability of the proposed strategy since it gives comparable results but using lower amounts of sample, partitioning salts and volume of the organic solvent, revealing of great potential and improvement of the proposed method.
- A non-invasive approach to explore the discriminatory potential of the urinary volatilome of invasive ductal carcinoma of the breastPublication . Taunk, Khushman; Taware, Ravindra; More, Tushar H.; Porto-Figueira, Priscilla; Pereira, Jorge A. M.; Mohapatra, Rajkishore; Soneji, Dharmesh; Câmara, José S.; Nagarajaram, H. A.; Rapole, SrikanthWorldwide, breast invasive ductal carcinoma (IDC) accounts for the majority of the reported cases of this form of cancer. IDC effective management, as for any form of cancer, would greatly benefit from early diagnosis. This, however, due to various socio-economic reasons, is very far for the reality in developing countries like India, where cancer diagnosis is often carried out at late stages when disease management is troublesome. With the present work, we aim to evaluate a simple analytical methodology to identify a set of volatile organic compounds (VOCs) in urine samples, as a biosignature for IDC. Using solid-phase microextraction followed by gas chromatography/mass spectrometry, a panel of 14 urinary VOCs was found to discriminate IDC (n = 65) from a healthy control (HC) group (n = 70) through multivariate statistical treatments. Furthermore, metabolic pathway analysis revealed various dysregulated pathways involved in IDC patients hinting that their detailed investigations could lead to novel mechanistic insights into the disease pathophysiology. In addition, we validated the expression pattern of five of these VOCs namely 2-ethyl-1-hexanol, isolongifolenone, furan, dodecanoic acid, 2-methoxy-phenol in another external cohort of 59 urinary samples (IDC = 32 and HC = 27) and found their expression pattern to be consistent with the primary sample set. To our knowledge, this is the first study exploring breast IDC volatome alterations in Indian patients.
- Exploring a volatomic-based strategy for a fingerprinting approach of Vaccinium padifolium L. berries at different ripening stagesPublication . Porto-Figueira, Priscilla; Figueira, José A.; Berenguer, Pedro; Câmara, José S.The effect of ripening on the evolution of the volatomic pattern from endemic Vaccinium padifolium L. (Uveira) berries was investigated using headspace-solid phase microextraction (HS-SPME) followed by gas chromato graphy/quadrupole-mass spectrometry (GC–qMS) and multivariate statistical analysis (MVA). The most sig nificant HS-SPME parameters, namely fibre polymer, ionic strength and extraction time, were optimized in order to improve extraction efficiency. Under optimal experimental conditions (DVB/CAR/PDMS fibre coating, 40 °C, 30 min extraction time and 5 g of sample amount), a total of 72 volatiles of different functionalities were isolated and identified. Terpenes followed by higher alcohols and esters were the predominant classes in the ripening stages – green, break and ripe. Although significant differences in the volatomic profiles at the three stages were obtained, cis-β-ocimene (2.0–40.0%), trans-2-hexenol (2.4–19.4%), cis-3-hexenol (2.5.16.4%), β-myrcene (1.9–13.8%), 1-hexanol (1.7–13.6%), 2-hexenal (0.7–8.0%), 2-heptanone (0.7–7.7%), and linalool (1.9–6.1%) were the main volatile compounds identified. Higher alcohols, carboxylic acids and ketones gradually increased during ripening, whereas monoterpenes significantly decreased. These trends were dominated by the higher alcohols (1-hexanol, cis-3-hexenol, trans-2-hexenol) and monoterpenes (β-myrcene, cis-β-ocimene and trans-β ocimene). Partial least squares regression (PLSR) revealed that ethyl caprylate (1.000), trans-geraniol (0.995), ethyl isovalerate (−0.994) and benzyl carbinol (0.993) are the key variables that most contributed to the successful differentiation of Uveira berries according to ripening stage. To the best of our knowledge, no study has carried out on the volatomic composition of berries from endemic Uveira.
- pH-sensitive Laponite®/doxorubicin/alginate nanohybrids with improved anticancer efficacyPublication . Gonçalves, Mara; Figueira, Priscilla; Maciel, Dina; Rodrigues, João; Qu, Xue; Liu, Changsheng; Tomás, Helena; Li, YulinThe efficacy of the anticancer drug doxorubicin (Dox) is limited by an insufficient cellular uptake and drug resistance, which is partially due to ion trapping in acidic environments such as the extracellular environment of solid tumors and the interior of endolysosome vesicles. Herein, we describe the preparation and in vitro evaluation of a new type of nanohybrid for anticancer drug delivery which is capable of carrying a high load of the cationic Dox through the cell membrane. In addition, the nanohybrids use the acidic environment of the endolysosomes to release the drug, simultaneously helping to disrupt the endolysosomes and diminishing endolysosome Dox trapping. Furthermore, as the nanohybrid carriers are capable of sustained drug delivery, those that remain in the cytoplasm and still contain Dox are expected to exert a prolonged anticancer activity. Briefly, Dox is loaded onto biocompatible anionic Laponite(®) (LP) nanodisks with a high aspect ratio (25 nm in diameter and 0.92 nm in thickness) through strong electrostatic interactions to get Dox-loaded LP disks. Alginate (AG), a biocompatible natural polymer, is then coated onto the Dox-loaded LP disks (LP/Dox/AG nanohybrids) to prevent the burst release of the drug. The results demonstrate that the nanohybrids have a high encapsulation efficiency (80.8 ± 10.6%), are sensitive to pH and display a sustained drug release behavior. Cell culture experiments indicate that the LP/Dox/AG nanohybrids can be effectively internalized by CAL-72 cells (an osteosarcoma cell line), and exhibit a remarkable higher cytotoxicity to cancer cells than the free Dox. The merits of Laponite(®)/alginate nanohybrids, such as biocompatibility, high loading capacity and stimulus responsive release of cationic chemotherapeutic drugs, render them as excellent platforms for drug delivery.
- Biogenic amines in food: occurrence and analytical challenges for their analysisPublication . Pereira, Jorge A. M.; Porto-Figueira, Priscilla; Andrade, Beatriz; Gonçalves, Patrícia; Pataca, Joanna; Câmara, José S.Biogenic amines (BAs) are naturally occurring nitrogenous organic compounds of low molecular weight organic bases with aliphatic, aromatic or heterocyclic structures. They are generally formed through microbiological activity during food processing and storage, and can be found in a wide variety of foods, particularly fermented foods and beverages as cheese, wine, beer, in addition to fishery products and meat. At low concentrations, BAs participate in the regulation of several physiological functions, but when present at high concentrations, they may cause several health problems in consumers, as headaches, hypo- or hypertension, nausea and cardiac palpitations, especially to sensitive persons. Therefore, the control of BAs levels in different food products is an important issue for food safety monitoring policies. Due to their low levels in complex food matrices, the analysis of BAs is not an easy task and several methods for their separation, identification, and determination have been described during the last decades. Overall, the chromatographic approaches are the most popular, although the recent trends points to the development of sensors able to measure BAs in food matrices without involving the laborious and complex laboratorial sample analysis methodologies. In this review we will essentially focus on the occurrence of BAs in different foodstuffs and in the correspondent challenges of their analysis.
- Avaliação de nova estratégia extrativa, baseada em NTDs, combinada com dados de análise cromatigráfica e quimiométrica para a deteção de biomarcadores de diferentes tipos de cancroPublication . Figueira, Priscilla Felício Porto; Câmara, José de Sousa; Pereira, Jorge Augusto MachadoA elevada incidência e mortalidade mundiais associadas ao cancro justificam o desenvolvimento e implementação de estratégias eficazes e não-invasivas conducentes a um diagnóstico precoce. Neste contexto, pretendeu-se avaliar a performance de uma metodologia inovadora, a microextração por “needle trap” (NTME), na extração de metabolitos voláteis (VOMs) da urina de pacientes oncológicos com diferentes tipos de cancro - cólon, pulmão e mama, e de indivíduos saudáveis, com a finalidade de identificar um conjunto de VOMs potenciais biomarcadores dos diferentes tipos cancros em estudo. De modo a maximizar a eficiência da extração dos VOMs, foram otimizados diferentes parâmetros experimentais, nomeadamente a natureza do sorvente, a temperatura, o tempo de equilíbrio, o volume de headspace, a força iónica, o pH do meio e o volume e a agitação da amostra. Usando como sorvente o DVB/Car1000/CarX, os melhores resultados foram obtidos com 4 mL de urina acidificada (pH= 2), 20% NaCl, 40 mL de headspace e 40 min de equilíbrio a 50 °C. Foi ainda avaliada a estabilidade dos VOMs no sorvente até 72 h após a extração. Nos quatro grupos em estudo foram identificados, por GC-MS, 259 VOMs pertencentes a diversas famílias químicas, nomeadamente cetonas, compostos sulfurados, furânicos e terpénicos. A matriz de dados obtida para cada grupo em estudo foi submetida a análise discriminante, usando o método dos mínimos quadrados parciais (PLS-DA), que resultou em clusters distintos diferenciadores de cada grupo. A aplicabilidade do modelo foi avaliada através do método de classificação SIMCA (modelagem suave e independente de analogias de classe), com elevadas taxas de classificação, sensibilidade e especificidade. Este foi o primeiro estudo usando NTME para o estabelecimento do padrão volatómico da urina. Os resultados obtidos revelam-se muito promissores originando perfis voláteis de maior expressividade, mais completos e abrangentes, que os obtidos usando metodologias de referência.
- Redox-responsive alginate nanogels with enhanced anticancer cytotoxicityPublication . Maciel, Dina; Figueira, Priscilla; Xiao, Shili; Hu, Dengmai; Shi, Xiangyang; Rodrigues, João; Tomás, Helena; Li, YulinAlthough doxorubicin (Dox) has been widely used in the treatment of different types of cancer, its insufficient cellular uptake and intracellular release is still a limitation. Herein, we report an easy process for the preparation of redox-sensitive nanogels that were shown to be highly efficient in the intracellular delivery of Dox. The nanogels (AG/Cys) were obtained through in situ cross-linking of alginate (AG) using cystamine (Cys) as a cross-linker via a miniemulsion method. Dox was loaded into the AG/Cys nanogels by simply mixing it in aqueous solution with the nanogels, that is, by the establishment of electrostatic interactions between the anionic AG and the cationic Dox. The results demonstrated that the AG/Cys nanogels are cytocompatible, have a high drug encapsulation efficiency (95.2 ± 4.7%), show an in vitro accelerated release of Dox in conditions that mimic the intracellular reductive conditions, and can quickly be taken up by CAL-72 cells (an osteosarcoma cell line), resulting in higher Dox intracellular accumulation and a remarkable cell death extension when compared with free Dox. The developed nanogels can be used as a tool to overcome the problem of Dox resistance in anticancer treatments and possibly be used for the delivery of other cationic drugs in applications beyond cancer.
- Exploring the potential of NTME/GC-MS, in the establishment of urinary volatomic profiles. Lung cancer patients as case studyPublication . Porto-Figueira, Priscilla; Pereira, Jorge; Miekisch, Wolfram; Câmara, José S.The growing cancer incidence and mortality worldwide claims for the development of novel diagnostic strategies. In this study we aimed to explore the potential of an innovative methodology, based on a needle trap microextraction (NTME), combined with gas chromatography-mass spectrometry (GC-MS), as new approach to isolate and profile urinary volatile organic metabolites (VOMs) from lung cancer (LC) patients and healthy individuals (CTRL). In this context, different experimental parameters with influence of NTME extraction efficiency including, temperature, equilibration time, headspace volume, ionic strength, pH, effects of sample volume and stirring, were investigated and optimized. For the DVB/CarX/Car1000 needle trap device (NTD), the best results were obtained using 40 mL headspace of a 4-mL acidified (pH = 2) urine sample with 20% NaCl and an extraction temperature of 50 °C for 40 min of equilibration time. The stability of the isolated VOMs was investigated up to 72 h after extraction. From the VOMs identified, belonging namely to ketones, sulphur and benzene derivatives, 98 presented a frequency of occurrence above 90%. Data were processed by discriminant analysis, retrieving differentiated clusters for LC and CTRL groups. As far we are aware, this is the first study using NTME/GC-MS to establish urinary volatomic profiles. Preliminary results are very promising, as broad and comprehensive volatile profiles were obtained. Moreover, the extended storage stability of the NTD devices opens new opportunities for sampling other matrices in a wide range of applications.