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Berenguer, Pedro Henrique Fernandes da Silva
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- 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.
- A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturizationPublication . Berenguer, Pedro Henrique Fernandes da Silva; Câmara, José de Sousa; Camacho, Irene Gomes CâmaraAsthma is a disease characterized by chronic airway inflammation and by its long term irreversible remodeling. The enzymatic peroxidation of the arachidonic acid leads to the formation of bioactive eicosanoids, key mediators of bronchial inflammation and response modulation in the airways contributing to the pathophysiology of this disease. The present work aimed to develop an improved ultra-high pressure liquid chromatography (UHPLC)-based strategy to characterize biomarkers of lipid peroxidation: leukotrienes E4 (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), present in urine of asthmatic patients and healthy individuals (control group). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) format was used to isolate the target analytes. Several experimental parameters with influence on this procedure and on the chromatographic resolution, were evaluated and optimized. The method was fully validated with compatible values under optimal extraction (R-AX sorbent, 3 conditioning-equilibration cycles with 250 µL of ACN-H2O at 0.1% FA, 10 extract-discard cycles of 250 µL of sample at a pH of 5.1, elution with 2 times 50 µL of MeOH and concentration of the eluate until half of its volume) and chromatographic conditions (14-min analysis at a flow rate of 300 µL min-1 in an UHPLC-PDA equipped with a BEH C18 column). The developed method was successfully applied to the urine of asthmatic patients and healthy individuals. On average, the urine of asthmatic patients present significantly higher concentrations of 11βPGF2α (112.96 ng mL-1 vs 62.56 ng mL-1 in control group), LTE4 (1.27 ng mL-1 vs 0.89 ng mL-1 in control group) and LTB4 (1.39 ng mL-1 vs 0.76 ng mL-1 in control group). These results suggest the potential of the target eicosanoids on asthma diagnosis and on the follow-up of the therapeutic response, even though a larger and more extensive study will be necessary, using a bigger number of cases, to confirm the data obtained and to guarantee a greater robustness to the approach.
- Madeira-a tourist destination for asthma sufferersPublication . Camacho, Irene; Grinn-Gofroń, Agnieszka; Camacho, Roberto; Berenguer, Pedro; Sadyś, MagdalenaMadeira Island is a famous tourist destination due to its natural and climatic values. Taking into account optimal weather conditions, flora richness and access to various substrates facilitating fungal growth, we hypothesised a very high risk of elevated fungal spore and pollen grain concentrations in the air of Funchal, the capital of Madeira. Concentration levels of the most allergenic taxa were measured from 2003 to 2009, using a 7-day volumetric air sampler, followed by microscopy analysis. Dependence of bioaerosols on the weather conditions and land use were assessed using spatial and statistical tools. Obtained results were re-visited by a comparison with hospital admission data recorded at the Dr. Nélio Mendonça Hospital in Funchal. Our results showed that despite propitious climatic conditions, overall pollen grain and fungal spore concentrations in the air were very low and did not exceed any clinically established threshold values. Pollen and spore peak concentrations also did not match with asthma outbreaks in the winter. Identification of places that are "free" from biological air pollution over the summer, such as Madeira Island, is very important from the allergic point of view.
- Determination of potential childhood asthma biomarkers using a powerful methodology based on microextraction by packed sorbent combined with ultra-high pressure liquid chromatography. Eicosanoids as case studyPublication . Berenguer, Pedro H.; Camacho, Irene C.; Câmara, Rita; Oliveira, Susana; Câmara, José S.Leukotrienes and prostaglandins are arachidonic acid bioactive derived eicosanoids and key mediators of bronchial inflammation and response modulation in the airways contributing to the pathophysiology of asthma. An easy-to-use ultra-high pressure liquid chromatography (UHPLC)-based strategy was developed to characterize biomarkers of lipid peroxidation: leukotrienes E (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), present in urine of asthmatic patients (N = 27) and healthy individuals (N = 17). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) was used to isolate the target analytes. Several experimental parameters with influence on the extraction efficiency and on the chromatographic resolution, were evaluated and optimized. The method was fully validated under optimal extraction (R-AX sorbent, 3 conditioning-equilibration cycles with 250 μL of ACN-water at 0.1% FA, 10 extract-discard cycles of 250 μL of sample at a pH of 5.1, elution with 2 times 50 μL of MeOH and concentration of the eluate until half of its volume) and chromatographic conditions (14-min analysis at a flow rate of 300 μL min-1 in an UHPLC-PDA equipped with a BEH C18 column), according to IUPAC guidelines. The findings indicated good recoveries (>95%) in addition to excellent extraction efficiency (>95%) at three concentration levels (low mid and high) with precision (RSDs) less than 11%. The lack-of-fit test, goodness-of-fit test and Mandel's fitting test, revealed good linearity within the concentration range. Good selectivity and sensitivity were achieved with a limits of detection ranging from 0.04 μg L-1 for LTB4 to 1.12 μg L-1 for 11βPGF2α, and limits of quantification from 0.10 μg L-1 for the LTB4 to 2.11 μg L-1 for 11βPGF2α. The successful application of the fully validated method shows that, on average, the asthmatic patients had significantly higher concentrations of 11βPGF2α (112.96 μg L-1vs 62.56 μg L-1 in normal controls), LTE4 (1.27 μg L-1vs 0.89 μg L-1 in normal controls), and LTB4 (1.39 μg L-1vs 0.76 μg L-1 in normal controls). The results suggest the potential of the target eicosanoids on asthma diagnosis, however, a larger and more extensive study will be necessary to confirm the data obtained and to guarantee a greater robustness to the approach.