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- Simultaneous determination of dapsone and clofazimine in nanoformulations by HPLCPublication . Fernandes, Sara R.; Fernandes, Sara; Chaves, Luíse L.; Lima, Sofia A. C.; Silva, Eduarda M. P.; Barreiros, Luísa; Reis, Salette; Segundo, Marcela A.The multidrug therapy with dapsone (DAP) and clofazimine (CLZ) is known as an effective treatment against Mycobacterium leprae. However, the low bioavailability and non-specific distribution can reduce therapy efficacy and produce side effects. The use of nanotechnological approaches was explored as a promising carrier for delivery enhancement of these drugs. Therefore, a simple and precise highperformance liquid chromatography (HPLC) method with UV/Vis detection has been developed and validated for the simultaneous determination of DAP and CLZ loaded in solid dispersion and poly(D,L-lactide-co-glycolic acid) nanoparticles, respectively, targeting therapy improvement. A reversed phase Kinetex core-shell C18 column at room temperature followed by UV/Vis detection at 280 nm was used for chromatographic separation. The elution was performed in gradient mode using aqueous acetate buffer (50 mol L-1, pH 4.8) and an increasing acetonitrile content from 27 to 63% (v/v), at a flow rate of 1.0 mL min-1. The injection volume was fixed at 20 µL and total run time was 23.0 min, with a retention time of 6.0 min for DAP and 14.0 min for CLZ. The method was validated according to EMA guideline and showed specificity, accuracy (between 99.6 and 114.0% of nominal values) and precision for intra-day (RSD ≤1.8%) and inter-day assays (RSD ≤12.5%). Calibration curves were linear (r2 >0.9979) and LOD ≤0.03 and LOQ ≤0.06 mg L-1 were obtained. Stability was studied after 24 h at room temperature and over three freeze-thaw cycles, and recovery values ≥86.2% were obtained. Precipitation of CLZ was observed at low temperatures (4 °C). Entrapment efficiency in nanoformulations was evaluated as 54.8 ± 0.1% for DAP and 24.9 ± 0.2% for CLZ. The developed method was successfully validated for the simultaneous determination of DAP and CLZ in nanoparticles.
- Quantification of tranexamic acid in human plasma: development and validation of UHPLC-MS/MS methodPublication . Barreiros, Luisa; Amoreira, Júlia L.; Machado, Sandia; Fernandes, Sara R.; Silva, Eduarda M. P.; Sá, Paula; Kietaibl, Sibylle; Segundo, Marcela A.; Fernandes, SaraTranexamic acid (TXA), an antifibrinolytic drug with the ability to inhibit lysine binding at plasminogen receptors, can be used in different settings such as trauma, cardiac surgery, major orthopedic surgery, obstetric when perioperative bleeding is concerned [1]. Effective methods for determination of TXA in biological samples are still required to understand the pharmacokinetics and pharmacodynamics of this drug in variable age groups undergoing surgeries with high blood loss [2]. The development and validation of a method based on ultra-high performance liquid chromatography coupled to triple quadrupole-tandem mass spectrometry (UHPLCMS/MS) to quantify TXA in human plasma is described herein. A simple, inexpensive and efficient sample treatment involving protein precipitation with acetonitrile containing 0.5% (v/v) formic acid was implemented using volumes within the microliter range. Separation was achieved using a hydrophilic interaction based stationary phase and ammonium bicarbonate in the mobile phase that permitted a more efficient separation of the analyte from the matrix interferences, thus reducing matrix effects and increasing method sensitivity. The method was validated according to the European Medicines Agency guideline [3]. Excellent linearity was achieved (r2 > 0.997) for TXA concentrations ranging from 30 to 600 ng mL-1 with LOD and LOQ of 3 and 6 ng mL-1 in plasma extracts, respectively. The developed method proved to be selective, sensitive, accurate (96.4-105.7% of nominal concentration values) and precise (RSD ≤ 4.5%).
- Bioacessibility of zinc in pet food determined by a dynamic leaching methodPublication . Fernandes, Sara R.; Pereira, Ana Margarida; Matos, Elisabete; Castanheira, Francisco; Baptista, Cláudia S.; Cabrita, Ana Rita J.; Segundo, Marcela A.; Fernandes, SaraIn dynamic leaching methods, portions of extractant reagents are continuously provided to the solid sample contained in flow-through microcolumns or chambers, enabling the renewal of extracting fluid and avoiding saturation effects from fluid stagnation. These methods are also suitable for fast measurements in real time with small extract manipulation, especially when coupled online with suitable detectors [1]. In this work, the bioaccessible fraction and kinetic leaching profile of zinc in pet food was determined using a robust flow-through device, composed by two filters placed in polypropylene holders to entrap the solid sample, designed for dynamic leaching experiments [2]. Continuous extraction flow was ensured by a peristaltic pump connecting the extraction reservoir and the extraction chamber, at a flow rate of 0.5 mL min-1. Synthetic fluids simulating digestive compartments were applied as extractants. The kinetic extraction profile of fast leachable Zn was evaluated by flame atomic absorption. Operational conditions, including filters’ composition and pore size, were tested. Preliminary results have shown that different extracting fluids (with and without digestive enzymes) had an influence on the total amount and on the leaching kinetic profile of Zn. In fact, higher values were obtained when enzymes were present in the extracting fluids. The proposed dynamic leaching method was suitable for evaluation of bioaccessible Zn in pet food. This information will be applied for the improvement of Zn supplementation in dog foods and for designing new products with enhanced mineral delivery.
- Evaluation of enzymatic digestion conditions for determination of immunoglobulins by tandem mass spectrometryPublication . Guerra, Gabriela S.; Ramos, Inês I.; Barreiros, Luísa; Silva, Eduarda M. P.; Segundo, Marcela A.Immunoassays, namely ELISA, have been the standard method for detecting clinically significant immunoglobulins (Igs). They are based on Ig-antigen interaction, often suffering interference from matrix components. New analytical approaches using detection by tandem mass spectrometry (MS/MS) search for fundamental structure information of target Igs based on protein features. In fact, there are few examples of quantitative assays achieved by liquid chromatography coupled with triple quadrupole (QqQ) mass analyzers. Due to the limited mass range of QqQ, the use of this mass analyzer requires previous tryptic digestion of IgG for analysis of highly specific surrogate peptides. In this work, initial studies on a LC-MS/MS method for the quantitative analysis of IgG are reported. The method relies upon the detection of the generic peptide DTLMISR (Fig. 1), originated from the fraction crystallizable (Fc) region of IgG after enzymatic cleavage. The multiple reaction monitoring transitions used for quantification and identification purposes were, respectively, m/z 418.20 506.10 and 418.20 619.30, corresponding to the fragmentation of double-charged molecular ions. In order to investigate the influence of trypsin concentration on digestion kinetics and efficiency, the trypsin-to-protein ratios 1:20, 1:50 and 1:100 were evaluated. Moreover, the performance of the digestion process was monitored for IgG standards and plasma samples over 18 h at 37 °C. Using a 1:50 ratio, two distinct kinetic profiles were observed for standards and plasma samples with a maximum signal intensity after 6 and 18 h, respectively.
- Determination of Vitamin C content in spinach plants grown under the use of cyanobacteria biomass as fertilizerPublication . Martins, Diogo; Massa, Anabella; Reimão, Mariana; Vasconcelos, Vítor; Campos, Alexandre; Pinto, Edgar; Freitas, Marisa; REIMÃO BORGES LOPES DA SILVA, MARIANA; Pinto, Edgar; Freitas, MarisaThe agricultural sector is continuously exploring sustainable and innovative approaches to enhance crop productivity, driving the quest for greener alternatives over synthetic fertilizers. Cyanobacteria have been reported as a promising agricultural fertilizer, with positive effects on crop yield. However, little is known about its potential effects on the produced vegetables' nutritional quality. Studies have documented that exposure of plants to cyanobacterial crude extracts stimulates the production of enzymatic and non-enzymatic antioxidants. Vitamin C, a non-enzymatic antioxidant, plays a crucial role in protecting cells from oxidative stress and spinach is known to be relatively high in vitamin C compared to other vegetables. The purpose of this study was to determine the content of vitamin C in spinach plants grown under the use of cyanobacterial biomass as fertilizer. Spinach plants were tested in soil, under six different conditions: (i) control, (ii) commercial chemical fertilizer, cyanobacterial biomass (iii) non-cyanotoxin producer, and cyanotoxin producer of (iv) Anatoxina (ANA), (v) Microcystin (MC), and (vi) Cylindrospermopsin (CYN). The analysis of vitamin C in spinach plants was performed according to EN 14130:2003. The results showed an increased content of vitamin C in all exposed groups when compared to the control (37 mg/Kg), with statistical differences in the conditions exposed to MC (56 mg/kg), ANA (76 mg/kg), and CYN (114 mg/kg). The results suggest that the rise in vitamin C may be related to a plant defence mechanism against oxidative stress potentially generated by exposure to cyanotoxins. Although the antioxidant content in spinach plants can be enhanced as a mechanism to cope with oxidative stress, prolonged exposure may increase plants' susceptibility to accumulate cyanotoxins, which may pose a risk to human health. Additional studies are needed to understand all the positive and negative effects of using cyanobacteria biomass as a biofertilizer.
- Segurança e eficácia do uso de inibidores da PARP no tratamento do cancro do ovário e mama metastizadoPublication . Jesus, Ângelo; Pinto, Rafaela; Jesus, ÂngeloO cancro da mama é um dos cancros mais comuns na mulher. O cancro do ovário, embora tenha uma taxa de incidência relativamente baixa, quando é detetado está geralmente numa fase muito avançada, o que resulta num mau prognóstico e, consequentemente, numa elevada taxa de mortalidade. Estes cancros têm uma forte componente genética de onde resulta uma perda da proteína funcional através da linha germinal ou mutação somática, levando a uma maior dependência de mecanismos de reparação de DNA mais propensos a erros, promovendo a carcinogénese. Os PARPies são uma classe de agentes de supressão da atividade das enzimas PARP. Objetivo: Analisar a segurança e eficácia dos inibidores PARP no tratamento do cancro do ovário e mama metastático, possuindo como medidas as reações adversas major a PFS.
- Unveiling the geographical influence on honey's physicochemical profile: a case study from Montesinho Natural ParkPublication . Soares, Sónia; Magalhães, Leandro; Moreira, Manuela M.; Rede, Diana; Fernandes, Virgínia Cruz; Viegas, Olga; Pinto, Edgar; Almeida, Agostinho; Azevedo, Rui; Delerue-Matos, CristinaIn recent years, global honey consumption has significantly increased due to the rising demand for natural products with health benefits. Consumers are particularly drawn to honey recognized for its superior quality, such as Protected Designation of Origin (PDO) honey or honey produced in protected areas, which is associated with distinctive characteristics. Portugal stands among the top producers of PDO honey, with Montesinho Natural Park (MNP) being a notable example of high-quality Portuguese honey. Despite its reputation, environmental challenges threaten both honey production and quality in this region. This study aims to evaluate the physicochemical parameters and nutritional characteristics of honey from MNP and investigate the correlation between these properties and the geographical location of the apiaries. Honey samples (n=13) were collected from local producers and supermarkets. Standard physicochemical parameters, including 5-hydroxymethylfurfural (HMF), diastase activity, moisture and ash content, free acidity, electrical conductivity, and pH, were analysed following honey legislation guidelines. Additional parameters, such as colour, protein content, low-molecular-weight carbohydrates, and mineral composition, were also assessed. The findings indicate that all honey samples complied with legal standards and align with previously published data, showing a high degree of homogeneity across the physicochemical properties analysed. Principal Component Analysis (PCA) revealed that parameters such as colour, HMF, and specific minerals (Mg, Ca, K, Mn, Sr, Cu, Ba) contributed to the clustering of honey samples, suggesting that geographical variations among apiary locations might influence these properties. However, these differences do not affect the overall quality of the PDO honey produced in MNP. This study represents the first comprehensive characterization of honey from different apiaries within MNP. While the results demonstrate the consistency and quality of MNP honey, further research is necessary to explore the relationship between geographic origin and quality under varying environmental conditions. These insights could be valuable for maintaining sustainable beekeeping practices in the region and preserving the unique attributes of MNP honey.
- Challenging invasive fungal infections: development of innovative electrochemical nanogenosensors to detect Candida spp.Publication . Castanheira, Michelle; Morais, Stephanie L.; Seguro, Isabel; Santos, Marlene; Lima, Luís; Pacheco, João; Barroso, M. FátimaDespite the considerable advances in the prevention and treatment of fungal infections, invasive fungus such as Candida spp., continues to be one of the major causes of morbidity and mortality. The Global Action Fund Infections reported that, annually, more than 300 million people are infected with fungal infection, from these, about 1.5 million ends up dying. Candida albicans is the most important fungal 66 opportunistic pathogen, it can cause superficial or invasive infections. Candida, often, causes superficial infections, per example in skin or mucous membranes with simple and effective treatment, however, also can break to the bloodstream and disseminate to internal organs. It has been observed among high-risk patients such as allogeneic stem-cell transplant recipients and with acute leukemia receiving highdose chemotherapy. These patients are at a heightened risk of developing infections due to the suppression of their immune system during the transplantation process. The diagnosis of systemic fungal infections persists as a problematic issue. Therefore, the development of more efficient, sensitive and specific methods for early diagnosis is need. In this study, an easy, rapid, and accurate detection methods for fungal infections in patients undergoing hematopoietic stem cell transplantation (HSCT) was designed. To address this challenge, it was developed an electrochemical nanogenosensor for the detection of Candida albicans.This nanogenosensor was assembled in an innovative low-cost electrochemical paper based analytical devices (ePAD). A sandwich hybridization reaction was used to enhaced the sensitivity of the electrochemical signal. Preliminary results demonstrated that using this nanogenosensors it was possible to detect Candida spp., in synthetic fungus sample. Despite these results, the optimization of the nanogenosensor in terms of quantifying Candida albicans is being carried out, which will be validated in future studies.The applicability in hospital environment relatively to sensitivity, accuracy, quickness response, challenges and opportunities will be discuss in future developments.
- Association between aluminium levels and cognitive decline in environmentally exposed eldersPublication . Gerardo, Bianca; Pinto, Marina Cabral; Nogueira, Joana; Almeida, Agostinho; Pinto, Edgar; Simões, Mário Rodrigues; Freitas, SandraNormal brain functioning depends on metals homeostasis, as imbalances may increase oxidative stress and cause other detrimental intracellular events that lead to cell loss. Furthermore, environmental exposure to toxic metals is hypothesized to induce neuroinflammation and neuropathology, paving the way to neurodegeneration. The aim of this study was to examine whether such Potentially Toxic Elements (PTE) are associated with cognitive performance over time in a sample of elderly individuals of environmentally contaminated regions. A total of 146 elders permanently residing in environmental risk areas were recruited. Participants completed the Mini Mental State Examination (MMSE) for cognitive assessment and were biomonitored for several PTE through hair and urine analysis. After 5 years, the cognitive assessment was repeated. Cognitive performance significantly declined from baseline to follow-up (p < 0.001), with participants scoring on average 4.41 less points on MMSE (Baseline: M = 25.79, SD = 3.520, Md = 27.00; Follow-up: M = 21.38, SD = 5.343, Md = 21.00). Regression analysis on the relationship between PTE and cognitive performance revealed that Aluminium (β = -0.322, p = 0.32) and Zinc (β = -0.372, p = 0.15) in hair and Aluminium in urine (β = -0.368, p = 0.010) significantly predicted MMSE scores past 5 years of biomonitoring (p = 0.002). Higher contents of these PTE were associated with worse cognitive performance at follow-up, after controlling the effects of age, education and supplement intake. Higher levels of Aluminium significantly predict worse general cognitive performance 5 years after biomonitoring across different biological samples. Environmental exposure to this metal may constitute a risk factor for cognitive decline.
- Bioaccessibility and changes on cylindrospermopsin concentration in edible mussels over storage and processing time.Publication . Freitas, Marisa; Azevedo, Joana; Carvalho, António Paulo; Mendes, Vera; Manadas, Bruno; Campos, Alexandre; Vasconcelos, VítorThe cyanotoxin cylindrospermopsin has been recognized of increased concern due to the global expansion of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that aquatic organisms, especially bivalves, can accumulate high levels of cylindrospermopsin. Based on the potential for human health risks, a provisional tolerable daily intake of 0.03 µg/kg body-weight has been recommended. However, human exposure assessment has been based on the cylindrospermopsin concentration in raw food items. This study aimed to assess the changes on cylindrospermopsin concentration in edible mussels over storage and processing time as well as cylindrospermopsin bioaccessibility. Mussels, (Mytilus galloprovincialis) fed cylindrospermopsin-producing C. raciborskii, were subjected to the treatments and then analyzed by LC-MS/MS. Mussels stored frozen allowed a significantly higher recovery of cylindrospermopsin (52.5%/48 h and 57.7%/one week). The cooking treatments did not produce significant differences in cylindrospermopsin concentration in mussel matrices (flesh), however, cylindrospermopsin was found in the cooking water, suggesting that heat processing can be used to reduce the availability of cylindrospermopsin in this food item. The in vitro digestion with salivary and gastrointestinal juices considerably decreased the cylindrospermopsin availability in uncooked and steamed mussels, highlighting the importance in integrating the bioaccessibility in the human health risk assessment.