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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.
- HPLC-MS/MS method for quantification of the neuropeptide Y Y1 receptor antagonist BIBP 3226 in cell extractsPublication . Barreiros, Luisa; Silva, Eduarda M. P.; Alencastre, Inês S.; Lamghari, Meriem; Segundo, Marcela A.; Barreiros, LuisaNeuropeptide Y (NPY) is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. NPY activates different receptors in several brain regions. Recently, Y1 receptor (Y1R) has arisen as a potential regulator in the local control of bone turnover suggesting that an antireceptor strategy may be a useful therapeutic approach to prevent and/or reverse bone loss. BIBP 3226 is a potent Y1R selective antagonist that has been successfully used in in vitro studies showing a positive impact in bone turnover and thus providing good perspectives towards its application as a pharmacological tool for bone regeneration. Hence, the major aim of the present work was to implement a method based on high performance liquid chromatography coupled to triple quadrupole-tandem mass spectrometry for quantification of BIBP 3226 in cellular internalization assays. Chromatographic separation was achieved using a reversed phase Kinetex coreshell C8 column at 30 C and elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25 mL min-1. Total run time was 5.0 min, with retention time of 3.7 min for the target compound. The MS/MS was operated in positive ionization mode (ESI+) and data were acquired in multiple reaction monitoring (MRM) mode (m/z 474>167 for quantification and m/z 474>107 for identity confirmation). Calibration curves were linear for concentrations ranging from 0.5 to 30 ng mL-1. BIBP 3226 was quantified in cell extracts obtained from internalization assays performed with bone marrow and breast cancer cells, after solvent evaporation and resuspension in mobile phase. LOD and LOQ were 0.04 and 0.1 ng mL-1, respectively, corresponding to values as low as 0.3 and 0.8 pg per wel
- 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.
- Monitoring tranexamic acid in human urine by automatic solid-phase extraction combined with liquid chromatography-mass spectrometryPublication . Barreiros, Luisa; Barreiros, Luisa; Sá, P.; Miró, M.; Segundo, Marcela A.; Fernandes, Sara R.; Fernandes, SaraTranexamic acid (TXA) is an important antifibrinolytic agent in the treatment of different haemorrhagic conditions.1,2 However, the information about pharmacokinetics and pharmacodynamics is scarce. Therefore, the development of analytical methods for the quantification of TXA in different types of biological samples is required, because this information will be relevant in the establishment of adequate doses. TXA has been determined in different biological matrices but the quantification in urine samples assumes particular importance because urinary excretion is the main route of elimination.1,2 Sample preparation is a critical step in analytical procedures for the elimination of interfering compounds and also for analyte pre-concentration.2,3,4 Among the different sample preparation techniques, solid-phase extraction (SPE) is one the most versatile sample-processing methods and the automation of this strategy increases precision by reducing human intervention, sources of error and also analysis time and cost.3,4 Hence, the main goal of the present work was the development of an automated micro-solid-phase extraction (SPE) methodology using bead injection (BI) in a mesofluidic lab-on-valve (LOV) flow system combined to liquid chromatography and mass spectrometry for the determination of TXA in urine samples. For the µSPE-BI-LOV methodology, three sorbents were tested, namely OASIS-HLB, -MCX and -MAX, and different parameters were evaluated, including eluent and carrier composition, composition of matrix removal solution and sample loading volume. All steps of SPE were defined and implemented by computer programming. The processed samples were analysed using a method based on ultra-highperformance liquid chromatography coupled to triple quadrupole-tandem mass spectrometry (UHPLC-MS/MS).5 Chromatographic separation was achieved using a BEH Amide column (50 × 2.1 mm; 1.7 µm particle size), maintained at 40 °C. The mobile phase consisted of a mixture of acetonitrile-aqueous ammonium bicarbonate (pH 7.4; 10 mM), at a flow rate of 0.1 mL min−1. The MS was operated in positive ionization mode (ESI+) and data was acquired in selected reaction monitoring (SRM) mode (m/z 158.25 > 95.15 for quantification, and m/z 158.25 > 123.20 for identification). Firstly, studies were performed using TXA standards, in order to establish the optimal conditions for SPE. The results revealed that OASISHLB sorbent permitted to achieve higher recovery percentages (ca. 80%) and higher repeatability compared to the other tested sorbents, particularly OASIS-MCX. Consequently, OASIS-HLB was selected for the further experiments. The eluent composition and the sample loading volume were also studied, and the best results were obtained using a mixture of acetonitrile-aqueous ammonium bicarbonate (pH 7.4; 10 mM) (75:25, v/v) and 1000 µL of sample, respectively. Furthermore, the use of 0.1% (v/v) of formic acid as washing solution and solvent for sample preparation permitted to increase analyte recovery from 55% to 80%. The use of aqueous ammonium bicarbonate (pH 7.4; 10 mM) or water as carrier was also tested, and the obtained analyte recoveries were similar. The method is currently under development targeting the application to urine samples recovered during scoliosis surgery and the implementation of a strategy for hyphenation of the automated SPE system with mass spectrometry.
- Analytical strategies based on tandem mass spectrometry detection for quantification of bioactive compounds in biological matricesPublication . Barreiros, Luisa; Fernandes, Sara R.; Machado, Sandia; Silva, Eduarda M. P.; Segundo, Marcela A.Fast and accurate analysis, providing reliable results at trace concentration levels, is a current demand of the modern world. This pressure is justifiable in limit situations but also in our daily life, for instance when waiting for a diagnosis based on lab results in a hospital or when wondering about the quality of water running from our taps. During the last years, tandem mass spectrometry (MS/MS) based techniques have become the method of choice for determination of chemical compounds in complex matrices due to their inherent high sensitivity and selectivity. MS/MS techniques allow the achievement of low limits of detection and therefore prompt for the quantification of trace analyte levels generally present in environmental and biological samples. The majority of applications rely on the coupling to a separative technique prior to MS/MS detection. In this work, relevant applications of the association HPLC-MS/MS for quantification of bioactive compounds in biological matrices will be critically discussed. The steps of sample preparation and analytical determination will be addressed. Moreover, the main analytical features of each developed method, including selectivity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), stability and matrix effects will be highlighted. First, despite the recognition of tranexamic acid (TXA) as an important antifibrinolytic drug, there is a lack of pharmacokinetic and pharmacodynamic data concerning variable age groups undergoing surgeries with high blood loss. Clinical trials performed so far suggest a wide variability in response to TXA and, therefore, the implementation of a methodology based on UHPLC-MS/MS for monitoring TXA in human plasma samples at sub-microgram per milliliter levels was pursued.1 In a different context, millions of people worldwide live with human immunodeficiency virus (HIV) infection raising the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs such as tenofovir (TFV) and efavirenz (EFV). An HPLC-MS/MS method was developed targeting the quantification of antiretrovirals in mice tissue and fluid samples recovered from a pharmacokinetics study with nanoparticles and it was fully validated for the different biological matrices.2 Finally, BIBP 3226 is a potent and selective neuropeptide Y Y1 receptor antagonist that has been successfully used in in vitro studies showing a positive impact in bone turnover and thus providing good perspectives towards its application as a pharmacological tool for bone regeneration. Having in mind the therapeutic potential of BIBP 3226 and also the need to elucidate receptor-antagonist internalization mechanisms, the challenge was to develop a methodology based on HPLC-MS/MS that permitted to quantify the low quantities of antagonist expected to be internalized by cells.
- 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.
- Protocolo de estudo para adaptação de ferramenta certificada para avaliação dos procedimentos de manipulação de medicamentos citotóxicos nos Países Africanos de Língua Oficial Portuguesa (PALOP)Publication . Pereira, Cátia; Jesus, Ângelo; Moreira, Fernando; Moreira, Fernando; Jesus, ÂngeloNos países de baixo e médio rendimento (PBMR), os recursos são muitas vezes inadequados e existem escassas oportunidades de formação sobre manipulação segura de citotóxicos. Para que seja possível a melhoria das condições em que trabalham estes profissionais, é indispensável a realização de avaliação do cumprimento das diretrizes internacionais. O objetivo deste trabalho é desenvolver um protocolo que permita identificar e adaptar uma ferramenta para avaliação da adequação da preparação de citotóxicos, em países africanos de língua oficial portuguesa (PALOP) considerados PBMR.
- 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.
- Estudo prospetivo para programa de pacientes simuladosPublication . Macedo, Rui; Trigueiro, Maria João; Gonçalves, Maria João; Faias, Joaquim; Jesus, Ângelo; Noites, Andreia; Araújo, AndréA Simulação abrange meios muito diversificados incluindo entre outros, manequins, pacientes virtuais, jogos sérios, roleplaying, pacientes simulados. Por todo o mundo, cresce o número de instituições de ensino superior (IES) de saúde que adotam estes recursos, constituem unidades de simulação e criam programas de pacientes simulados (PS). Comum em medicina e enfermagem, PS tem pouca expressão noutras profissões de saúde, apesar do seu crescente uso no treino interprofissional. Desconhecem-se estudos sobre o uso seja da simulação senso lato ou de PS nessas profissões em Portugal. Inventariar estas atividades em cursos na área da saúde das IES Politécnicas públicas portuguesas (IESPPP) permitirá identificar boas práticas e facilitar a sua adoção.