Percorrer por autor "Fernandes, Sara R."
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- Acetonitrile adducts of tranexamic acid as sensitive ions for quantification at residue levels in human plasma by UHPLC-MS/MSPublication . Silva, Eduarda M. P.; Barreiros, Luísa; Fernandes, Sara R.; Sá, Paula; Ramalho, João P. Prates; Segundo, Marcela A.The quantitative analysis of pharmaceuticals in biomatrices by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is often hampered by adduct formation. The use of the molecular ion resulting from solvent adducts for quantification is uncommon, even if formed in high abundance. In this work, we propose the use of a protonated acetonitrile adduct for the quantitative analysis of tranexamic acid (TXA) by LC-MS/MS. The high abundance of the protonated acetonitrile adduct [M + ACN + H]+ was found to be independent of source-dependent parameters and mobile phase composition. The results obtained for TXA analysis in clinical samples were comparable for both [M + ACN + H]+ and [M + H]+ , and no statistically significant differences were observed. The relative stability and structure of the [M + ACN + H]+ ions were also studied by analyzing probable structures from an energetic point of view and by quantum chemical calculations. These findings, and the studied fragmentation pathways, allowed the definition of an acetimidium structure as the best ion to describe the observed acetonitrile protonated adduct of TXA.
- 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.
- ANTI-VEGF therapies in neovascular age-related macular degeneration: a scoping review of functional and morphological outcomes in older adultsPublication . Queirós, Marta; Teixeira, Daniela; Jesus, Ângelo; Santos, Marlene; Moreira, Fernando; Fernandes, Sara R.; Santos, Marlene; Moreira, Fernando; Fernandes, SaraAge-related macular degeneration (AMD) is one of the leading causes of blindness in individuals over 60 years of age and is classified into two main subtypes: geographic atrophy (dry AMD) and neovascular AMD (wet AMD). Antivascular endothelial growth factor (anti-VEGF) agents are the standard treatment for neovascular AMD, although guidelines diverge regarding first-line choices. This scoping review aims to identify and characterize current scientific evidence on the use of anti-VEGF agents in the treatment of neovascular AMD. A systematic search was conducted in PubMed for observational or experimental studies published between 2020 and April 2025. Eligible studies included individuals aged ⩾60 years, reported on the use of anti-VEGF therapy for neovascular AMD, and assessed both functional (visual acuity) and morphological outcomes. Sixty-five studies met the inclusion criteria – 56 observational studies (prospective cohorts and cross-sectional designs), and nine clinical trials. Aflibercept (34%), faricimab (18%), and ranibizumab (15%) were the most commonly agents. Ranibizumab and aflibercept consistently improved visual acuity, with average gains of 7–15 ETDRS letters. Bevacizumab yielded more variable outcomes. In refractory patients, brolucizumab or faricimab showed anatomical improvements; however, brolucizumab was associated to higher risk of intraocular inflammation. The findings suggest that clinical and anatomical factors, such as persistent subretinal fluid, central retinal thickness, and initial treatment response, may help guide a more individualised selection of anti-VEGF. While ranibizumab and aflibercept remain well-established options, faricimab shows potential, in patients with suboptimal response to conventional agents.
- 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.
- Determination of tranexamic acid in human plasma by UHPLC coupled with tandem mass spectrometry targeting sub-microgram per milliliter levelsPublication . Barreiros, Luisa; Amoreira, Júlia L.; Machado, Sandia; Fernandes, Sara R.; Silva, Eduarda M.P.; Sá, Paula; Kietaibl, Sibylle; Segundo, Marcela A.Tranexamic acid (TXA) is an antifibrinolytic drug, with the ability to inhibit lysine binding at plasminogen receptors, used in adult trauma patients with on-going or at risk of significant haemorrhage. To understand the pharmacokinetics and pharmacodynamics of this drug in variable age groups undergoing surgeries with high blood loss, effective methods for determination of TXA in biological samples at sub-μg mL−1 are still required. We describe herein the development and validation of a method based on ultra-high performance liquid chromatography coupled to triple quadrupole-tandem mass spectrometry to quantify TXA in human plasma. An inexpensive, simple and efficient sample clean-up was implemented, not requiring matrix-matching calibration. Sample preparation consisted in protein precipitation using acetonitrile containing 0.5% (v/v) formic acid, followed by hydrophilic interaction based chromatographic separation, with elution in isocratic mode using a combination of acetonitrile and water (75:25, v/v), with quantification of TXA based on selected reaction monitoring. Good linearity was achieved (r2 > 0.997) for TXA concentrations ranging from 30 to 600 ng mL−1, with LOD of 18 ng mL−1 in plasma. The developed method proved to be selective, sensitive, accurate (96.4–105.7% of nominal values) and precise (RSD ≤ 4.5%). TXA was found to be stable in plasma extracts standing 24 h at room temperature (20 °C) or in the autosampler, and after three freeze-thawing cycles. Mean recovery values of TXA spiked plasma samples were ≥91.9%. No significant matrix effects were observed. The proposed methodology was successfully applied to the clinical study of plasma samples recovered during scoliosis surgery of pediatric patients pretreatment with TXA.
- E-Health strategies to promote treatment adherence in asthma and COPD: A scoping reviewPublication . Almeida, Helena; Gonçalves, Olívia; Oliveira, Vânia; Fernandes, Sara R.; Santos, Marlene; Moreira, Fernando; Fernandes, Sara; Santos, Marlene; Moreira, FernandoThe control of asthma and chronic obstructive pulmonary disease (COPD) depends on adherence to treatment, often administered via inhalation devices [1]. The digitalisation of healthcare has been suggested as a viable strategy to promote treatment adherence [2,3]. This scoping review aimed to identify eHealth strategies promoting adherence to asthma and COPD treatment and to map the methodologies adopted in studies addressing this topic. A systematic search was conducted in PubMed and ScienceDirect to identify studies reporting the use of digital interventions applied to patients with asthma or COPD. Experimental and observational studies published between 2015 and 2025, in English or Portuguese, were included. A total of 61 studies were identified, describing digital strategies with variable technology, interactivity, and integration with face-to-face healthcare services – 10 addressing the treatment of COPD, 47 for asthma treatment and four for both asthma and COPD. Most studies focused on mobile applications (n=31), followed by smart digital inhalers (n=13) and web-based platforms (n=12). Regarding outcomes, the assessment of the treatment adherence rates was performed in 85% of the studies, while 10% evaluated correct inhaler use. The heterogeneity of assessment methods (personalised feedback, automated reminders, and intake monitoring) limits direct comparison across studies. The use of digital strategies to enhance COPD and asthma treatment adherence has been consistently demonstrated. Effective integration of eHealth to improve treatment adherence in asthma and COPD requires personalized feedback, user-centered design, clinical follow-up, and equitable technology access.
- Flow-through 3D-printed device for automatic microsampling and handling of dried urine spotsPublication . Fernandes, Sara R.; Cunha, Diana R.; Guidetti, Federica; Barreiros, Luísa; Miró, Manuel; Segundo, Marcela A.; Barreiros, Luisa; Fernandes, Sara3D printing has revolutionized analytical chemistry by allowing the development of miniaturized and high- precision devices. In bioanalysis, sample collection and pre-treatment can be facilitated using 3D printing combined with flow analysis and mass spectrometry. Hence, a customized 3D-printed device was designed for sampling, clean-up, and target retrieval using filter paper as a sample collection material and sorbent. This device was integrated into a flow network for fully automatic extraction and applied to detect three anticoagulants in human urine. Different printing materials, designs and other factors related to fused deposition modeling 3D printing such as the infill percentage were evaluated to achieve the configuration that allowed the implementation of sampling and separation procedures in the same device. After establishing the final design of the device, several parameters such as the eluent composition and the hydrodynamic conditions were studied to enhance the recovery of the target analytes, namely apixaban, rivaroxaban, and warfarin. The processed samples were analyzed by UHPLC-MS/MS in positive ionization mode using a BEH C18 column. The method demonstrated good linearity (r2 >0.998) for quantification of the target analytes at concentrations ranging from 0.20 to 20 μg L 1 for apixaban and warfarin, and from 0.50 to 20 μg L 1 for rivaroxaban. LOD and LOQ values of 0.06–0.2 μg L 1 and 0.1–0.5 μg L 1, respectively, for undiluted urine were obtained. The method was found to be accurate (97.0–102 %) and precise (CV ≤6.0 %). This new approach, according to the scores obtained by applying the AGREEprep (0.70), AGREE (0.65) and BAGI (70) metrics, can be described as environmentally friendly, practical and suitable for bioanalytical purposes.
- 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.
- 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%).
- Sample preparation and chromatographic methods for the determination of protein-bound uremic retention solutes in human biological samples: An overviewPublication . Fernandes, Sara R.; Meireles, Andreia N.; Marques, Sara S.; Silva, Luís; Barreiros, Luisa; Sampaio-Maia, Benedita; Miro, Manuel; Segundo, Marcela A.Protein-bound uremic retention solutes, such as indole-3-acetic acid, indoxyl sulfate, p-cresol and p-cresol sulfate, are associated with the development of several pathologies, namely renal, cardiovascular, and bone toxicities, due to their potential accumulation in the human body, thus requiring analytical methods for monitoring and evaluation. The present review addresses conventional and advanced sample treatment procedures for sample handling and the chromatographic analytical methods developed for quantification of these compounds in different biological fluids, with particular focus on plasma, serum, and urine. The sample preparation and chromatographic methods coupled to different detection systems are critically discussed, focusing on the different steps involved for sample treatment, namely elimination of interfering compounds present in the sample matrix, and the evaluation of their environmental impact through the AGREEprep tool. There is a clear trend for the application of liquid-chromatography coupled to tandem mass spectrometry, which requires protein precipitation, solid-phase extraction and/or dilution prior to analysis of biological samples. Furthermore, from a sustainability point of view, miniaturized methods resorting to microplate devices are highly recommended.