Browsing by Author "Segundo, Marcela A."
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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.
- Analysis of 17- β -estradiol and 17- α -ethinylestradiol in biological and environmental matrices — A reviewPublication . Barreiros, Luisa; Queiroz, Joana F.; Magalhães, Luís M.; Silva, Adrián M.T.; Segundo, Marcela A.The estrogens 17-β-estradiol (E2) and 17-α-ethinylestradiol (EE2) are reported as highly endocrine-disrupting agents, being recently included in an EU watch list regarding emerging aquatic pollutants. Therefore, the monitoring of these chemicals in the different environmental compartments assumes great importance. Moreover, due to the possible adverse effects on living beings, their occurrence on animal tissues and fluids must also be addressed. In recent years, a significant number of studies have described and proposed different analytical methodologies to detect and/or quantify E2 and EE2 mostly in environmental aqueous samples, including sludge and sediments and also in biological matrices such as plasma and tissues. Taking into account the complexity of real matrices and that both estrogens are generally present at trace levels, the development of accurate and reliable techniques for their determination can be quite a challenge. The present review aims at describing the main characteristics of the analytical methods recently used for E2 and EE2 determination in environmental and biological samples. The steps for sample preparation such as analytes extraction, preconcentration and clean-up are discussed and the instrumental based analytical techniques are compared. Furthermore, the application of biological tools to determine the total estrogenicity of environmental samples, as well as their potential combination with instrumental analyses, is highlighted.
- Analytical methods for quantification of tranexamic acid in biological fluids: A reviewPublication . Silva, Eduarda M.P.; Barreiros, Luisa; Sá, Paula; Afonso, Carlos; Kozek-Langenecker, Sibylle; Segundo, Marcela A.Tranexamic acid (TXA) is a synthetic derivative of the amino acid lysine with antifibrinolytic properties. There is still a lack of pharmacokinetic and pharmacodynamic data concerning variable age groups undergoing surgeries with high blood loss. The optimum dose and administration schedules of TXA are still subject of research, aiming at a safe inhibition of fibrinolysis in the perioperative period. Hence, effective methods for determination of TXA in biological samples are needed. The aim of this review is to discuss the required sample treatment procedures and the analytical methods applied for quantification of TXA, focusing on selected derivatisation agents and internal standards. Methods comprising a separative step (GC, LC or CZE) coupled to spectrophotometric, fluorimetric and mass spectrometry detection were considered, showing a tendency for implementation of MS/MS methods in more recent reports. Detection limits ranging from 0.01 to 0.5 μg mL− 1 in blood plasma were so far attained using LC-MS/MS.
- 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.
- Assessing the differences of two vineyards soils’ by NIR spectroscopy and chemometricsPublication . Machado, Sandia; Barreiros, Luísa; Graça, António R.; Madeira, Manuel; Páscoa, Ricardo N. M. J.; Segundo, Marcela A.; Lopes, João A.Soil properties influence greatly the status of vine plants which consequently influences the quality of wine. Therefore, in the context of viticulture management, it is extremely important to assess the physical and chemical parameters of vineyards soils. In this study, the soils of two vineyards were analysed by near-infrared (NIR) spectroscopy and established analytical reference procedures. The main objective of this study was to verify if NIR spectroscopy is a potential tool to discriminate the soils of both vineyards as well as to quantify differences of soil’s parameters. For that, a total of eight sampling spots were selected at each vineyard taking into consideration the soil type and sampled at different depths. The data analysis was performed using analysis of variance (ANOVA), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) and partial least squares (PLS) regression. The ANOVA results revealed that 12 out of the 18 parameters analysed through the reference procedures can be considered statistically different (p < 0.05). Regarding PCA, the obtained results revealed a clear separation between the scores of both vineyards either considering NIR spectra or the chemical parameters. The PLS-DA model was able to obtain 100 % of correct predictions for the discrimination of both vineyards. PLS regression analysis using NIR spectra revealed R2 P and RER values higher than 0.85 and 10, respectively, for 8 (pH (H2O), N, Ca2+, Mg2+, SB, CEC, ECEC and GSB) of the 18 chemical parameters evaluated. Concluding, these results demonstrate that it is possible to discriminate the soils of the different vineyards through NIR spectroscopy as well as to quantify several chemical parameters through soils NIR spectra in a rapid, accurate, cost-effective, simple and environmentally friendly way when compared to the reference procedures.
- Assessment of immunoglobulin capture in immobilized protein A through automatic bead injectionPublication . Ramos, Inês I.; Marques, Sara S.; Magalhães, Luís M.; Barreiros, Luisa; Reis, Salette; Lima, José L.F. C.; Segundo, Marcela A.The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 μg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 μL) from serum and saliva.
- Automatic and renewable micro-solid-phase extraction based on bead injection lab-on-valve system for determination of tranexamic acid in urine by UHPLC coupled with tandem mass spectrometryPublication . Fernandes, Sara; Barreiros, Luísa; Sá, Paula; Miró, Manuel; Segundo, Marcela A.An automatic micro-solid-phase extraction (μSPE) method using on-line renewable sorbent beads followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was established for the determination of tranexamic acid (TXA) in urine. The μSPE method was based on the bead injection (BI) concept combined with the mesofluidic lab-on-valve (LOV) platform. All steps of the μSPE–BI–LOV were implemented by computer programming, rendering enhanced precision on time and flow events. Several parameters, including the type of sorbent, volume and composition of the conditioning solution, washing solution, and eluent composition, were evaluated to improve the extraction efficiency. The best results were obtained with a hydrophilic–lipophilic balanced mixed-mode sorbent, decorated with sulfonic acid groups (Oasis MCX), and 99% acetonitrile–water (50:50, v/v)–1% ammonium hydroxide as eluent. Chromatographic separation was performed using a BEH amide column coupled to MS/MS detection in positive ionization mode. Good linearity was achieved (R2 > 0.998) for TXA concentrations in urine ranging from 300 to 3000 ng mL−1, with LOD and LOQ of 30 and 65 ng mL−1, respectively. Dilution integrity was observed for dilution factors up to 20,000 times, providing the extension of the upper limit of quantification to 12 mg mL−1. The method was validated according to international guidelines and successfully applied to urine samples collected during scoliosis surgery of pediatric patients treated with TXA.
- Automatic solid-phase extraction by programmable flow injection coupled to chromatographic fluorimetric determination of fluoroquinolonesPublication . Peixoto, Patricia S.; Silva, Eduarda M. P.; Osório, Marcelo V.; Barreiros, Luisa; Lima, José L. F. C.; Segundo, Marcela A.Fluoroquinolones are broad-spectrum bactericidal agents applied for the treatment of human and veterinary diseases. Their common use and their incorrect disposal foster environmental contamination, namely in water resources, increasing the risk of antimicrobial resistance. Hence, a method based on automatic solid-phase extraction coupled to liquid chromatography and fluorimetric detection is proposed for the determination of fluoroquinolones in environmental waters. For the solid-phase extraction procedure, a commercially available molecularly imprinted polymer targeting fluoroquinolones was trapped inside a flow-through extraction column, integrated into a programmable flow injection system using multisyringe flow injection analysis, where all steps concerning sorbent conditioning, sample loading, matrix removal, and analyte elution were performed under computer control. The eluate resulting from the sample preparation was collected and transferred at-line to chromatographic analysis using a reversed-phase monolithic column coupled to a fluorimetric detector, and isocratic elution with methanol-phosphoric acid (pH 3.0; 5.0 mM) (17.5:82.5, v/v) at a flow rate of 3.5 mL min-1. Sample treatment and chromatographic analysis were performed in tandem, with sample throughput limited by the sample treatment step. Calibration curves based on fluorescence intensity vs. analyte mass were obtained in the range of 10 to 1000 pg for norfloxacin, ciprofloxacin, and enrofloxacin with LOD values of 6-19 ng L-1 for a sample volume of 100 mL, and RSD < 11% at 0.7 ¿g L-1. The method was successfully applied to estuarine river water analysis.
- 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.
- Chromatographic method for the simultaneous quantification of dapsone and clofazimine in nanoformulationsPublication . Machado, Sandia; Fernandes, Sara; Chaves, Luise L.; Lima, Sofia A. C.; Silva, Eduarda M. P.; Barreiros, Luisa; Reis, Salette; Segundo, Marcela A.The low bioavailability and nonspecific distribution of dapsone and clofazimine, commonly applied in combination for the treatment of leprosy, can produce toxic effects. Nanotechnological approaches enhance the delivery of these drugs. Therefore, a high-performance liquid chromatography method was developed for the simultaneous determination of dapsone and clofazimine loaded in nanoformulations for quality control purposes. Chromatographic separation was achieved on a reversed-phase Kinetex core-shell C18 column, followed by spectrophotometric detection at 280 nm. Considering the different physicochemical properties of dapsone and clofazimine, elution was performed in gradient mode using an aqueous acetate buffer (50 mmol/L, pH 4.8) and an increasing acetonitrile content from 27 to 63% v/v at a flow rate of 1.0 mL/min with retention times of 6.2 and 14.0 min, respectively. The method was validated according to the European Medicines Agency guideline and it was found to be specific, accurate (99.6-114.0%), and precise for intra- (RSD ≤ 1.8%) and interday assays (RSD ≤ 12.5%). Both drugs showed stability after 24 h at room temperature and over three freeze-thaw cycles with recoveries ≥86.2%. Low temperature (4°C) in the autosampler caused the precipitation of clofazimine and must be avoided. The validated method was successfully applied in the quantification of both drugs in nanoformulations.
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