Browsing by Author "Silva, Eduarda M. P."
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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.
- 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.
- 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.
- 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.
- Exploiting kinetic features of ORAC assay for evaluation of radical scavenging capacityPublication . Carvalho, Joana R. B.; Meireles, Andreia N.; Marques, Sara S.; Gregório, Bruno J. R.; Ramos, Inês I.; Silva, Eduarda M. P.; Barreiros, Luisa; Segundo, Marcela A.The analysis and interpretation of data retrieved from Oxygen Radical Absorbance Capacity (ORAC) assays represent a challenging task. ORAC indexes originate from different mathematical approaches often lacking correct elucidation of kinetic features concerning radical scavenging reactions by antioxidant compounds. In this work, the expression of ORAC values as area under fluorescein (FL) decay curves (AUC) and lag time are critically compared. This multi-parametric analysis showed the extension of radical scavenging reactions beyond the lag time period for caffeic acid, gallic acid, reduced glutathione and quercetin, extending their antioxidant protection of FL. Ethanol delayed the reaction of both FL and antioxidant compounds with free radical species generated from 2,20 -azobis(2-amidinopropane) dihydrochloride thermolysis. Trolox equivalent values, commonly used to express ORAC values, were more affected by the differences in radical scavenging kinetics between the reference and the tested antioxidant compounds when calculated from AUC than from lag time. These findings stressed the importance of choosing calibrator compounds presenting ORAC kinetics similar to samples to prevent biased estimation of the antioxidant capacity. Additionally, the framework proposed here provides a sustainable analytical method for the evaluation of antioxidant capacity, with an AGREE score of 0.73.
- 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%).
- 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.