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.
- 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.