Browsing by Author "Machado, Sandia"
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- 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.
- 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.
- A data mining tool for untargeted biomarkers analysis: Grapes ripening applicationPublication . Machado, Sandia; Barreiros, Luisa; Graça, António R.; Páscoa, Ricardo N.M.J.; Segundo, Marcela A.; Lopes, João A.In metabolomics, data generated by untargeted approaches can be very complex due to the typically extensive number of features in raw data (with and without chemical relevance), dependence on raw data preprocessing methods, and lack of selective data mining tools to appropriately interpret these data. Extraction of meaningful information from these data is still a significant challenge in metabolomics. Moreover, currently available tools may overprocess the data, eliminating useful information. This work aims at proposing a data mining tool capable of dealing with metabolomics data, specifically liquid chromatography-mass spectrometry (LC-MS) to enhance the extraction of meaningful chemical information. The algorithm construction intended to be as general as possible in highlighting chemically relevant features, discarding non-informative signals specially background features. The proposed algorithm was applied to an LC-MS data set generated from the analysis of grapes collected over a developmental period encompassing a 4-month period. The algorithm outcome is a short list of features from metabolites that are worth to be further investigated, for example by HRMS fragmentation for subsequent identification. The performance of the algorithm in estimating potentially interesting features was compared with the commercial MZmine software. For this case study, the MZmine output yielded a final set of 37 features (out of 1543 initially identified) with noise features while the proposed algorithm identified 99 systematic features without noise. Also, the algorithm required 2 times less user-defined parameters when compared to MZmine. Globally, the proposed algorithm demonstrated a higher ability to pin-point features that may be associated with grapes developmental and maturation processes requiring minimal parameters definition, thus preventing user uncertainty and the compromise of experimental information.
- 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.
- Fast monolith-based chromatographic method for determination of methotrexate in drug delivery studiesPublication . Barbosa, Ana Isabel; Fernandes, Sara; Machado, Sandia; Sousa, Patrícia; Sze, Ong Yong; Silva, Eduarda M.P.; Barreiros, Luisa; Lima, Sofia A.C.; Reis, Salette; Segundo, Marcela A.Methotrexate (MTX) is a derivative of aminopterin, used as an anticancer or an anti-inflammatory agent. The development of suitable drug delivery systems containing MTX is an active area of research, requiring suitable analytical methods. Therefore, a high-throughput HPLC method is proposed for determination of MTX in the delivery system and permeation studies. Chromatographic separation was achieved on a reversed phase monolithic C18 column using isocratic elution (phosphate buffer (pH 7.0, 10 mM)-ACN (91:9, v/v)) and spectrophotometric detection at 302 nm. Total run time was 3.5 min, with MTX retention time of 2.1 min, providing 17 determinations per hour. The method was found to be specific, accurate (99.2–110%) and precise for intra-day (RSD ≤ 3.5%) and inter-day assays (RSD ≤ 3.4%). MTX showed stability after 24 h at room temperature or in the autosampler (4 °C) and over three freeze-thaw cycles with recoveries ≥94.2%. The validated method was successfully applied to establish in vitro drug release profile of MTX delivered by lipid nanoparticles. Application to pig skin permeation media provided mean recovery values ranging from 94.1 to 101.6% (RSD ≤ 1.1%).
- Searching for the most variable m/z values in grape development in a Portuguese vineyardPublication . Machado, Sandia; Barreiros, Luisa; Graça, António R.; Páscoa, Ricardo N. M. J.; Lopes, João A.; Segundo, Marcela A.Each vineyard is known to have a strong impact on the metabolic compounds of grapes due to its external factors, named terroir [1]. Furthermore, knowledge on the metabolic behavior of vines in response to the terroir effect can help to assess, in advance, the optimal maturity of grapes. The aim of this work was to obtain a metabolic profile of vines in different locations and consequently associate it with the external conditions present during grapes’ development using an untargeted approach. Samples were collected in eight sites of a Portuguese vineyard during different developmental stages and analyzed using a metabolomic protocol based on liquid chromatography coupled to tandem mass spectrometry [2]. Briefly, samples were grounded and extracted using a mixture of water/methanol/chloroform (20:40:40, v/v/v). The aqueous methanol fraction was used for further analysis. An Agilent Eclipse plus C18 column (RRHD 1.8 µm, 2.1 mm × 100 mm) was used for chromatographic separation and elution was achieved in gradient mode. Water and acetonitrile both containing 0.1% (v/v) formic acid were used as mobile phase. Mass spectrometry analysis was performed in positive and negative ionization mode and data were acquired in scan mode to maximize the number of detected m/z values. MZmine software was chosen for data analysis due to its robustness in fragment selection. A baseline correction was applied to equalize baselines and an alignment algorithm was used to equalize retention times aiming to compare m/z values from different samples. Statistical and chemometric tools were used to exclude m/z values attributed to blanks and to establish a metabolic profile, respectively. Preliminary results confirm that the methodology chosen for data analysis is fast and accurate for the viable selection of the most significant m/z features. Regarding currently analyzed samples, the proposed methodology allowed the identification of several m/z features presenting a statistically significant variation among sampling points, which will be further investigated as indicators of the maturity state.