Browsing by Author "Lima, Sofia A. C."
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- 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.
- Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in NanocarriersPublication . Marques, Sara S.; Ramos, Inês I.; Fernandes, Sara; Barreiros, Luisa; Lima, Sofia A. C.; Reis, Salette; Domingues, M. Rosário M.; Segundo, Marcela A.The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.
- 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.