Total analysis systems based on mesofluidics coupled to tandem mass spectrometry

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Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers

Publication . 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.

2020-04Journal article

Open access

Chemistry, bioactivities, extraction and analysis of azadirachtin: State-of-the-art

Publication . Fernandes, Sara; Barreiros, Luisa; Ferraz Oliveira, Rita; Cruz, Agostinho; Prudêncio, Cristina; Oliveira, Ana Isabel; Pinho, Cláudia; Santos, Nuno; Morgado, Joaquim

Azadirachta indica A. Juss. (Neem) is an Indian tree recognized for its activity as pesticide, as well as several pharmacological properties. Among the various compounds already isolated and studied from Neem tree, azadirachtin (AZA) was identified as the main bioactive compound. Azadirachtin can be found at different parts of the Neem plant but assumes its maximum concentration at the seed level. This compound features a quite complex chemical structure, which justifies the 20-plus-year difficulty to identify the synthetic pathway that subsequently permitted to carry out its artificial synthesis. Azadirachtin is widely used as a basis for production of biopesticides; nevertheless, other properties have been recognized for this substance, among which the anticancer and antimalarial activity stand out. The methods available for azadirachtin extraction are diverse, including solid-liquid extraction and extraction with solvents at high or low temperatures. Alcohol based solvents are associated with higher extraction yields and are therefore preferred for the isolation of azadirachtin from plant parts. Clean-up of the extracts is generally required for further purification. The highest azadirachtin levels have been obtained from Neem seeds but concentration values present a large variation between batches. Therefore, in addition to extraction procedures, it is essential to establish routine methods for azadirachtin identification and quantification. Chromatography-based techniques are preferably selected for detection and quantification of azadirachtin in plant matrices. Overall, this process will guarantee a future reproducible, safe and effective use of the extracts in formulations for commercial applications.

2019Journal article

Open access