Evolution 300

Dissolution tests of VAN and HA-VAN₂₅, were carried out following USP Method I (SOTAX AT 7 Smart, Westborough, MA, USA) using 500 mL of phosphate-buffered solution (PBS) at pH = 7.4 and 37.0 ± 0.5 °C as dissolution medium, with a basket at 100 rpm speed.
Dissolution tests of new formulations are usually compared to a reference. However, there are no commercial formulations of VAN for the inhalation route. For this reason, an amount of powder containing 150 mg of VAN as raw material and the equivalent of VAN in the HA-VAN₂₅ complex were tested. The latest formulation was also sieved using a 560–630 μm sieve prior to the test. Each sample was incorporated into the dissolution medium and 0 and 2 mL aliquots were taken at predetermined times (5, 10, 15, 30, 45, 60, 90 and 120 min); these extracted volumes were replaced with a fresh thermostated medium. The aliquots were filtered through a cellulose filter before dilution. The concentration of dissolved VAN was determined by UV-Vis spectrophotometry (Evolution 300, Thermo Electron Corporation, Waltham, MA, USA) at 280 nm. A calibration curve, in triplicate, was designed with six different concentrations of VAN between 55 and 280 µg/mL dissolved in deionized water, obtaining an R² of 0.9985.
The results were expressed as the mean of three determinations with their SD.

This method is based on the reduction of stable DPPH nitrogen radicals in the presence of antioxidants. Antioxidant capacity was determined according to the method described by Brand-Williamset al. (23 (link)) in triplicate. A volume of 50 µL of diluted extract, 800 µL of 80% methanol and 200 µL of methanol solution of 6·10^-4 mol/L DPPH were mixed. Then, the mixture was incubated at 37 °C for 1 h in the absence of light. The absorbance was measured at 515 nm (Evolution™ 300; Thermo Fisher Scientific), and compared to a Trolox calibration curve in the concentration range from 100 to 600 µmol/L. The results were expressed as Trolox equivalents in mg/L.