Comparative Study of Reversed-Phase High-Performance Liquid Chromatography and Ultraviolet-Visible Spectrophotometry to Determine Doxorubicin in pH-Sensitive Nanoparticles

Analytical methods by reversed-phase high-performance liquid chromatography (HPLC) and ultraviolet-visible spectrophotometry were developed and validated to determine doxorubicin in pH-sensitive chitosan nanoparticles. Chromatographic separation was performed on a reversed-phase C-18 column, with ultraviolet detection at 254nm and a mobile phase composed by 90% (v/v) acetonitrile in water and water pH 3.0 (33:67, v/v). The spectrophotometry method had the wavelength set at 480nm and pH 3.0 water was used as a diluent. Calibration curves were linear from 1 to 30 mu g/mL (r>0.9995) and the specificity was demonstrated by verifying the absence of interferences from nanoparticle components. The values of accuracy and precision were within the acceptable limits, and robustness studies were performed by a two-level full factorial design. The validated methods were further tested to assess doxorubicin content in six different batches of pH-sensitive chitosan nanoparticles. The comparative analyses showed nonsignificant differences (p>0.05). Likewise, the HPLC method was successfully applied to determine the drug encapsulation efficiency as well as to measure doxorubicin during in vitro release assays and degradation kinetic studies under ultraviolet light C irradiation. Both methods fulfilled all validation parameters and were shown to be suitable for the characterization of doxorubicin-loaded pH-sensitive chitosan nanoparticles, without interferences from nanoparticle matrix.