Box-Behnken assisted HPLC development of simultaneous determination of doxorubicin and vorinostat encapsulated into polymeric nanoparticles

The objects of the present study are nanoparticles (NPs) based on a copolymer of lactic and glycolic acids (PLGA), loaded with the anticancer drug doxorubicin (DOX-NP) and histone deacetylase inhibitor vorinostat (SAHA-NP), developed for the breast cancer treatment. Drug encapsulation into PLGA matrix improve drug safety profile and allow to overcome multidrug resistance. In the current study, we developed a high-performance liquid chromatography method for the simultaneous determination of DOX-NP and SAHA-NP using Box-Behnken design, followed by the validation and NPs stability determination after sterilization treatment with γ-irradiation.

The separation was performed using a Nucleodur C-18 Gravity column (250 mm × 4.6 mm × 5 µm). Samples were prepared by precipitating PLGA with dimethyl sulfoxide. Three independent variables were analyzed to determine the most optimal conditions: methanol concentration (0-20%), pH (2.5-4.5) and flow rate (0.8 -1.2 mL /min). We evaluated contributions of these variables to the peak resolution and the retention time of the last peak of the analyte using Box-Behnken design. Next, we simultaneously optimized all dependent variables and established their most optimal values using the desirability function.

The optimized method was accurate, precise and linear in the range of 4.2–52.0 µg/mL for both drugs (R² = 0.9999 for vorinostat and R² = 0.9988 for doxorubicin). γ-irradiation at a dose of 25 kGy resulted in degradation of DOX-NP less than 95%, while the amount of SAHA-NP impurities was 88%.

Thus, the developed method is suitable for simultaneous analysis of DOX-NP and SAHA-NP, including the analysis of impurities.

Funding: This study was supported by the Russian Science Foundation research grant No. 22-25-00293, https://rscf.ru/project/22-25-00293/