Validated HPLC method for paclitaxel determination in PLGA submicron particles conjugated with α-fetoprotein third domain: Sample preparation case study.

Objectives: The goal of this study was to develop sample preparation method and validate the HPLC method for precise determination of paclitaxel (Ptx) in PLGA submicron particles conjugated with protein vector molecule.

Methods: Ptx loaded PLGA submicron particles were formulated by a single emulsification method. PLGA submicron particles were conjugated with alpha fetoprotein third domain (rAFP3d) via standard carbodiimide technique. The obtained conjugate was analyzed using 1525 binary pump and 2487 UV-VIS detector system (Waters, USA) and Reprosil ODS C-18 analytical column with the dimensions of 150mm×4.6mm ID×5μm (Dr. Maisch GmbH, Germany). Sample preparation method was developed utilizing guard cartridge with С18 stationary phase (Phenomenex, USA). HPLC method was validated according to the international conference on harmonization guidelines.

Results: Efficient sample preparation was achieved using 4% of DMSO pre-dissolution, following by 10min of centrifugation at 4500g. Ptx determination was performed using acetonitrile/0.1% phosphoric acid (50:50 v/v) mobile phase at a flow rate of 1.0mL/min, injection volume of 10μL, and at 227nm. The developed method showed linearity, accuracy and precision in the range from 0.03 to 360μg/mL, with LOD and LOQ values of 0.005 and 0.03μg/mL, respectively. The intra- and inter-day precisions presented RSD values of lower than 2%.

Conclusion: The validated method was successfully applied to calculate Ptx encapsulation efficacy and drug loading in the developed formulation.