Reversed-Phase High-Performance Liquid Chromatography Assisted Simultaneous Estimation and Validation as per ICHQ2(R2) Guidelines: Application of Method in Quantification of Paclitaxel and Berberine in Liposomes-Based Delivery System.

Abstract

Berberine (BER) is an antioxidant, anti-inflammatory, and antitumor agent, while paclitaxel (PTX) is a widely used synthetic chemotherapeutic agent for breast cancer. Several reports have suggested the use of a PTX and BER combination for the effective treatment of breast cancer, and many pharmaceutical scientists are working to develop a novel drug delivery system incorporating this combination. However, the literature lacks a reliable simultaneous estimation method for this combination. Therefore, in the present study, we aimed to develop a robust reversed-phase high-performance liquid chromatography method for the simultaneous estimation of PTX and BER in free drug form in liposomal formulation. The method employed a C₁₈ column (250 × 4.6 mm, 5 µm) with acetonitrile and water (70:30, v/v) as the mobile phase at a flow rate of 0.8 mL/min and detection at 250 nm. Retention times were 2.84 and 5.62 min for PTX and BER, respectively. Theoretical plates >2000 were demonstrated, and peak tailing of <2 in validation as per ICH Q2(R2) was observed. In the 10-50 ppm range, linearity was found with R² values of 0.9979 (PTX) and 0.9975 (BER). Furthermore, the method achieved within acceptable limits precision (<2% relative standard deviation) and accuracy (90%-110%). Robustness assessments checked method reliability in small variations. In addition, using the method effectively, entrapment efficiencies of 85.27 ± 1.74% and 78.62 ± 2.38% were obtained for PTX and BER in liposomal formulations. Moreover, in vitro release studies revealed 98.83 ± 2.94% (PTX) and 96.56 ± 1.92% (BER) release over 24 h. The validated method was precise, accurate, and reliable, making it suitable for application in drug formulation analysis.