Development and Validation of an HPLC Method for Quantifying Endoxifen in pharmaceutical formulation: Analytical and Forced Degradation Studies

Abstract

This study presents the development and validation of a high-performance liquid chromatography (HPLC) method for the quantification of Endoxifen, a potent active metabolite of Tamoxifen, in pharmaceutical formulation. Endoxifen, known for its enhanced therapeutic potential in treating estrogen receptor-positive (ER+) breast cancer, requires precise analytical methods for accurate measurement in plasma, serum, and tissue samples. The method was optimized using a Zodiac C18 column and a mobile phase consisting of 0.1% trifluoroacetic acid buffer and acetonitrile in a 78:22 ratio, with UV detection at 278 nm. The chromatographic conditions included a flow rate of 1.0 mL/min and a column temperature of 30°C. Validation of the method was conducted following ICH Q2 (R1) guidelines, demonstrating excellent specificity, linearity, precision, and accuracy. The method exhibited a linear response over the concentration range of 2.5 to 15 μg/mL, with a correlation coefficient of 1. Precision studies, including repeatability and intra-day and inter-day variations, showed low % RSD values, confirming the method’s consistency. Accuracy was affirmed through recovery studies, with mean recoveries close to 100% at all tested levels (50%, 100%, and 150% of the label claim). The robustness of the method was evaluated by varying the detection wavelength, showing negligible impact on performance. Forced degradation studies revealed that Endoxifen is susceptible to degradation under acidic, basic, oxidative, thermal, and photolytic conditions. Oxidative degradation had the most significant effect, with a 17.24% reduction in Endoxifen’s peak area. These findings provide insights into Endoxifen’s stability and highlight the need for stability-indicating methods. The method’s limit of detection (LOD) was 0.017 μg/mL and limit of quantitation (LOQ) was 0.051 μg/mL, demonstrating high sensitivity. In conclusion, the validated HPLC method offers a reliable approach for Endoxifen quantification in biological matrices, crucial for pharmacokinetic studies and patient safety. The forced degradation analysis supports the development of stability-indicating methods and informs strategies to maintain Endoxifen’s efficacy throughout its lifecycle.