DEVELOPMENT AND MULTIVARIATE OPTIMIZATION OF A STABILITY-INDICATING UHPLC-QTOF-MS METHOD FOR SIMULTANEOUS QUANTIFICATION OF STRUCTURALLY RELATED IMPURITIES IN A NOVEL ANTINEOPLASTIC DRUG UNDER ACCELERATED STRESS CONDITIONS

Abstract

Background

Comprehensive impurity profiling and stability evaluation are critical for ensuring the safety, efficacy, and regulatory compliance of novel antineoplastic agents. Apoptinib, a newly developed targeted anticancer drug, possesses structural features that render it susceptible to chemical degradation, necessitating the development of a robust stability-indicating analytical method.

Objective

The objective of this study was to develop and validate a stability-indicating ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS) method for the simultaneous quantification and structural characterization of Apoptinib and its structurally related impurities under accelerated stress conditions using a multivariate optimization approach.

Methods

Chromatographic separation was achieved using UHPLC coupled with QToF-MS operated in positive electrospray ionization mode. Method optimization was performed using Design of Experiments (DoE) based multivariate analysis to evaluate critical method parameters and their interactions. Forced degradation studies were conducted under acidic, alkaline, oxidative, thermal, photolytic, and humidity stress conditions in accordance with ICH guidelines. The method was validated as per ICH Q2(R2) for specificity, linearity, accuracy, precision, sensitivity, and robustness.

Results

The optimized method provided excellent resolution between Apoptinib and its degradation products with high mass accuracy (< ±5 ppm). Linearity was established over the tested concentration ranges with correlation coefficients greater than 0.998. The method demonstrated high precision (%RSD < 2%) and satisfactory accuracy (98–102% recovery). Significant degradation of Apoptinib was observed under acidic, alkaline, and oxidative stress conditions, while moderate degradation occurred under thermal and photolytic stress. High-resolution mass spectrometry enabled confident identification and quantification of structurally related impurities formed through hydrolytic, oxidative, and elimination pathways.

Conclusion

A robust, sensitive, and stability-indicating UHPLC-QToF-MS method was successfully developed and validated for Apoptinib. The integration of multivariate optimization significantly enhanced method performance and robustness. The proposed method is suitable for routine quality control, stability testing, and regulatory submission of Apoptinib.