Reverse Phase HPLC Methodology for the Determination of Bay K8644

Abstract

Following ICH guidelines for analytical validation, we report a common C18 column stability indicating isocratic reverse phase high performance liquid chromatography method for the determination of the ion channel modulator Bay K8644. Two main forced degradation products and a minor impurity were also tentatively identified by Mass Spectrometry. The mobile phase consisted of a 50/50 acetonitrile/buffer mixture at a flow rate of 2 mL/min. Mean retention time for Bay K8644 was 3.030 minutes. Excellent linearity (r = 0.9998) was achieved in the range 0.10-1.40 μg/mL at 274 nm wavelength. Analytical limits were 16.56 ± 1.04 ng/mL for detection and 55.21 ± 3.48 ng/mL for quantitation respectively. Accuracy and precision studies showed good results (95-105%). Robustness was assessed by varying ±3%, both temperature and flow rate. Five different stress conditions were applied to assess Bay K8644's stability. Only basic and photolytic treatments yielded degradation products, both correctly resolved in a total runtime of 4 minutes. In conclusion, we developed a fast, simple, sensitive, accurate, precise, reliable and stability indicating method for detecting/quantifying Bay K8644, and tentatively characterized its main impurities/forced degradation products.