Quantitative analysis of ripasudil hydrochloride hydrate and its impurities by reversed-phase high-performance liquid chromatography after precolumn derivatization: Identification of four impurities.

ABSTRACT

We report the development and validation of a stability-indicating reversed-phase high-performance liquid chromatography method with precolumn derivatization for the separation and identification of the impurities of ripasudil hydrochloride hydrate, a novel protein kinase inhibitor. 2,3,4,6-Tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate was chosen as the derivatizing reagent and triethylamine was added as catalyst. 200 µL sample solution (1 mg/mL), 600 µL derivatizing reagent (1 mg/mL), and 200 µL triethylamine solution (1%, v/v) were mixed and reacted at 40°C for 30 min. The separation was achieved on an Inertsil C18 ODS-3 (250 mm × 4.6 mm, 5 µm) column using mobile phases including 10 mmol monopotassium phosphate buffer (pH 3.0) and methanol in gradient mode. The column temperature was adjusted at 25°C and the flow rate at 1 mL/min. The detection was carried out at 220 nm. Different precolumn derivatization conditions as well as the high-performance liquid chromatography conditions were optimized. Ripasudil hydrochloride hydrate and its four impurities were detected and quantitated, among which two new compounds were characterized. The proposed method was validated and proven to be selective, accurate, and precise and suitable for the quantitative analysis of ripasudil hydrochloride hydrate.