Validation of an Ion-Pair Reverse Phase High-Performance Liquid Chromatography Method for the Detection of Major Components and Related Substances in Diquafosol Sodium Eye Drops.

Abstract

A simple, feasible, isocratic elution, and stable reversed-phase high performance liquid chromatography method was established and verified. The chromatographic conditions are as follows: EF-C18H, 4.6 × 250 mm, 5 μm column; column temperature 30 °C; for the mobile phase 27.2 g of KH2PO4 and 8.5 g of tetrabutylammonium hydrogen sulfate were taken, 2500 mL of water was added to dissolve, and the pH was adjusted to 6.7 with phosphoric acid:methanol solution with a ratio of 84:16 (V:V). The flow rate was 1.0 mL/min; the injection volume was 10 μL; and the wavelength was 262 nm. According to the current ICH guidelines, the developed method was verified, and the system suitability, specificity, LOD, LOQ, linearity, range, accuracy, repeatability, durability, and solution stability of the proposed method were verified. The validation results demonstrated that the LOQ for the method was 0.05% and the LOD was 0.02%. The content was detected within the concentration range of 300 to 900 μg/mL. The relationship between concentration and measurement was linear, with an r2 of >0.999. The concentration of impurities ranged from 0.3 to 4.5 μg/mL. A good linear correlation was observed within the range of g/mL, with a coefficient of determination r2 greater than 0.999. The accuracy and repeatability met the specified criteria.