A Stability Demonstrating HPLC Method Development and Validation for Related Substances in Oxtriphylline using Quality by Design

Abstract

A new reverse phase HPLC procedure, stability demonstrating for estimating related substances in oxtriphylline, was developed and validated according to ICH guidelines by applying Analytical Quality by Design (AQbD) principles. The three critical method parameters identifi ed were mobile phase fl ow rate, column temperature, and mobile phase pH. Based on these factors, the resolution between oxtriphylline and theophylline related compound F and the tailing of oxtriphylline peak were selected as critical method attributes. XBridge BEH C18 with measurements 150 x 4.6 mm, 5 μ was used to achieve peak separation. The mobile phase fl ow rate was at 1.0 mL/min and 40°C was the column temperature. The injection volume was 10 μL, the autosampler temperature was 25°C, and UV detection was done at 270 nm. The elution was a gradient. Analytical method validation parameters like precision, accuracy, specifi city, linearity, and solution stability of solution were proved. The linearity of the method was established from 0.05 to 1% of sample concentration. The correlation coeffi cient was more than 0.999. The percentage recoveries for all impurities were from 100.71 to 102.45%, and the relative standard deviation (RSD) was as less than 1% proving the procedure is precise. The recoveries of all impurities were proven from LoQ to 150% level of sample concentration. The recoveries ranged from 98.40 to 101.70% at LoQ, 101.56 to 101.88 at 100% level, and 101.28 to 102.54 at 150% level of spiking. Robustness studies proved that small and deliberate changes to fl ow (± 0.2 mL/min), column temperature (± 5°), and mobile phase pH variation (± 0.2 units) does not impact the method. Also, the stability-indicating feature of the method was demonstrated by performing forced degradation studies. The method developed by AQbD approach was accurate, precise, linear specifi c, robust, and stability-demonstrating to quantify oxtriphylline and its related substances.