RP-HPLC Method Development and Validation for the Determination of Pemigatinib using Design of Experiments Approach

Aim: To develop and validate a simple, precise, accurate and robust RP-HPLC method for the determination of Pemigatinib by using Design of Experiments (DoE) approach.

Study Design: A 23 Factorial design consisting of three factors at two levels was considered for the experimental plan initially to select the initial chromatographic conditions and optimization was done using Box-Behnken Design. The critical method parameters selected for optimization were % Organic phase composition, pH of the buffer and flow rate. The critical quality attributes investigated were retention time, theoretical plates and tailing factor.

Methodology: Chromatographic separation was achieved on Agilent Zorbax XDB C18 (250×4.6 mm, 5 µm) column maintained at ambient temperature and PDA-UV detection set at 262nm. The optimized and predicted data from the Design Expert® (12.0.12.0) modelling software (Stat-Ease Inc., Minneapolis, MN, USA) consisted of mobile phase 0.1% OPA pH 2.5 buffer (60%): Acetonitrile (40%) pumped at a flow rate of 1.06ml/min gave the highest desirability.

Results: The retention time of the drug was found to be 3.258 min. The developed method was linear over the concentration range of 25-150 µg/mL with correlation coefficient of 0.999. The optimized method was validated as per ICH Q2 (R1) guidelines.

Conclusion: Based on the ANOVA results, the selected models for the responses retention time and tailing factor were found to be significant with P=0.05. 2D Contour plots were used to visualize the effect of factors and their interactions on the responses. Design validation was done using predicted vs. actual plots for the responses. The results of the validation parameters were within the acceptable limit. The stability of the drug was examined under different stress conditions forcibly and significant degradation was found in reductive condition.