Analytical Box-Behnken Design Approach for Method Optimization and Validation for Concurrent Determination of Doxecitine and Doxribtimine by RP-HPLC in Bulk and Powder Dosage Form With Degradation Assessment

Abstract

Purpose

The goal of the present research was to create, refine, and validate a stability-indicating isocratic RP-HPLC technique based on AQbD for concurrent measurement of Doxecitine and Doxribtimine in pharmaceutical powder dosage form and bulk medications, with a AQbD approach utilizing a Box–Behnken Design was applied for chromatographic method optimization.

Method

Optimization was performed by selecting critical method variables like flow rate, the quantity of acetonitrile, and a volume of 0.1% perchloric acid as independent variables, while determining responses such as tailing factor along with retention time. Using mobile phase 0.1% acetonitrile and 30:70 % v/v perchloric acid with a 1.0 mL/min flow rate, a standard chromatographic differentiation was achieved on a X-Terra RP-18 column (250 × 4.6 mm, 5 µm, Waters) with detection at 249 nm and a runtime of 7 minutes.

Result

For Doxecitine and Doxribtimine, the retention times were determined to be roughly 2.52 and 4.73 minutes, respectively. With correlation values (r²) of 0.999 for the two analytes, the technique demonstrated outstanding linearity. According to ICH requirements, parameters such as system suitability, linearity, precision, specificity, accuracy, robustness, LOQ and LOD were determined to be within acceptable bounds. Studies on forced degradation verified the technique’s capability to indicate stability.

Conclusion

The established RP-HPLC technique based on AQbD provides a suitable approach for the simultaneous determination and stability testing of Doxecitine and Doxribtimine in pharmaceutical formulation, contributing significantly to the method optimization and regulatory compliance in pharmaceutical analysis.

Graphical Abstract