RP-HPLC Method Development and Validation for Simultaneous Quantitation of Sitagliptin Phosphate and Empagliflozin: QbD Driven

Abstract

The recently approved combination of Sitagliptin Phosphate and Empagliflozin (September 2024) is widely used to improve glycemic control in adults with type 2 diabetes mellitus. Reported HPLC method for the quantification of Sitagliptin Phosphate and Empagliflozin lack the required robustness for regulatory approval and ignore interaction effects between method parameters. Also there is a lack of structured, risk-based methodology in the literature to optimize the separation of both drugs. Till date, no robust Quality by Design (QbD) -based RPHPLC method has been systematically developed for their simultaneous estimation, creating a gap in reliable and optimized analytical procedures for routine quality control. This study aims to develop and validate a simple, robust, and efficient RP-HPLC method using a QbD approach for the simultaneous quantification of these drugs in a laboratory-prepared blend. Critical method parameters like percentage of acetonitrile (ACN), percentage of o-phosphoric acid (OPA), and flow rate were initially screened using a Plackett–Burman design and further optimized using a Box–Behnken design with response surface methodology. Key analytical responses, including retention time, theoretical plates, and tailing factor, were evaluated and optimized. Chromatographic separation was achieved on an XR ODS II C18 column (150 × 3 mm, 5 μm) at 25 °C, using a mobile phase of ACN and 0.1% OPA in water (70:30, v/v), at a flow rate of 1.0 mL/min, with detection at 254 nm. The retention times were 2.626 min for Sitagliptin Phosphate and 4.041 min for Empagliflozin. In AQbD multiple factors can be varied simultaneously to optimise it as well as enables regulatory flexibility in comparison to traditional HPLC methods where one factor is varied at a time. Validation results complied with ICH Q2(R2) guidelines. Its robustness and efficiency make it particularly valuable for simultaneous estimation of this newly approved drug combination in bulk and formulation studies.