Analytical Method Development and Validation of Treosulfan and Its Impurities by Ultra Performance Liquid...

Treosulfan is a bifunctional alkylating agent widely used as a conditioning drug in haematopoietic stem cell transplantation. In this study, a robust reverse-phase UPLC method was developed and validated for the quantitative determination of treosulfan and its related substances. The optimised method employed a Waters Acquity UPLC BEH shield RP-18 (50 × 1.0 mm, 1.7 μm) column with an isocratic mobile phase of acetonitrile and ammonium acetate (pH 2.5)/formic acid in a 40:60 ratio, a flow rate of 0.2 mL/min, injection volume of 5 μL and detection at 239 nm. Validation, performed according to ICH Q2 (R2), confirmed excellent system suitability, specificity, precision (%RSD < 1%), linearity (r² > 0.999), accuracy (recoveries 99%-101%) and sensitivity, with LODs and LOQs meeting acceptable criteria. Forced degradation studies under acid, alkali, oxidative, reductive, photolytic, hydrolytic and thermal conditions confirmed the method's stability-indicating capability, with all purity angles below their respective purity thresholds and acceptable mass balance. LC-MS/MS analysis further enabled structural characterisation of major degradation products. Overall, the developed RP-UPLC method is selective, reproducible and suitable for routine quality control and stability evaluation of treosulfan in bulk drugs.