Analytical Method Development and Validation of Treosulfan and Its Impurities by Ultra Performance Liquid Chromatography (UPLC) and Identification of Degradation Products by LC–MS/MS

ABSTRACT 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 ( r 2 > 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.