Estimation of related substances by using high-performance liquid chromatographic method was developed and validated for the determination of Bendamustine hydrochloride. Reversed-phase chromatography was performed on Waters 2489 UV 2695 pump, Waters 2998 PDA 2695 pump Software Empower2 photodiode array detector using ACE C18 (250 mm × 4.6 mm, 5 μm particle size) column with pH 7.0 buffer: methanol as mobile phase at a flow rate of 1.0 mL/min. with UV detection at 235 nm. Linearity was observed in the concentration range of Monohydroxy impurity 0.05–1.16 μg/mL (R2 = 0.998), the concentration range of BND-VI impurity 0.06–1.18 μg/mL (R2 = 0.998), the concentration range of Bendamustine HCl 0.08-0.79 μg/mL (R2 = 0.997) and the concentration range of Isopropyl ester 0.05-1.18 /mL (R2 = 0.998). The limit of quantitation (LOQ) and limit of detection (LOD) were found to be Monohydroxy impurity 0.05&0.02 μg/mL, BND-VI impurity 0.06&0.02μg/mL, Bendamustine HCl 0.08&0.03μg/mL and Isopropyl ester 0.05&0.02μg/mL respectively. The method was validated as per ICH guidelines. The RSD for intra-day (0.14-0.32) and inter-day (0.47-0.66) precision were found to be less than 1 %. The percentage recovery was in good agreement with the labeled amount in the pharmaceutical formulations and the method is simple, specific, precise and accurate for the determination of Bendamustine hydrochloride in pharmaceutical formulations.
