Development and validation of RP-HPLC method for determination of related substances of bendamustine hydrochloride in bulk drug

Three known impurities in Bendamustine Hydrochloride bulk drug were detected by a simple, sensitive and accurate gradient reverse phase high performance liquid chromatography (HPLC). These impurities were isolated from crude sample of Bendamustine Hydrochloride using reverse phase HPLC. The IUPAC names of impurities were Impurity-A is 4-6-[2-chloro ethyl)-2-hydroxy ethyl amino)3 methyl-benzimidazolyl-2)-butyric acid (HP-1),Impurity- B is 4-(-5-[(4-5-(bis(2-hydroxyethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid and Impurity - C Isopropyl 4-(5-(bis(2-chloroethyl)amino-1-methyl-1-H-benzol[d]imidazol-2yl)butanoate. The effective separation was achieved on a Zorbax SB-C18; (4.6 mm x 25 cm) 5μm using a gradient mode by the Mobile phase A : 0.1% Trifluroacetic acid in Water: Acetonitrile (90:10) and mobile phase B : 0.1% Trifluroacetic acid in Water: Acetonitrile (50:50). The flow rate of the mobile phase was 1.0 mL/min and the total elution time, including the column equilibration was approximately 60.01minutes.The retention times of Bendamustine Hydrochloride and its impurities are 24.109,24.109,15.309,30.521 and 38.638. The UV detection wavelength was carried at 230 nm and experiments were conducted at 30°C. The developed method was validated in terms of system suitability, selectivity, linearity, range, precision, accuracy, limits of detection and quantification for the impurities following the ICH guidelines. Therefore, the proposed method was suitable for the simultaneous determination of Bendamustine Hydrochloride and its three related impurities.

Author(s): E. Sasi Kiran Goud and V. Krishna Reddy

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