Detection and Quantification of Two Potential Genotoxic Impurities in Telbivudine Active Pharmaceutical Ingredient by Targeted Ultra Performance LC-MS/MS Analysis

Abstract

A new selective, rapid and highly sensitive method using reversed-phase ultra-performance liquid chromatography (RP-UPLC) coupled with mass analyzer was developed for the detection and quantification of two genotoxic impurities viz. 4-dimethyl aminopyridine (GTI-1) and 2-bromo-3,5-diacetyl thymidine (GTI-2) in Telbivudine drug substance. The separation of two genotoxic impurities was accomplished using Kromasil C8 column (100 mm × 4.6 mm, 3.5 μm) maintained at 45 ºC using formic acid (0.1%) as buffer solution and gradient grade acetonitrile with 0.5 mL/min flow rate. Both GTIs were quantified using positive electrospray ionization (ESI) mode on MS/MS Analyser. ICH guidelines were followed for validation of genotoxic impurities. % RSD for system precision was found to be 2.71 and 2.94 and the overall %RSD for ruggedness was 3.11 and 2.89, respectively for GTI-1 and GTI-2. Two GTIs and Telbivudine drug were well resolved at retention times: 1.76 min for GTI-1, 3.81 min for Telbivudine and 6.62 min for GTI-2. The linearity relationship holds good in the range of concentrations 0.5 ppm to 7.5 ppm for both the impurities with a correlation coefficient of 0.999. Accuracy ranged from 95.1% to 105.1%. The GTI-1 and GTI-2 were shown, respectively detection limits: 0.07 ppm and 0.09 ppm and quantification limits: 0.22 ppm and 0.27 ppm and the solutions were stable upto 24 h at room temperature. The proposed and validated method is sensitive, reproducible, specific and linear and can be successfully applied for the quantification of both genotoxic impurities in Telbivudine active pharmaceutical ingredient and formulation.