SCIENTIFIC STUDY FOR DETERMINATION OF BENZYL CHLORIDE AND GUANINE CONTENT IN ENTECAVIR BY HPLC TECHNIQUE

Abstract A highly sensitive analytical HPLC method for the simultaneous quantification of trace level impurities of Benzyl chloride and guanine in the active pharmaceutical ingredient Entecavir has been developed. These genotoxic impurities have demonstrated to induce genetic mutation, r chromosomal rearrangement and have potential to cause cancer. The analysis was accomplished on an Supelco Discovery HS F5 (25 cm x 4.6mm, 5µ)using 0.1% Triethyl amine in 100ml of water, pH adjusted to 3.0 with orthophosphoric acid , Acetonitrile, Methanol Isocratic mode Solution A : Solution B: Solution C (50:25:25 v/v) .The flow rate was set at 1.0 ml/minute. with a total analysis time of about 30 minutes. The method was validated for the analytical parameters such as system suitability, specificity, linearity and range, accuracy, limit of detection (LOD), limit of quantification (LOQ), precision, and solution stability. The limit of detection and limit of quantification were found to be 0.016% for benzyl chloride and 0.012% for Guanine respectively with respect to Entecavir sample and limit of quantification. were found to be 0.04%for benzyl chloride and 0.03%for Guanine respectively with respect to Entecavir sampl