Highly sensitive method for the determination of genotoxic impurity such as 3-Chloro acetanilide impurity  in acetaminophen drug substance using RP-HPLC method has been presented in the paper. Quantification of  3-chloro acetanilide content in acetaminophen samples by HPLC with UV Detector (254 nm). 3-chloro acetanilide was determined by RP-HPLC method using Zorbax SB-C8 (250x4.6mm, 5µm) column as stationary phase. Flow rate was 0.8 mL/min, column temperature maintained 35°C, sample cooler temperature 25°C, injection volume 10µL  and run time was 73 minutes. Mobile phase-A was used as 0.1% formic acid in water and methanol in the ratio of (950:50 v/v) and Mobile phase-B was used as 0.1% formic acid in water and methanol in the ratio of  (500:500 v/v). The method validation has been carried as per international conference on harmonization guidelines (ICH). Limit of quantitation (LOQ) and Limit of detection (LOD) was found 3.219 ppm and 1.062 ppm for 3-chloro acetanilide.

Key words: Genotoxic impurity, 3-chloro acetanilide, Acetaminophen, RP-HPLC method, Validation and Limit of quantitation and Limit of detection .