Optimization and validation of RP-HPLC method for the estimation of chlorzoxazone and paracetamol with its genotoxic impurity (4-amino phenol) in bulk and pharmaceutical drug product using PDA detector

Abstract

A simple, accurate, precise, reproducible RP-HPLC method has been developed for simultaneous estimation of chlorzoxazone (CZX) and paracetamol (PCM) with its genotoxic impurity, p-amino phenol (4-AP) in bulk and combined dosage form (tablet). The method was validated in compliance with ICH guidelines [1-2]. The LC separation was achieved on LiChrospher RP-18e (250X4.6mm), 5µm column at 279 nm in isocratic mode using mobile phase composition methanol: acetate buffer (70:30 v/v), pH adjusted to 5.5 by acetic acid. Flow rate employed was 1.0 ml/min. The retention time for paracetamol, chlorzoxazone and 4-amino phenol were found to be, 3.76, 6.20 and 2.75 minutes respectively. Linearity ranges for paracetamol, chlorzoxazone and 4-amino phenol were established in the range of 10-50 µg/ml, 10-50 µg/ml and 0.8-2.8 µg/ml respectively with correlation coefficient of 0.997, 0.996 and 0.999 respectively. The % recoveries for paracetamol, chlorzoxazone and 4-amino phenol impurity were found in range with relative standard deviation (RSD) less than 1. The LOD and LOQ were found to be 1.1483 and 3.4798 for paracetamol, 1.3890 and 4.2092 for chlorzoxazone and 0.01459 and 0.0442 for p-amino phenol respectively in µg/ml. The proposed method is successfully applied for the quantification of paracetamol, chlorzoxazone and 4-amino phenol impurity in bulk and formulations.     

Key words: Chlorzoxazone, Paracetamol, 4-Amino phenol impurity, Photodiode array detector