Development and validation of HPLC method for simultaneous determination of Chlorhexidine and Para-Chloroaniline

Abstract

A simple, precise and rapid isocratic HPLC-UV method for simultaneous determination of chlorhexidine (CHD) and its degradation product, para chloroaniline (pCA) in their pharmaceutical formulations was developed. Simple isocratic elution was selected, the optimized mobile phase was composed of methanol and acetate buffer solution at 55: 45 ratio, with flow rate of 1.0 ml/min, injection volume was 20μl, and the separation was performed using C18 column (200 × 4.6 mm, 5 μm particle size) at ambient temperature. Both components were determined at 254nm. Linearity of this method was checked using concentration range of 20 –160μg/ml for chlorhexidine and 0.3 –1.2μg/ml for p-chloroaniline, the linearity correlation was (R2 =1), for both components. The limit of detection was (1.07 and 0.012μg/ml) for chlorhexidine and pchloroaniline respectively. The limit of quantitation was (3.25 and 0.038μg/ml) for chlorhexidine and p-chloroaniline respectively. The specificity tests were checked to find that there was no interference between the excipients used and the active ingredient and its impurity. The average percentage of accuracy for chlorhexidine and p-chloroaniline was 99.82 (0.34 RSD) and 100.37 (0.38 RSD), respectively (Not more than 2.0, USP and ICH acceptable limit). For intraday precision for 80%, 100% and 120%, the RSD for recovery percentage for chlorhexidine and pchloroaniline was 0.08, 0.09 and 0.18, and 0.04, 0.21 and 0.21, respectively. For interday precision, was 0.81, 0.24 and 0.95, and 0.24, 0.35 and 0.28, respectively. (Not more than 2.0 acceptable limit). System suitability parameters at all different conditions were also found to be within the accepted limit