[A study on the simultaneous HPLC determination of chlorhexidine and its impurity 4-chloroaniline]

This paper reports a new RP-HPLC method using phenacetin internal standard (IS) for simultaneous determination of chlorhexidine (CH) and its impurity 4-chloroaniline (4-CA). We use the LC-3A HPLC instrument, a Zorbax-C8 column (25cm x 4.6mm id, 10 microns), a variable wave length UV detector and C-RIA chromatographic data processor. A mixture of methanol and 0.2 mol/L NaH2PO4 (50.5 : 49.5, pH 3.0) is used as mobile phase, whose flow rate is 1.0 ml/min. Sample solution of 15 microliters is injected onto the column, which is maintained at 30 degrees C. The eluent is monitored at 240nm. The retention times are 16.72 min for CH and 5.54 min for 4-CA. The standard curves of CH and 4-CA are established using peak area ratio of CH/IS and 4-CA/IS to concentration respectively. They are both linear (r = 0.9999) within 15-200 micrograms/ml for CH and 60-1200 micrograms/ml for 4-CH. The limits of detection are 1.33 micrograms/ml for CH AND 7.5 micrograms/ml for 4-CA (S/N = 3). The analytical recovery rates are 100.1% for CH and 103.1% for 4-CA. This method has been used for investigating the stability of CH preparation.