HPLC Determination of the Antiseptic Agent Chlorhexidine and its Degradation Products 4-Chloroaniline and 1-Chloro-4-Nitrobenzene in Serum and Urine

Chlorhexidine (1,1-hexamethylene-bis-5-(p-chlorophenyl)-biguanide]-digluconate; CHD) is one of the most frequently used antiseptics. In spite of this, its resorption and elimination has hardly been investigated. One of the main reasons for this is the lack of suitable procedures for its analysis in biological materials. Samples of CHD also may contain the carcinogen 4-chloroaniline which also may be formed as a degradation product in preparations.A HPLC method with end-capped stationary phase was developed which separates CHD, 4-chloroaniline and 1-chloro-4-nitrobenzene (a degradation product of 4-chloroaniline). Methods of solid-phase extraction and deproteinization for the purpose of isolating the analytes in serum and urine were also tested. In the latter process, isolation of all three compounds from the biological material using a single procedure was unsuccessful. Isolation of CHD, on the other hand, both from serum and from urine using solid-phase extraction with weak cation exchangers was successful and showed reliable retrieval rates.