New stability-indicating high performance liquid chromatography assay and proposed hydrolytic pathways of...

Chlorhexidine (CHD) is an antiseptic agent widely used in mouth rinses and ophthalmic solutions. Its assay in these solutions has been commonly performed with high performance liquid chromatography (HPLC). Published HPLC assays were unable to detect the many possible hydrolysis products of CHD, with the exception of p-chloroaniline. This raises concerns regarding the stability-indicating capabilities of the HPLC assays. In addition, the reliability of these assays was affected by the irreversible absorption of CHD on silica-based reversed phase (RP) columns. Special attention must be paid to the mobile phase or sample matrices in order to obtain reliable assay results. This paper presents a polymer-based RP-HPLC method that is stability-indicating. The method detected six UV-absorbing hydrolysis products of CHD. The identities of the products, which were elucidated by UV and mass spectral analyses, are consistent with the proposed hydrolytic pathways of CHD. Quantitation problems observed with silica-based RP-HPLC assays do not arise with the new method. Preliminary data indicate that the new HPLC method can be developed into a reliable, stability-indicating assay for CHD in ophthalmic solutions.