High Performance Liquid Chromatographic (HPLC) Assay for the Determination of Chlorhexidine in Saliva Film

Abstract

Abstract An HPLC assay for the determination of chlorhexidine in small samples (<1 μl) of saliva is described. A base deactivated reverse phase C-18 narrow bore column (ODS-B Exsil) was used for the analysis. Saliva samples were collected on Periopaper strips and chlorhexidine was extracted with 0.1 ml mobile phase. The optimal mobile phase comprised 55%v/v acetonitrile, 0.2%v/v glacial acetic acid, 7 mM sodium lauryisuiphate and column temperature was maintained at 55°C. Benzethonium was included as the internal standard and a dual wavelength UV absorbance detector was used to analyse chlorhexidine at 254 nm and benzethonium at 275 nm. Triplicate standard curves were linear for the range of concentration 1 to 15 ng/sample (R2 < 0.99). If 0.5 μl of saliva is collected then 2 to 30 μg/ml chlorhexidine can be measured. The intra-assay variability, determined from repeated injections of quality control standards containing 1, 3.75 and 12.5 ng chlorhexidine/tube was 12.9, 4.4 and 1.5% respectively.