High-performance liquid chromatographic analysis of chlorhexidine in saliva after mouthrinsing

A high-performance liquid chromatographic method was developed to quantify chlorhexidine in human saliva. After addition of an internal standard and acidic-deproteinization, saliva samples were chromatographed by the reversed-phase ion-pair system using pentanesulfonate as a counterion and the eluates were detected by a diode array detector. Under optimal conditions, the baseline separation of analytes was achieved within 4.5 min without interference from components in saliva matrix. The method showed high selectivity to salivary chlorhexidine, quantitative range (50.0 ng/ml-50.0 mu g/ml), recovery (96.0-97.8%) and analytical precision (intra- and interassay CVs within 0.41%), permitting the effective application to both saliva and aqueous solutions. Chlorhexidine was found in saliva at microgram per milliliter levels for at least 8 h after mouthrinsing with 10 ml of an aqueous solution of chlorhexidine (0.1 or 1.0 mg/ml) for 1 min, The concentrations of salivary chlorhexidine were reduced by ingesting the acidic beverage and food, but not by ingesting the neutral beverage. The adsorption experiments in artificial saliva revealed that chlorhexidine was significantly adsorbed to saliva-coated hydroxyapatite, buccal epithelial cells and mucin. The proposed method will be a useful tool to study salivary chlorhexidine after oral application and its retention in the oral cavity.