Assay of stability, free and total concentration of chlorhexidine in saliva by solid phase microextraction

This paper presents the development and application of a solid phase microextraction method for the full investigation of chlorhexidine interaction with saliva during a pharmacokinetic study: chemical stability, binding to proteins, free concentration, total concentration and kinetics of elimination after oral administration. Only 0.1 mL sample were needed for each time point and the concentration of salivary proteins was determined as well. It was shown that chlorhexidine remained stable in the oral cavity for at least 9 h and high concentrations of the drug (2 microg/mL total) were still present even 8 h after mouthrinsing. Supplementary facts were uncovered: while the total concentration followed first-order elimination kinetics, the free concentration remained almost constant for several hours; this showed that the oral cavity acted like a reservoir that slowly released the drug. It was also revealed that following oral administration of chlorhexidine, the normal composition of saliva changed for a few hours, probably as a physiological response to the bitter taste of the medicine. The method had a wide linear range (0.1-40 microg/mL free chlorhexidine) that was perfectly suitable for the study of chlorhexidine retention in the oral cavity. Separation and quantitation were achieved by liquid chromatography coupled to mass spectrometry; no interference from endogenous compounds was observed. This selective and sensitive solid-phase microextraction (SPME) approach for monitoring the free and total concentration of a drug, as well as the concentration of proteins that bind that drug, should prove to be more useful for pharmacokinetic studies than classic methods that only provide the total concentration as a final result.