A HPLC method for simultaneous quantification of chlorhexidine and thymol using Box-Behnken design for robustness of the method assessment

Abstract The antimicrobial chlorhexidine is considered the gold standard in dentistry. Thymol is a phenol found in the essential oils of various plant species and also stands out for its antimicrobial potential. Synergistic effects can be promoted by applying these two active pharmaceutical ingredients together in technological products, for example in controlled release microparticles. The objective of this work was to develop and validate an analytical methodology applying a Box-Behnken experimental design and using High Performance Liquid Chromatography capable of quantifying chlorhexidine and thymol simultaneously in a matrix including pharmaceutical excipients. After optimization, the mobile phase consisted of methanol and 0.03 M monobasic sodium phosphate buffer (60:40), with 0.4% triethylamine and octylsilane as the applied stationary phase. The method proved selective, even in the presence of chlorhexidine and thymol degradation products. For chlorhexidine, the method was linear from 4.8 to 19.2 µg/mL, and for thymol from 8.0 to 32.0 µg/mL. Accuracy was close to 100%, and the precision assessment yielded coefficient variation values of <5%. Being based on the Box-Behnken design, the method was robust and therefore validated for assisting in quality control processes involving these active pharmaceutical ingredients. Graphical Abstract