A new method for chlorhexidine (CHX) determination: CHX release after application of differently concentrated CHX-containing preparations on artificial fissures

Aims of the study were (1) to establish a method for quantification of chlorhexidine (CHX) in small volumes and (2) to determine CHX release from differently concentrated CHX-containing preparations, varnishes, and a CHX gel applied on artificial fissures. CHX determination was conducted in a microplate reader using polystyrene wells. The reduced intensity of fluorescence of the microplates was used for CHX quantification. For verification of the technique, intra- and inter-assay coefficients of variation were calculated for graded series of CHX concentrations, and the lower limit of quantification (LLOQ) was determined. Additionally, artificial fissures were prepared in 50 bovine enamel samples, divided into five groups (A–E, n = 10) and stored in distilled water (7 days); A: CHX-varnish EC40; B: CHX-varnish Cervitec; C: CHX-gel Chlorhexamed; D: negative control, no CHX application; and E: CXH-diacetate standard (E1, n = 5) or CHX-digluconate (E2, n = 5) in the solution. The specimens were brushed daily, and CHX in the solution was measured. The method showed intra- and inter-assay coefficients of variation of <10 and <20%, respectively; LLOQ was 0.91–1.22 nmol/well. The cumulative CHX release (mean ± SD) during the 7 days was: EC40 (217.2 ± 41.8 nmol), CHX-gel (31.3 ± 8.5 nmol), Cervitec (18.6 ± 1.7 nmol). Groups A–C revealed a significantly higher CHX release than group D and a continuous CHX-release with the highest increase from day 0 to 7 for EC40 and the lowest for Chlorhexamed. The new method is a reliable tool to quantify CHX in small volumes. Both tested varnishes demonstrate prolonged and higher CHX release from artificial fissures than the CHX-gel tested

Pitfalls and success of experimental design in the development of a mixed MEKC method for the analysis of budesonide and its impurities

A mixed MEKC method for the analysis of budesonide and its related substances is presented. The micelles were formed from sodium cholate (CHOL) and 3-(N,N-dimethylmyristylammonio) propanesulfonate (MAPS). A multivariate optimisation was carried out with the aim of obtaining a baseline separation of all compounds. The influence of voltage, borate concentration, cholate concentration, MAPS concentration and pH was evaluated on the responses, corresponding to critical resolution values. Problems with the investigated experimental design were encountered due to the complexity of the separation process. As a consequence, a first design was not sufficient to reach the optimal conditions, but was needed in order to obtain the necessary information to successfully plan a second in-depth study by means of response surface methodology. The optimal conditions were as follows: capillary total and effective lengths of 48.5 and 40.0 cm, respectively, with 50 \u3bcm id; 70 mM borate buffer (pH 8.8) containing 65 mM CHOL and 10 mM MAPS; temperature 20\ub0C and voltage 16 kV. Separation of all the compounds, including R- and S-epimers of budesonide, was obtained in a reasonable time. Validation of the method was performed for both drug substances and drug product