Abstract:
Box - Behnken experimental designs do not appear to be extensively used in optimisation of analytical methods
using capillary electrophoresis (CE). This paper describes the use of the Box-Behnken experimental design to
optimise the factors affecting the separation of ethambutol hydrochloride (EB), its impurity 2-amino-l-butanol and
the internal standard (phenylephrine hydrochloride) in a CE method for a pharmaceutical tablet assay. The three
factors studied simultaneously were: buffer pH, buffer concentration and applied electric field. each at three levels.
The method was optimised with respect to three responses: resolution between peaks, theoretical plate count and the
migration time of the EB peak. A statistical programme, which applies a multiple response optimisation algorithm,
was used to calculate and optimise the three responses simultaneously. The optimum conditions were established to
be 58.0 mM sodium borate buffer at pH 9.50 and an applied electric field of 412 Vjcm. The robustness of the method
was also determined and confirmed using a second Box-Behnken design, as part of the validation exercise. System
suitability values for the method were derived from the regression surface analysis. The CE method for a
pharmaceutical tablet formulation was further validated according to current regulatory requirements, with respect to
linearity and range, precision, specificity, accuracy and limit of quantitation. The optimised method gives a fast and
efficient separation under 4 min, with complete resolution between the three peaks, and represents an improvement
over the existing USP method. It can be concluded that the Box-Behnken experimental design provides a suitable
means of optimising and testing the robustness of a CE pharmaceutical method.
