Chromatographic assay of pharmaceutical compounds under column overloading

A HPLC assay method utilizing overloaded chromatography and dual-wavelength detection was developed for a pharmaceutical formulation containing an antibacterial (clotrimazole) and a steroid (mometasone furoate) at widely different concentrations. In order to meet the limit of quantitation (LOQ) objective of not less than 0.05% of assay concentration simultaneously for both actives in the HPLC assay method, the assay concentration of the antibacterial falls into the non-linear range of its equilibrium isotherm, but still in the linear dynamic range of an ultraviolet detector. Although the analytical column is overloaded with the antibacterial and a non-symmetric elution peak is obtained, the HPLC assay method exhibits good linearity, recovery and reproducibility.