Chromatographic assay of pharmaceutical compounds under column overloading
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Updated: Oct 16, 2026

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
Published on: April 26, 2016
1Analytical Development, Schering-Plough Corporation, Kenilworth, NJ 07033-0530, USA.
Abstract:
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.
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