Ion-exchange liquid chromatographic analysis of bisphosphonates by on-line post-column photochemical reaction and spectrophotometric detection

A simple ion-exchange high-performance liquid chromatographic method was developed and employed for the analysis of bisphosphonate compounds in dosage formulations using on-line post-column photochemical reactions. The method used molybdate as the post-column reagent to react with the photolyzed bisphosphonate to form phosphomolybdate for enhanced spectrophotometric detection. A bisphosphonate compound, 2-thioethane-1,1-bisphophonic acid, was selected to evaluate the separation using both isocratic and gradient elution methods, along with the effects of other experimental parameters including mobile phase composition, flow-rate and post-column reagent concentration. The gradient elution method showed improved resolution and detection sensitivity compared to the isocratic elution method. The optimized gradient method was simple, reproducible, and specific to bisphosphonate compounds. It was successfully employed for the stability study of the bisphosphonate compound in pharmaceutical dosage formulations.