Extraction and measurement of pamidronate from bone samples using automated pre-column derivatization, high-performance liquid chromatography and fluorescence detection

A method for the determination of 3-amino-1-hydroxylpropylidene-1,1-bisphosphonic acid (pamidronate) in bone samples is described. This method combines and modifies parts of previous procedures. Pamidronate is extracted from finely ground bone with dilute hydrochloric acid, Amine-containing contaminants are removed by co-precipitation of pamidronate with calcium. Excess calcium is removed with EDTA and an ion-exchange resin. Pamidronate is automatically derivatized at the primary amine and quantified by high-performance liquid chromatography with fluorescence detection. The method assay was linear in the concentration range 7.5-600 ng/mg bone (20-1000 pmol/mg). The imprecision for repeat analyses were 16.5 and 7.8%, at pamidronate levels of 7.5 and 600 ng/mg bone, respectively. The method has been used to analyze bone samples from pharmacokinetic animal studies involving both acute and chronic dosages.