Determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid in urine by automated pre-column derivatization with 2,3-naphthalene dicarboxyaldehyde and high-performance liquid chromatography with fluorescence detection.

A sensitive (5 ng/ml) method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid in human urine is described. The procedure includes (1) the isolation of the drug from urine by co-precipitation of its calcium salt with endogenous phosphates in the presence of base, (2) a solid-phase anion-exchange sample clean-up and (3) automated pre-column derivatization of the primary amino group with 2,3-naphthalene dicarboxyaldehyde-cyanide reagent followed by fluorescence detection of the N-substituted cyanobenz[f]isoindole derivative. The derivative of the drug was synthesized and its spectral and fluorescence properties were evaluated. The fluorescence quantum efficiency was determined to be 0.82 in the mobile phase used for the assay. The derivative is also capable of accepting energy in an oxalate ester-hydrogen peroxide chemiluminescence system.