Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection

An improved method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid (alendronate) in human urine and an assay in human plasma are described. The methods are based on co-precipitation of the bisphosphonate with calcium phosphates, automated pre-column derivatization of the primary amino group of the bisphosphonic acid with 2,3-naphthalene dicarboxyaldehyde (NDA)—N-acetyl- d-penicillamine (NAP) or cyanide (CN −) reagents, and high-performance liquid chromatography (HPLC) with electrochemical (ED) or fluorescence detection (FD). The feasibility of ED of the NDA—CN − derivative of alendronate has been demonstrated, and a HPLC—ED assay in human urine has been validated in the concentration range 2.5–50.0 ng/ml. In order to eliminate the cyanide ion from the assay procedure, several other nucleophiles in the NDA derivatization reaction were evaluated. An NDA—NAP reagent was found to produce highly fluorescent derivatives of alendronate. The assay in urine based on NDA—NAP derivatization and HPLC—FD has been developed and fully validated in the concentration range 1–25 ng/ml. Based on the same NDA—NAP derivatization, an assay in human plasma with a limit of quantification of 5 ng/ml has also been developed. Both HPLC—FD assays were utilized to support various human pharmacokinetic studies with alendronate.