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 aldendronate 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.
Kline
W F
WF
Merck Research Laboratories, West Point, PA 19846.
Matuszewski
B K
BK
D019386:Alendronate; D001681:Biological Assay / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003486:Cyanides / Q000737:chemistry; D004164:Diphosphonates / Q000032:analysis* / Q000097:blood / Q000652:urine; D004563:Electrochemistry; D006801:Humans; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D013438:Sulfhydryl Compounds / Q000737:chemistry
D003486:Cyanides; D004164:Diphosphonates; D013438:Sulfhydryl Compounds; D019386:Alendronate