title: A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates.
abstract: A new reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method has been developed for the determination of the following bisphosphonic acids: alendronic acid (ALEN), etidronic acid (ETID), ibandronic acid (IBAN) and risedronic acid (RISE). Separation was achieved on a C(18) column using a mixture of 50 mmol L(-1) borate buffer pH 9.0 containing 0.25 mmol L(-1) tetrabutylammonium chloride and 0.5 mmol L(-1) EDTA and acetonitrile (97:3) as the mobile phase. The sensitive detection of the above bisphosphonic acids was based on their oxidation to orthophosphate by the on-line peroxydisulfate-assisted photolysis followed by post-column reaction with molybdate to yield phosphomolybdate. This subsequently reacted with thiamine to generate thiochrome and, finally, the fluorescence of thiochrome was measured at 440 nm with excitation at 375 nm. The developed method is precise with a mean relative standard deviation of 1.3%, sensitive (with a detection limit at the nmol L(-1) level), accurate, specific, rapid (analysis time approximately 13 min) and inexpensive because to the low cost of the reagents. The assay was applied to the analysis of the four bisphosphonic acids in commercial dosage formulations, in which the excipients did not interfere with the determination. The method was also applied to the determination of etidronate, risedronate and ibandronate in human urine. Sample preparation involves precipitation of the analytes from urine along with endogenous phosphates such as calcium salts by addition of calcium chloride at alkaline pH and dissolution of the precipitate in 0.05 mol L(-1) ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid.
authors.affiliation: Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain. tpr@um.es
authors.forename: Tomás
authors.initials: T
authors.lastname: Pérez-Ruiz
authors.forename: Carmen
authors.initials: C
authors.lastname: Martínez-Lozano
authors.forename: María Dolores
authors.initials: MD
authors.lastname: García-Martínez
mesh_terms: D050071:Bone Density Conservation Agents / Q000032:analysis* / Q000302:isolation & purification / Q000652:urine; D002122:Calcium Chloride / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004164:Diphosphonates / Q000032:analysis* / Q000302:isolation & purification / Q000652:urine; D004867:Equipment Design; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D008982:Molybdenum / Q000737:chemistry; D010756:Phosphoric Acids / Q000737:chemistry; D010777:Photochemistry / Q000379:methods*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013607:Tablets / Q000032:analysis; D013831:Thiamine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry
chemical_list: D050071:Bone Density Conservation Agents; D004164:Diphosphonates; D010756:Phosphoric Acids; D000644:Quaternary Ammonium Compounds; D013607:Tablets; C005793:thiochrome; D008982:Molybdenum; C009405:tetrabutylammonium; D002122:Calcium Chloride; C003125:phosphomolybdic acid; D013831:Thiamine