Anion-exchange separation and determination of bisphosphonates and related analytes by post-column indirect fluorescence detection

Bisphosphonic acids and their salts can be detected after their liquid chromatographic separation by post-column indirect fluorescence detection (IFD). After separation the analyte is combined with the highly fluorescent Al³⁺–morin (2′,3,4′,5,7-pentahydroxyflavone) solution and fluorescence decreases because of the formation of the nonfluorescent Al³⁺–bisphosphonate complex. The decrease in fluorescence is proportional to the amount of bisphosphonate present. Separation of the multivalent anionic bisphosphonate analytes from other anions and sample matrix is achieved on a strong base anion-exchange column with a strong, basic eluent. The post-column reaction variables, which influence IFD, are identified and optimized for the detection of the bisphosphonates after separation on the anion exchanger. The method is selective, since only a few anions will undergo a reaction with the Al³⁺–morin solution, and sensitive, detection limit for difluoromethylene bisphosphonate, F₂MDP, is 4 ng for S/N=3. The separation–IFD method can be applied to the determination of bisphosphonates, such as F₂MDP, ethane-1-hydroxy-1,1-bisphosphonic acid, dichloromethylene bisphosphonic acid, 4-amino-1-hydroxybutane-1,1-bisphosphonic acid, in biological samples. The separation–IFD method is also applicable to the detection of inositol phosphate anions.