Chromatographic analysis of bisphosphonates.

Chromatographic analysis of bisphosphonates in the past has been based primarily on reversed-phase liquid chromatography (RPLC) and ion-exchange chromatography. Gas chromatography (GC) and recently even capillary electrophoresis have also been employed. For bioanalysis, pre-treatment of the sample is a major part of the analysis; protein precipitation, calcium precipitation, solid-phase extraction (SPE) and derivatization have demonstrated to play an important role in bisphosphonate assays. For some of these treatments, for example SPE and derivatization, automation may be possible. Derivatization is a prerequisite for GC analysis of bisphosphonates; a volatile derivative has to be formed. For liquid chromatography, two types of derivatization are known for bisphosphonates. First, the bisphosphonate side chain can be modified by a chemical reaction to yield a derivative with advantageous chromatographic and spectroscopic properties. Secondly, by complexation of both phosphonate groups or of phosphate after decomposition of the analyte, a coloured complex can be formed. The most sensitive bioanalytical methods are based on RPLC and fluorescence detection, if necessary after derivatization. If low detection limits are not required, for example for analysis of pharmaceutical preparations, non-specific detection methods can be applied.