Derivatization of pamidronate and other amino(bis)phosphonates with different isothiocyanates prior to ion-pair liquid chromatography

The derivatization of the amino group of pamidronate and other amino(bis)phosphonates with UV absorbing or fluorescent isothiocyanates (ITC) has been investigated. After derivatization at 80°C and pH> 12 for 5-30 min and after liquid-liquid (LL) extraction, the reaction mixtures are analysed by ion-pair liquid chromatography with UV-Vis and/or fluorescence detection. The derivatization reaction results in the formation of both a thiocarbamyl and a carbamyl derivative of the respective amino(bis)phosphonates. Conversion of a thiocarbamyl-amino(bis)phosphonate into its carbamyl derivative can be achieved by oxidation using diluted hydrogen peroxide. The identity of the reaction products of pamidronate has been confirmed by fast atom bombardment mass spectrometry. A possible reaction mechanism for the oxidative desulphuration during derivatization, based on an autocatalytic effect, is postulated; supportive evidence is obtained by chromatographic analysis of different amino(bis)phosphonate derivatives after a reaction with phenylisothiocyanate. The most promising ITC reagent for bioanalysis of pamidronate is 1-naphthylisothiocyanate.