Determination of zoledronic acid in human urine and blood plasma using liquid chromatography/electrospray mass spectrometry

A new method for the analysis of 1-hydroxy-2-imidazol-1-yl-phosphonoethyl phosphoric acid (zoledronic acid) in urine and blood samples has been developed. It consists of a derivatisation of the bisphosphonate with trimethylsilyl diazomethane under multiple methylester formation. The formed derivative can, in contrast to the non-derivatised analyte, easily be separated by reversed phase liquid chromatography due to its reduced polarity. Detection is performed by electrospray tandem mass spectrometry. For calibration purposes, a deuterated internal standard has been synthesised in a three-step synthesis starting with d4-imidazole. For human urine, the limit of detection (LOD) is 1.2 x 10−7 mol/L, limit of quantification (LOQ) is 3.75 × 10−7 mol/L in the MRM mode. For human blood plasma, a LOD of 1 × 10−7 mol/L and a LOQ of 2.5 × 10−7 mol/L were determined. The linear dynamic range comprised 3.5 decades starting at the limit of quantification. The method was successfully applied for the analysis of spiked urine and blood plasma samples as well as samples from two osteoporosis patients.