Quantitation of zoledronic acid in murine bone by liquid chromatography coupled with tandem mass spectrometry.

ABSTRACT

An in vitro method for extraction and quantification of zoledronic acid (ZA) from murine bone was developed. Whole mouse bones were incubated in ZA solutions with predetermined concentrations and bound ZA was subsequently extracted from bone with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetra-methyl phosphonate was quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This resulted in a sensitive, accurate, and precise method that was linear over three orders of magnitude (0.0250-50.0µg/mL ZA). For quality control (QC) samples, intra-and inter-day coefficients of variance were calculated and were less than 10%. This method was then applied to an in vivo model to quantitate ZA from the femur and mandible of three mice treated with ZA for two weeks. The mean ZA extracted from the mandible was four fold higher than that extracted from the femur (3.06±0.52 vs. 0.76±0.09ng/mg, respectively) indicating that ZA did not distribute equally in the skeleton and had a preference to the mandible. In conclusion, a highly sensitive method to measure ZA from mouse skeleton was developed, which can be easily adapted to multiple mammalian models including humans receiving ZA treatment.