Trimethylsilyldiazomethane derivatization coupled with solid-phase extraction for the determination of alendronate in human plasma by LC-MS/MS

Alendronate is an important representative of bisphosphonates, strongly polar compounds that lack chromophores. With rare exceptions, derivatization of the analytes is necessary for bioanalysis. In this study, a rapid liquid chromatography–tandem mass spectrometry method employing pre-column derivatization was developed and validated for the determination of alendronate concentrations in human plasma. The procedure was based on derivatization with trimethylsilyldiazomethane during solid-phase extraction on a weak anion-exchange solid-phase cartridge, which integrated sample purification and derivatization into one step. The alendronate derivative was eluted with methanol. Chromatographic separation was performed on a Capcell PAK-C18 column. The total run time was 6.5 min. The calibration curve was linear in the range 1.00–1,000 ng/mL using d6-alendronate as the internal standard. The lower limit of quantification was 1.00 ng/mL. The intra- and inter-assay precision (in RSD) calculated from quality control samples was less than 15%, and the accuracy was between 98.1% and 100.2%. The validated method was successfully applied to characterize the pharmacokinetic profiles of alendronate following the intravenous infusion of 5 or 10 mg alendronate sodium to healthy volunteers.