LC–MS/MS method for the determination of clodronate in human plasma

Abstract

Clodronate belongs to the class of bisphosphonates which are used for the treatment of bone disorders. Due to its high polarity it has a low and highly variable oral bioavailability which results in low plasma con- centrations and requires sensitive bioanalytical methods to characterize its pharmacokinetics in human. Here, we describe for the first time the development and validation of a LC–MS/MS assay for the quan- tification of clodronate in human plasma. The bisphosphonate was isolated from the biological matrix by protein precipitation using perchloric acid (10%), and derivatized with trimethylorthoacetate prior sample clean-up with liquid–liquid extraction using methyl tert-butyl ether. The chromatography was performed using an isocratic elution with ammonium acetate 5 mM (85% v/v, pH 3.8) and acetonitrile (15% v/v) as mobile phase with a flow rate of 300 l/min on a reversed-phase column (Supelco Ascentis® , C18) temporized at 50 ◦ C. The mass spectrometric detection was done using the API4000 triple quadruple mass spectrometer monitoring the mass/charge transitions 301.0/145 for clodronate and 305.2/137.1 for the internal standard etidronate. The analytical range was set to 5–800 ng/ml, allowing an evaluation of the plasma concentration–time profiles of clodronate for approximately 7–8 half-life (∼24 h). The method was validated according to cur- rent FDA/EMA guidelines on bioanalytical method validation with respect to specificity, linearity, intra- and inter-day accuracy and precision, matrix effect, recovery as well as stability. The precision of the assay was 0.6–6.9% and 0.6–8.1% for the intra-day and inter-day variability, respectively. The intra-day and inter-day accuracy (error) was 0.6–8.8% and 2.2–4.5%. The recovery of the analyte was low (2–3%) but reproducible over the entire validation range and sufficient to monitor the target concentrations in human plasma. The drug was shown to be stable in plasma at room temperature for at least 3 h (96.0 ± 6%) and for at least 24 h when stored in the cooled autosampler at 4 ◦ C (102.4 ± 4.5%). Clodronate can also undergo up to three freeze–thaw cycles without impaired stability. Thus, the method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to measure plasma concen- trations of clodronate. Finally, the developed method was successfully applied to study the clodronate serum levels in a pharmacokinetic study in healthy volunteers.