title: 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 concentrations 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 quantification 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 5mM (85% v/v, pH 3.8) and acetonitrile (15% v/v) as mobile phase with a flow rate of 300μl/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 5-800ng/ml, allowing an evaluation of the plasma concentration-time profiles of clodronate for approximately 7-8 half-life (∼24h). The method was validated according to current 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 3h (96.0±6%) and for at least 24h 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 concentrations of clodronate. Finally, the developed method was successfully applied to study the clodronate serum levels in a pharmacokinetic study in healthy volunteers.
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany; Department of Pharmaceutical Chemistry, Future University, Cairo, Egypt.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Hasan
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany.
authors.forename: Gitta
authors.initials: G
authors.lastname: Schumacher
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Seekamp
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Taedken
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Siegmund
authors.affiliation: Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine, Greifswald, Germany. Electronic address: stefan.oswald@uni-greifswald.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Oswald
mesh_terms: D000284:Administration, Oral; D000328:Adult; D050071:Bone Density Conservation Agents / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics; D002138:Calibration; D056148:Chromatography, Reverse-Phase / Q000592:standards; D004002:Clodronic Acid / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics; D016903:Drug Monitoring / Q000379:methods* / Q000592:standards; D004355:Drug Stability; D005260:Female; D006207:Half-Life; D006801:Humans; D016014:Linear Models; D059625:Liquid-Liquid Extraction; D008297:Male; D010865:Pilot Projects; D012015:Reference Standards; D015203:Reproducibility of Results; D012997:Solvents / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D050071:Bone Density Conservation Agents; D012997:Solvents; D004002:Clodronic Acid
keywords: Clodronate; Etidronate; LC–MS/MS; Plasma; Quantification