title: Development and validation of a sensitive solid-phase-extraction (SPE) method using high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) for determination of risedronate concentrations in human plasma.
abstract: Risedronate is a commonly prescribed bisphosphonate for the treatment of bone disorders. Due to its high polarity and low oral bioavailability, low concentrations of risedronate are expected in human plasma and therefore a sensitive assay is required to serve in pharmacokinetic studies. Here, we describe the development and validation of an LC-MS/MS assay for the measurement of risedronate concentrations in human plasma. Risedronate and the internal standard, risedronate-d4, were derivatized on an anion exchange solid-phase extraction cartridge. Trimethylsilyl-diazomethane which is a thermally stable and relatively non-toxic derivatization agent was used to methylate the risedronate phosphonic acid groups and decrease analyte polarity. Following extraction, the analytes were separated on a Phenomenex Gemini C18 column (150 mm×2.0 mm, 5 μm), using a gradient of ammonium acetate 10 mM and acetonitrile with a flow rate of 300 μL/min. The assay calibration range was 0.2-25 ng/mL. The calibration curve of risedronate standards spiked in six individual plasma samples was linear (r²=0.9998). Accuracy (percent deviation from nominal) and precision (percent coefficient of variation) at concentrations 0.5, 5 and 20 ng/mL, and at the lower limit of quantification (LLOQ) of 0.2 ng/mL were excellent at <6%. Mean recovery was 54% for risedronate and 51% for the internal standard. Risedronate was stable in human plasma samples for at least 5 h at room temperature, 101 days frozen at -80°C, 72 h in an autosampler at 10°C, and for three freeze/thaw cycles. The validated assay method successfully quantified the concentrations of risedronate in plasma samples from informed consenting healthy volunteers administered a single 35 mg risedronate tablet.
authors.affiliation: Centre for Integrated Preclinical Drug Development, The University of Queensland, Brisbane, QLD, Australia. s.ghassabian@uq.edu.au
authors.forename: Sussan
authors.initials: S
authors.lastname: Ghassabian
authors.forename: Linda A
authors.initials: LA
authors.lastname: Wright
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Dejager
authors.forename: Maree T
authors.initials: MT
authors.lastname: Smith
mesh_terms: D000082:Acetaminophen / Q000097:blood / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003978:Diazomethane / Q000031:analogs & derivatives; D004355:Drug Stability; D012968:Etidronic Acid / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D006461:Hemolysis; D006801:Humans; D007052:Ibuprofen / Q000097:blood / Q000302:isolation & purification; D012044:Regression Analysis; D015203:Reproducibility of Results; D000068296:Risedronic Acid; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014297:Trimethylsilyl Compounds
chemical_list: D014297:Trimethylsilyl Compounds; D000082:Acetaminophen; D003978:Diazomethane; D000068296:Risedronic Acid; D012968:Etidronic Acid; C060070:trimethylsilyldiazomethane; D007052:Ibuprofen