title: Evaluation of a LC-MS method for everolimus preclinical determination in brain by using [(13)C2D4]RAD001 internal standard.
abstract: Isotopic internal standards are increasingly frequent in LC-MS analysis to control biological matrix effects in the quantitation of immunosuppressant drugs, such as everolimus (RAD001). Here we present the evaluation of a LC-MS method, exploiting [(13)C2D4]RAD001 as internal standard, for preclinical determination of RAD001 in mice brain tissue. Samples were purified by solid phase extraction. Brain and blood were collected from vehicle-treated and RAD001-treated mice. The QTOF MS detector was set to select RAD001 ammonium adducts (m/z 975.6152) and [(13)C2D4]RAD001 (m/z 981.6481). Two different UHPLC columns were preliminarily tested. The method showed linear behavior between 4 and 100ng/mL (r(2)=0.99943) and linearity was preserved in the presence of blood (r(2)=0.99107) and brain (r(2)=0.99098) matrix components. Intra-day and inter-day precision (3-19%) and accuracy (82-109%) were comparable between standards and spiked blood and brain samples. As resulting from recovery comparison (82-98%), [(13)C2D4]RAD001 compensated ion suppression phenomena maintaining method performance over a wide range of consecutive analytical runs. The comparison with a HPLC-UV method showed reliability of the method with good correlation between blood (r(2)=0.94319) and brain (r(2)=0.97773) samples and acceptable biases (<15%). This validation suggests that the investigated method could be useful for the preclinical monitoring of RAD001 brain therapeutic concentrations in animal models.
authors: Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy. Electronic address: stefano.giovagnoli@unipg.it.
authors: Stefano
authors: S
authors: Giovagnoli
authors: Department of Clinical and Experimental Medicine, Medical School, University of Foggia, viale Luigi Pinto, 1, 71100 Foggia, Italy.
authors: Tommaso
authors: T
authors: Cassano
authors: Department of Clinical and Experimental Medicine, Medical School, University of Foggia, viale Luigi Pinto, 1, 71100 Foggia, Italy.
authors: Lorenzo
authors: L
authors: Pace
authors: Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy.
authors: Alessandro
authors: A
authors: Magini
authors: Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy.
authors: Alice
authors: A
authors: Polchi
authors: Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy.
authors: Brunella
authors: B
authors: Tancini
authors: Department of Biochemical Sciences, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.
authors: Marzia
authors: M
authors: Perluigi
authors: Laboratory of Virology - Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy.
authors: Federico
authors: F
authors: De Marco
authors: Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy.
authors: Carla
authors: C
authors: Emiliani
authors: Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy.
authors: Diego
authors: D
authors: Dolcetta
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002247:Carbon Isotopes / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D002853:Chromatography, Liquid / Q000379:methods*; D003903:Deuterium / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D000068338:Everolimus; D016014:Linear Models; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D002247:Carbon Isotopes; D000068338:Everolimus; D003903:Deuterium; D020123:Sirolimus
keywords: Biological samples; Brain levels; Everolimus; Isotopically labeled internal standard; LC–MS