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J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Mar 15;985:155-63. doi: 10.1016/j.jchromb.2015.01.035. Epub 2015 Feb 3.

Evaluation of a LC-MS method for everolimus preclinical determination in brain by using [(13)C2D4]RAD001 internal standard.

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Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy. Electronic address:
Department of Clinical and Experimental Medicine, Medical School, University of Foggia, viale Luigi Pinto, 1, 71100 Foggia, Italy.
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy.
Department of Biochemical Sciences, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.
Laboratory of Virology - Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy.


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.


Biological samples; Brain levels; Everolimus; Isotopically labeled internal standard; LC–MS

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