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

Stefano Giovagnoli, Tommaso Cassano, Lorenzo Pace, Alessandro Magini, Alice Polchi, Brunella Tancini, Marzia Perluigi, Federico De Marco, Carla Emiliani, Diego Dolcetta

Research output: Contribution to journalArticlepeer-review


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 [13C2D4]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 [13C2D4]RAD001 (m/z 981.6481). Two different UHPLC columns were preliminarily tested. The method showed linear behavior between 4 and 100ng/mL (r2=0.99943) and linearity was preserved in the presence of blood (r2=0.99107) and brain (r2=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%), [13C2D4]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 (r2=0.94319) and brain (r2=0.97773) samples and acceptable biases (

Original languageEnglish
Pages (from-to)155-163
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Publication statusPublished - Mar 5 2015


  • Biological samples
  • Brain levels
  • Everolimus
  • Isotopically labeled internal standard
  • LC-MS

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Cell Biology
  • Clinical Biochemistry
  • Medicine(all)


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