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

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.


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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain Chemistry*
  • Carbon Isotopes / analysis*
  • Carbon Isotopes / chemistry
  • Carbon Isotopes / pharmacokinetics
  • Chromatography, Liquid / methods*
  • Deuterium / analysis*
  • Deuterium / chemistry
  • Deuterium / pharmacokinetics
  • Everolimus
  • Linear Models
  • Male
  • Mass Spectrometry / methods*
  • Mice
  • Mice, Inbred C57BL
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sirolimus / analogs & derivatives*
  • Sirolimus / analysis
  • Sirolimus / chemistry
  • Sirolimus / pharmacokinetics


  • Carbon Isotopes
  • Everolimus
  • Deuterium
  • Sirolimus