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

• Samples were obtained from vehicle and everolimus-treated C57BL/6 mice.
• (13C2D4)RAD001 was evaluated for the first time on brain tissue samples.
• Good method performance on a wide range of concentrations.
• The use of (13C2D4)RAD001 limits biological matrix effects.
• The method could be used for preclinical monitoring of everolimus.

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 100 ng/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 (<15%). This validation suggests that the investigated method could be useful for the preclinical monitoring of RAD001 brain therapeutic concentrations in animal models.