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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.

Author information

1
Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy. Electronic address: stefano.giovagnoli@unipg.it.
2
Department of Clinical and Experimental Medicine, Medical School, University of Foggia, viale Luigi Pinto, 1, 71100 Foggia, Italy.
3
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy.
4
Department of Biochemical Sciences, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.
5
Laboratory of Virology - Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy.

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.

KEYWORDS:

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

PMID:
25682337
DOI:
10.1016/j.jchromb.2015.01.035
[Indexed for MEDLINE]

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