Abstract 5749: Determination of the mTOR inhibitor temsirolimus and its active metabolite sirolimus in human blood, plasma, cerebrospinal fluid, and brain tumor tissue using a validated LC-MS/MS assay

Abstract Temsirolimus is an FDA approved mTOR (mammalian target of rapamycin) inhibitor that is presently under investigation in multiple clinical trials for various cancers, including for recurrent high-grade glioma. Here, we report A robust, sensitive, and extremely rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of total concentrations of temsirolimus and its active metabolite sirolimus in human blood, plasma, brain tumor and cerebrospinal fluid (CSF) samples. The method was validated for the simultaneous determination of both analytes over a concentration range of 1-1000 nM within a total run time of 2.6 min. Simple one-step protein precipitation with methanol was employed to extract analytes from biomatrices. Chromatographic separation of analytes was achieved on Kinetex™ EVO C18 (50 × 2.1 mm, 1.7 µm) column using gradient elution with mobile phases of 5 mM ammonium formate and methanol at a flow rate of 0.5 mL/min and an injection volume of 5 µL. Detection was performed with a Sciex QTRAP 6500+ mass spectrometer, utilizing multiple reaction monitoring mode to register the precursor-to-product ion transitions under positive electrospray ionization. The maximum coefficient of variation for both intra- and inter-day precision was 12.3%, with accuracy ranging from 85.6% - 114.2% in all matrices. Combined temsirolimus and sirolimus samples were determined to be stable at room temperature (RT) in whole blood and brain homogenate for at least 4 hours and in plasma for at least 1 hour. At -80±15°C in a freezer, samples in plasma, brain homogenate, and whole blood were stable for at least 132 days. Stock and working solution stability was also evaluated to be stable for at least 4 hours (RT) and 113 days (-20°C nominal for stock solutions and 4°C for working solutions). The validated method is currently employed in a clinical trial evaluating the efficacy of super-selective intra-arterial infusion for the CNS delivery of temsirolimus in recurrent high-grade glioma patients (NCT05773326) to determine temsirolimus and sirolimus pharmacokinetics in blood, brain tumor, plasma, and CSF. Citation Format: Gerold S. Kaplan, Tigran Margaryan, Nader Sanai, Artak Tovmasyan. Determination of the mTOR inhibitor temsirolimus and its active metabolite sirolimus in human blood, plasma, cerebrospinal fluid, and brain tumor tissue using a validated LC-MS/MS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5749.