LC-MS/MS assay of riluzole and etoricoxib in rat plasma and brain tissue with applications for sampling and evaluation in pre-clinical rat model of traumatic brain injury

Abstract

A sensitive and robust liquid chromatography – tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify the concentration levels of selective cyclo-oxygenase- 2 inhibitor, etoricoxib, and glutamate antagonist, riluzole, in rat plasma and brain tissue. Labeled internal standards (IS) etoricoxib D4 and [13C,15N2] riluzole were used for the quantifications of etoricoxib and riluzole, respectively. Analytes riluzole and etoricoxib, as well as their IS's, were separated from rat plasma and brain tissue homogenate by means of liquid – liquid extraction using organic solvent of ethyl acetate. A Waters Acquity UPLC BEH C18 (1.7 µm) column was used for chromatographic separation of analytes on a SCIEX ExionLC AD using a binary solvent system. In conjunction, a SCIEX QTRAP 5500 mass spectrometer was used to quantify etoricoxib at m/z of 359 → 244 (IS of m/z 363 → 248) and riluzole at m/z 235 → 166 (IS of m/z 238 → 169) mass transitions using positive mode electrospray ionization (ESI) with multiple reaction monitoring (MRM). The bioanalytical method was validated over a linear concentration range of 0.25 (LLOQ at average signal to noise >10) – 600 ng/ml in plasma and 0.625 – 1500 ng/g in brain tissue for both analytes. The methods intra/inter-day accuracy and precision are well within the FDA validation recommendations range for chromatographic assays for both riluzole and etoricoxib in rat plasma. A partial validation was conducted within rat brain biomatrix, yielding an acceptable intra-day accuracy and precision for both riluzole and etoricoxib. For riluzole and etoricoxib the average matrix effect and recovery from rat plasma and brain tissue were negligible. This bioanalytical method was effectively applied to perform a pharmacokinetic study of each compound administered concurrently in healthy male Long-Evan (L.E.) rats. This method was developed as a pre-requisite for the pre-clinical pharmacokinetic and pharmacodynamic evaluations of co-treatment with riluzole and etoricoxib in rats with a controlled cortical impact (CCI) induced traumatic brain injury.