Bioanalytical considerations for quantification of ondansetron in rat microdialysis study using LC-MS/MS

Background: Ondansetron is an anti-emetic drug and has been recently identified as a therapeutic for neuropathic pain. Permeability of ondansetron into the nervous system may be impacted by the presence of P-glycoprotein (Pgp) at the blood brain barrier, since ondansetron is a substrate of Pgp, which means it may not easily cross into the brain. In order to determine a drug concentration within the brain, microdialysis can be used, which measures unbound analyte concentrations in tissues or body fluids. Aim: The purpose of this study was to develop and validate an LC-MS/MS method to measure ondansetron concentrations in serum and brain microdialysates for pharmacokinetic studies in rats. To the best of our knowledge, a fully validated method for the quantification of ondansetron in brain microdialysis studies has not been reported so far. Methods: Chromatographic separation was achieved at a flow rate of 0.6 mL/min using the Gemini C18 column (5 µm; 50 × 4.6 mm) and a mobile phase consisting of 10 mM ammonium formate with 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile (55:45, v/v). Serum samples were prepared by protein precipitation; for microdialysate, no specific cleaning procedure was needed, they were only diluted with the internal standard solution. Results: The presented method requires only a small volume of serum (2.5 µL) and microdialysate (15–20 µL), which allows for frequent sampling in animals. Moreover, it enables high throughput due to a short run time and straightforward sample preparation. Artificial cerebrospinal fluid (aCSF) was confirmed to be useful as a surrogate matrix for brain microdialysate. The lower limit of quantitation for ondansetron was 0.01 µg/mL for serum and 0.025 ng/mL for microdialysate. A significant ion suppression for ondansetron was observed in brain microdialysate and aCSF, but the IS-normalized matrix factor was close to 1.0. Conclusion: The developed LC-MS/MS method met the FDA and EMA validation criteria and was successfully applied to the in vivo pilot pharmacokinetic study. It was sensitive enough to capture the low ondansetron concentrations in brain microdialysate and universal enough to measure high ondansetron concentrations in rat serum.