Quantitation of celecoxib and four of its metabolites in rat blood by UPLC-MS/MS clarifies their blood distribution patterns and provides more accurate pharmacokinetics profiles.

A sensitive UPLC-MS/MS method was established and validated for the quantitation of celecoxib and its metabolites in rat blood. The analytes were extracted from rat blood samples by a salting-out liquid-liquid extraction method followed by the UPLC chromatography. The mass analysis of effluent was performed on an API 5500 Qtrap mass spectrometer via multiple reactions monitoring (MRM). The linear response ranges were 0.3-20000nM for celecoxib, and 1.2-20000nM, 0.3-20000nM, 2.0-2000nM, 1.5-6000nM for its metabolites carboxycelecoxib (M2), hydroxycelecoxib (M3), hydroxycelecoxib glucuronide (M1), and carboxycelecoxib glucuronide (M5), respectively. The inter-day and intra-day accuracies were within 85-115%, and the inter-day and intra-day precision were acceptable (<12%) for all analytes. Recoveries were above 70% and no obvious matrix effects were observed. The validated UPLC-MS/MS method was successfully applied to a pharmacokinetics study of oral celecoxib (20mg/kg) in Sprague-Dawley rats, and the rat blood concentrations (0-48h) of celecoxib and two of its metabolites M2 and M3 were successfully determined. Using the same method, we also showed preferential distributions of celecoxib, M2 and M3 in the blood cells as compared to the plasma. In conclusion, our results showed that our validated LC-MS/MS method can be successfully used for the pharmacokinetic studies of celecoxib and that the blood cells are a very important compartment for this drug such that profiles of celecoxib and its metabolites in whole blood will be more comprehensive and accurate representation of their profiles in vivo than the plasma.