A simple and sensitive assay for eflornithine quantification in rat brain using pre-column derivatization and UPLC-MS/MS detection

摘要

Eflornithine (-difluoromethylornithine) has been used to treat second-stage (or meningoencephalitic-stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for 5min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse-phase C-18 UPLC column with a 6-min gradient; elution occurred at approximately 1.5min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (similar to 70% recovery). Derivatized eflornithine was stable in the autosampler (6 degrees C) for at least 24h. This new assay had acceptable intra- and interday accuracy and precision over a wide dynamic range (5000-fold) and excellent sensitivity with a lower limit of quantification of 0.1 mu m (18ng/mL) using only 10L of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright (c) 2014 John Wiley & Sons, Ltd.