ABSTRACT
Carmofur is an acid ceramidase inhibitor with superior efficacy in suppressing and killing fatally aggressive glioblastoma cell lines compared to the FDA-approved drug temozolomide. In addition to brain tumors, carmofur also gained attention as a potential lead inhibitor of the main protease (MPRO) of SARS-CoV-2. It is also reported efficacious against numerous other cancers and non-cancerous diseases including acute lung injury, dementia, Parkinson's disease, childhood ependymoma, and Krabbe disease etc. Carmofur also possesses antifungal and antimicrobial properties. Therefore, a sensitive bio-analytical method is needed in order to support further in vivo pharmacological investigation, pre-clinical and clinical studies. Herein, we report a sensitive, and reliable LC-MS/MS method for quantitative bioanalysis of carmofur using mouse plasma. The samples were prepared employing liquid-liquid extraction (LLE) technique using ethyl acetate and 2-propanol (85:15). Chromatographic separation was achieved on an XBridge BEH C18 XP column (100 mm × 3 mm, 2.5 µm) with a runtime of eight minutes. Quantification was performed in multiple reaction monitoring (MRM) mode with precursor to product ion transition of m/z 256.25 â m/z 129.01 for carmofur and m/z 145.53 â m/z 42.00 for 5-chlorouracil (IS) in negative electrospray ionization. Carmofur showed good linearity over the range of 5-1,000 ng.mL-1. The method was validated in terms of specificity, linearity, carry-over, matrix effect, recovery efficiency, accuracy, precision, dilution integrity, and stability. Finally, the method was successfully employed in a pharmacokinetic study in mouse plasma after intraperitoneal administration of the drug solution. To the best of our knowledge, this is the first report of an LC-MS/MS method for carmofur bioanalysis.