The development and validation of sensitive LC-MS/MS method for quantitative bioanalysis of carmofur in mouse plasma and its application to pharmacokinetic study

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). Chromato-graphic separation was achieved on an XBridge BEH C18 XP column (100 mm x 3 mm, 2.5 mu m) 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-* m/z 129.01 for carmofur and m/z 145.53-* 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.