Yuan_2022_Biomed.Chromatogr__e5380

Remdesivir (RDV), a phosphoramidate prodrug, has broad-spectrum antiviral activity. It is the first antiviral drug approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19. RDV is rapidly metabolized in the body to produce derivatives: alanine metabolite (RM-442) and RDV C-nucleoside (RN). Here, phosphatase inhibitor PhosSTOP and carboxylesterase inhibitor 5,5'-dithiobis-2-nitrobenzoic acid were used to improve stability of RDV in mouse blood. We developed a rapid and sensitive LC-MS/MS method to simultaneously quantify RDV, RM-442 and RN in mouse blood. Chromatographic separation was achieved by gradient elution on an ACQUITY HSS T(3) column. The run time was 3.2 min. The linearity ranges of the analytes were 0.5-1000 ng/mL for RDV, 5-10000 ng/mL for both RM-442 and RN, respectively. The method had an acceptable precision (RSD < 8.4% for RDV, RSD < 10.7% for RM-442, and RSD < 7.2% for RN) and accuracy (91.0%-106.3% for RDV, 92.5%-98.6% for RM-442, and 87.5%-98.4% for RN). This method was successfully applied to analyze RDV, RM-442 and RN in blood of normal and diabetic nephropathy DBA/2J mice after intravenous injection of RDV 20 mg/kg. The AUC(0-t) of RN between the normal and diabetic nephropathy mice had significant difference (P < 0.01).