Development and validation of an LC–MS/MS method for simultaneous determination of remdesivir and its hydrolyzed metabolite and nucleoside,and its application in a pharmacokinetic study of normal and diabetic nephropathy mice

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. Remdesivir is rapidly metabolized in the body to produce derivatives: alanine metabolite (RM-442) and RDV C-nucleoside (RN). Here, the 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 T₃ column. The run time was 3.2 min. The linearity ranges of the analytes were 0.5–1,000 ng/ml for RDV and 5–10,000 ng/ml for both RM-442 and RN. 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 the blood of normal and diabetic nephropathy DBA/2 J mice after intravenous injection of RDV at 20 mg/kg. The area under the concentration–time curve of RN between the normal and diabetic nephropathy mice showed a significant difference (P < 0.01).