Simultaneous Analysis of Remdesivir and Metabolites in Human Plasma
Remdesivir (brand name: Veklury®), which was developed by Gilead Science (U.S.) for treatment of Ebola virus disease, is a prodrug having antiviral activity against single-strand RNA viruses. It is known to be partly metabolized to activated GS-441524, the main metabolite of remdesivir, in vivo1). In this article, we present the results of research into an analytical system that can analyze remdesivir and its metabolites simultaneously using LC/MS/MS, an analytical method that demonstrates outstanding selectivity (Fig. 1).
Analysis Conditions and Sample Pretreatment
Remdesivir (P/N: C8799*) and GS-441524 (P/N: C8847*), as the target compounds, and [U-Ring-13C6]-remdesivir (P/N: C8845*) and [13C5]-GS-441524 (P/N: C8855*), as their stable isotopes, were purchased from Alsachim, one of the companies of the Shimadzu Group. [U-Ring-13C6]-remdesivir and [13C5]-GS- 441524 were used as materials of the internal standard. The structural formulas of remdesivir and GS-441524 are shown in Fig. 2. To commercially available human plasma treated with EDTA 2K, remdesivir and GS-441524 were added. Following this, the calibration curves were prepared. Analysis was performed using the LC and MS analysis conditions shown in Table 1 and the multiple reaction monitoring (MRM) data acquisition parameters shown in Table 2. Shim-pack ScepterTM C18-120 (50 mm×2.1 mm I.D., 1.9 μm) was used as the analytical column. Fig. 3 shows theMS chromatograms. Calibration was performed using 5 calibration points at concentrations of 100, 500, 1000, 2500 and 5000 ng/mL for remdesivir and 5 calibration points at concentrations of 5, 25, 50, 250 and 500 ng/mL for GS-441524 (n = 5 for each calibration point). [U-Ring-13C6]-remdesivir (2.5 μg/mL) and [13C5]-GS- 441524 (0.25 μg/mL) were mixed with methanol to be used as the internal standard (ISTD).
The samples are prepared in the following sequence. After 20 μL of 75% IPA, 50 μL of plasma, 10 μL of ISTD and 100 μL of acetonitrile were added and mixed well, the mixture was centrifuged. The supernatant obtained by centrifuging was transferred into an LC vial for analysis.
Preparation of Calibration Curves
Calibration curves of plasma-spiked samples are shown in Table 3. For Remdesivir and GS-441524, good linearity was obtained in the set calibration range. The precision (reproducibility) of remdesivir and GS-441524 in the entire concentration range, including the quantitative lower limit was %RSD 0.8 %－1.8 % and %RSD 2.2 %－5.0 %, respectively. Similarly, the accuracy of remdesivir and GS-441524 was 92.0 %－107 % and 94.8 %－106 %, indicating that the accuracy of both was within 100 ± 15 %.