Simultaneous Analysis of Remdesivir and Metabolites in Human Plasma

Importance of the Topic

Remdesivir is an approved antiviral prodrug that is rapidly metabolized to GS-441524, its active agent against RNA viruses. Accurate quantification of both compounds in human plasma is critical for pharmacokinetic studies, therapeutic drug monitoring, and clinical research on dosing optimization and safety.

Objectives and Overview of the Study

The study aimed to develop and validate a robust LC-MS/MS method for simultaneous determination of remdesivir and GS-441524 in human plasma. Key goals included achieving high sensitivity, selectivity, and long-term stability suitable for high-throughput clinical analysis.

Used Instrumentation

• UHPLC system: Shimadzu Nexera X2
• Mass spectrometer: Triple quadrupole LCMS-8060 with heated ESI interface
• Analytical column: Shim-pack Scepter C18-120 (50 mm×2.1 mm I.D., 1.9 μm)

Methodology and Sample Preparation

An aliquot of human plasma was spiked with remdesivir, GS-441524, and their stable isotopic internal standards ([U-Ring-13C6] remdesivir, [13C5] GS-441524). Proteins were precipitated by adding isopropanol, plasma, internal standard mix, and acetonitrile, followed by centrifugation. The supernatant was injected (2 μL) under gradient elution (0.05% formic acid in water/acetonitrile) at 0.4 mL/min and 40 °C.
Multiple reaction monitoring (MRM) transitions were optimized for each analyte. Calibration curves ranged from 100–5000 ng/mL for remdesivir and 5–500 ng/mL for GS-441524, with five concentration levels and five replicates per level.

Main Results and Discussion

• Linearity: R² > 0.9992 for remdesivir; R² > 0.9993 for GS-441524.
• Accuracy: 92.0 %–107 % for remdesivir; 94.8 %–106 % for GS-441524.
• Precision: %RSD 0.8 %–1.8 % (remdesivir); 2.2 %–5.0 % (GS-441524).

A robustness study with 100 repeated injections over two days demonstrated %RSD of 2.2 %–3.9 %, confirming excellent long-term stability and consistent sensitivity.

Benefits and Practical Applications

• Simultaneous quantification accelerates pharmacokinetic and drug-interaction studies.
• High throughput and stability support large clinical sample sets.
• Robust method reduces downtime and maintenance costs.

Future Trends and Potential Applications

• Extension to additional metabolites or combination antiviral therapies.
• Miniaturized or microflow LC for reduced solvent use and higher sensitivity.
• Automation and integration with clinical laboratory information systems.
• Application to other biological matrices such as tissue homogenates or cerebrospinal fluid.

Conclusion

The validated LC-MS/MS assay enables precise, accurate, and reproducible measurement of remdesivir and GS-441524 in human plasma. Its outstanding stability and sensitivity make it a valuable tool for clinical and research applications in antiviral drug development.

References

1. Richard T et al., “Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19”, ACS Cent. Sci.