Simultaneous Analysis of Remdesivir and Metabolites in Human Plasma

Importance of the topic

Remdesivir is a key antiviral prodrug used in the treatment of RNA virus infections, including Ebola and COVID-19. Accurate and simultaneous measurement of remdesivir and its active metabolite GS-441524 in human plasma is essential for pharmacokinetic studies, therapeutic drug monitoring and ensuring the safety and efficacy of antiviral treatments.

Objectives and Overview

This study aimed to develop and validate a highly selective and robust LC-MS/MS analytical method capable of quantifying remdesivir and GS-441524 simultaneously in human plasma. The method was evaluated for linearity, accuracy, precision and long-term stability to support high-throughput bioanalysis in research laboratories and clinical settings.

Methodology

A protein-precipitation protocol was used for sample preparation. Human plasma spiked with known concentrations of remdesivir, GS-441524 and their stable isotope-labeled internal standards ([U-Ring-13C6]-remdesivir and [13C5]-GS-441524) was treated with isopropanol and acetonitrile, mixed, centrifuged and the supernatant analyzed.
Calibration curves were constructed over five concentration levels for each analyte: 100–5000 ng/mL for remdesivir and 5–500 ng/mL for GS-441524. Multiple reaction monitoring (MRM) in positive electrospray ionization mode was employed for quantitation and confirmation.

Instrumentation

• Ultrahigh-performance liquid chromatography: Shimadzu Nexera X2 UHPLC system with Shim-pack Scepter C18-120 column (50 × 2.1 mm, 1.9 μm).
• Mobile phases: 0.05% formic acid in water (A) and in acetonitrile (B); gradient from 5% to 90% B over 2.8 minutes, flow rate 0.4 mL/min, column temperature 40 °C, injection volume 2.0 μL.
• Mass spectrometry: Shimadzu LCMS-8060 triple quadrupole with heated ESI, MRM acquisition; interface temperature 300 °C, heat block 400 °C, desolvation and nebulizing gas flows optimized for sensitivity.

Main Results and Discussion

Calibration curves exhibited excellent linearity (r2 > 0.999) across the tested ranges. Precision (%RSD) was 0.8–1.8% for remdesivir and 2.2–5.0% for GS-441524. Accuracy ranged from 92.0–107% and 94.8–106%, respectively, all within ±15% acceptance criteria.
A robustness study involving 100 consecutive injections of plasma spiked at mid-range concentrations demonstrated stable response with %RSD of 2.2–3.9% for both analytes, confirming the method’s suitability for extended analytical batches.

Benefits and Practical Applications

• High sensitivity and selectivity enable reliable quantification at low ng/mL levels.
• Rapid chromatographic run time (<5 min) supports high sample throughput.
• Robust performance over extensive injection series reduces instrument downtime and maintenance.
• Suitable for pharmacokinetic profiling, clinical PK/PD studies and quality control in drug development.

Future Trends and Potential Applications

• Extension to additional remdesivir metabolites or related antiviral compounds in multiplex assays.
• Integration with automated sample preparation platforms for large-scale clinical trials.
• Miniaturization and microflow LC-MS approaches to further enhance sensitivity and reduce solvent consumption.
• Coupling with pharmacometric modeling to inform dose optimization and personalized therapy.

Conclusion

The developed UHPLC-MS/MS method provides a rapid, accurate and highly reproducible approach for simultaneous analysis of remdesivir and GS-441524 in human plasma. Its robust performance and high throughput make it well suited for support of antiviral drug research and clinical monitoring.

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 Central Science.