Simultaneous Analysis of Remdesivir and Metabolites in Human plasma Using Fully Automated Sample Preparation LC/MS/MS System

Importance of the topic

The antiviral prodrug remdesivir and its primary metabolite GS-441524 are critical targets for pharmacokinetic and therapeutic drug monitoring in the treatment of RNA virus infections. A reliable, high-throughput analytical method supports drug development, dose optimization, and safety evaluation in clinical and research laboratories.

Objectives and overview of the study

This work aimed to develop and validate a fully automated sample preparation and LC-MS/MS workflow for simultaneous quantification of remdesivir and GS-441524 in human plasma. The goals were to reduce manual handling, decrease analytical variation, and accelerate sample throughput while maintaining regulatory-level accuracy and precision.

Methodology

Human plasma samples were spiked with remdesivir and GS-441524 along with their stable isotope-labeled internal standards ([U-Ring-13C6]-remdesivir and [13C5]-GS-441524). Sample pretreatment was performed automatically by mixing plasma with isopropanol, internal standard solution and acetonitrile, followed by protein precipitation, shaking and filtration through a PTFE membrane.

Used Instrumentation

• Automated sample preparation system (CLAM + LC/MS/MS).
• UHPLC system: Shimadzu Nexera X2.
• Analytical column: Shim-pack Scepter C18-120 (50 × 2.1 mm, 1.9 μm).
• Triple quadrupole mass spectrometer: Shimadzu LCMS-8060 with heated ESI interface.

Main results and discussion

Chromatographic separation achieved clear peaks for remdesivir, GS-441524 and their isotopically labeled analogs within a seven-minute cycle time. Calibration curves (5 levels) exhibited excellent linearity (R2 > 0.998). Intra- and inter-day precision (%RSD) ranged from 0.5 % to 2.9 % for remdesivir and 2.4 % to 4.9 % for GS-441524 across the calibration range. Accuracy values were within 87.8 %–108 % for remdesivir and 94.5 %–105 % for GS-441524. QC samples at low, medium and high concentrations confirmed repeatability and reproducibility within acceptance criteria (100 ± 15 %).

Benefits and practical applications

• Elimination of manual protein precipitation steps reduces variation and risk of sample cross-contamination.
• Parallel sample prep and analysis shorten turnaround time to under seven minutes per sample.
• Robust accuracy and precision meet regulatory guidelines for bioanalytical assays.
• Applicable to pharmacokinetic studies, therapeutic drug monitoring, and high-throughput antiviral research.

Future trends and possibilities

Advances in automation and multiplexed LC-MS/MS analysis will enable broader panels of antiviral and metabolic markers in a single run. Integration with laboratory information management systems (LIMS) can further enhance traceability. Emerging technologies such as microflow LC and novel ionization sources may improve sensitivity and reduce solvent consumption.

Conclusion

The fully automated sample preparation LC-MS/MS method provides a rapid, robust and reproducible approach for simultaneous quantification of remdesivir and GS-441524 in human plasma. High throughput and regulatory-grade performance make it suitable 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.