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

Importance of the topic

Quantitative analysis of antiviral agents such as remdesivir and its active metabolite GS-441524 in human plasma is crucial for pharmacokinetic profiling, therapeutic drug monitoring and clinical research. Automated sample preparation minimizes manual errors, reduces exposure risk and increases throughput.

Objectives and study overview

This work presents the development and validation of a fully automated LC/MS/MS method for simultaneous measurement of remdesivir and GS-441524 in human plasma. Key goals included reducing manual pretreatment workload, improving reproducibility between runs and ensuring robust sensitivity across clinically relevant concentration ranges.

Methodology and instrumentation

Samples were prepared using a CLAMTM+ LC/MS/MS automated system that mixes human plasma with internal standards ([U-Ring-13C6]-remdesivir and [13C5]-GS-441524), isopropanol, acetonitrile and then performs PTFE membrane filtration. Chromatographic separation was achieved on a Shim-pack Scepter C18-120 column (50 mm × 2.1 mm I.D., 1.9 μm) using a NexeraTM X2 UHPLC with a gradient of 0.05% formic acid in water and acetonitrile at 0.4 mL/min and 40 °C. Detection employed a Shimadzu LCMS-8060 triple quadrupole with heated electrospray ionization in positive MRM mode. Specific transitions were monitored for parent and labeled compounds.

Main results and discussion

Calibration curves were linear over 100–5000 ng/mL for remdesivir and 5–500 ng/mL for GS-441524 (r² > 0.9986). Intra- and inter-day precision (%RSD) ranged from 0.5% to 7.8% and accuracy remained within ±15% across most levels and ±20% at LLOQ. The total cycle time from pretreatment to analysis was approximately seven minutes per sample, demonstrating high throughput and consistent performance.

Benefits and practical applications

• Automated workflow reduces hands-on time and minimizes operator variability
• High sensitivity and reproducibility support pharmacokinetic and clinical studies
• Reduced risk of sample mix-up and laboratory exposure
• Scalable platform suitable for high-throughput drug monitoring in research or diagnostic settings

Future trends and opportunities

Advances may include integration of additional antiviral or small-molecule assays into the same automated workflow, miniaturization for microsampling, real-time data processing and application in decentralized or point-of-care environments. Machine-learning-driven optimization of MS parameters could further enhance sensitivity and selectivity.

Conclusion

The fully automated sample preparation LC/MS/MS method delivers robust, high-throughput quantification of remdesivir and GS-441524 with excellent validation metrics. This approach streamlines antiviral drug monitoring and supports reliable pharmacokinetic assessment.

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