Comprehending COVID-19: LC-MS/MS Analysis of Small Molecule Anti-Viral and Anti-Inflammatory Drugs in Plasma in Clinical Research

Significance of the topic

Clinical research into repurposed small-molecule antivirals and anti-inflammatory agents requires robust quantification in plasma for pharmacokinetic and pharmacodynamic studies, guiding therapeutic strategies and ensuring patient safety.

Objectives and study overview

This study aimed to develop and validate a rapid LC-MS/MS assay for simultaneous measurement of nine compounds in plasma: favipiravir, remdesivir, GS-441524, chloroquine, hydroxychloroquine, desethylchloroquine, lopinavir, ritonavir, and dexamethasone.

Methodology

A 50 µL plasma sample underwent protein precipitation with ZnSO4/methanol containing internal standards. After centrifugation, the supernatant was diluted and injected (15 µL) onto an ACQUITY UPLC I-Class system fitted with a CORTECS T3 (2.1 x 50 mm, 2.7 µm) column at 45 °C. Gradient elution employed ammonium formate/formic acid in water (mobile phase A) and methanol (mobile phase B) over a 3.3-minute run. Detection used a Xevo TQ-S micro mass spectrometer in ESI+ MRM mode. Data processing utilized MassLynx v4.2 and TargetLynx XS.

Used instrumentation

• ACQUITY UPLC I-Class PLUS System
• Xevo TQ-S micro Triple Quadrupole MS
• CORTECS T3, 2.1 mm x 50 mm, 2.7 µm column
• MassLynx v4.2 with TargetLynx XS

Main results and discussion

The method showed linear calibration (r2 > 0.995) and high sensitivity (S/N >25:1 at the lowest calibrator). Precision (≤9.1%) and accuracy (94–108%) were maintained over low, mid, and high QC levels across five days. Room temperature stability was limited for remdesivir, while freeze-thaw and autosampler stability (124 hours at 8 °C) were acceptable. Matrix effects were mitigated by internal standards, and extraction efficiencies were reproducible, although non-polar analytes exhibited lower recovery.

Benefits and practical application

• High-throughput sample preparation suitable for large clinical studies
• Simultaneous quantitation of multiple COVID-19 drug candidates in a single short run
• Robust sensitivity and wide dynamic range to support dose-escalation and PK/PD profiling

Future trends and opportunities

Further work may focus on quantifying phosphorylated metabolites of nucleoside analogues within PBMCs, addressing analytical challenges via metal chelators or high-performance surface (HPS) technologies. Expansion to new antiviral classes and incorporation of automated sample preparation will enhance throughput and data reliability.

Conclusion

A rapid, robust LC-MS/MS assay has been established for nine therapeutic agents in plasma, offering a reliable tool for clinical research on COVID-19 treatments. Its performance characteristics support accurate PK/PD evaluation, guiding repurposing efforts and future antiviral development.

References

• FDA. Bioanalytical Method Validation Guidance for Industry, 2018.
• Avantaneo V. et al. Development and Validation of a UHPLC-MS/MS Method for Quantification of Remdesivir and GS-441524. J Antimicrob Chemother. 2020;75(7):1772–1777.
• Warren TK. et al. Therapeutic Efficacy of GS-5734 Against Ebola Virus in Rhesus Monkeys. Nature. 2016;531:381–385.