Analysis of Favipiravir in Human Plasma Using Fully Automated Sample Preparation LC/MS/MS System

Significance of the topic

Favipiravir is an RNA polymerase inhibitor used in antiviral therapy, requiring precise quantification in human plasma for pharmacokinetic and therapeutic monitoring. Automation of LC-MS/MS sample preparation reduces manual error, increases throughput, and enhances laboratory safety.

Objectives and Study Overview

The study aimed to develop and validate a fully automated sample preparation workflow coupled to LC-MS/MS for sensitive and reproducible quantification of favipiravir in human plasma. Key goals included reducing sample-to-sample variability, minimizing operator workload, and establishing robust calibration and QC procedures.

Methodology

A Shimadzu CLAM automated sampler performed plasma deproteinization by mixing 50 µL of EDTA-treated human plasma with internal standard ([13C,15N]-favipiravir), isopropyl alcohol, and acetonitrile, followed by PTFE membrane filtration. Prepared samples were injected (1 µL) into a Nexera X2 UHPLC system with a Shim-pack Scepter C18 column (50 × 2.1 mm, 1.9 µm). Mobile phases were 0.05 % formic acid in water (A) and acetonitrile (B) under a gradient elution. Detection used an LCMS-8060 triple quadrupole in positive MRM mode with transitions m/z 157.7→85.1 and 157.7→113.2 for favipiravir, and m/z 159.7→85.1 and 159.7→113.2 for the isotopic internal standard.

Used Instrumentation

• CLAM™ automated sample preparation system
• Shimadzu Nexera™ X2 UHPLC
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer
• Shim-pack Scepter™ C18-120 column (50 × 2.1 mm, 1.9 µm)

Main Results and Discussion

A seven-point calibration curve (1–100 µg/mL) demonstrated excellent linearity (R2 = 0.9987). Intra-assay precision (%RSD) ranged from 1.0 % to 5.6 % with accuracy between 95 % and 105 %. QC samples at 3, 50, and 90 µg/mL showed repeatability precision of 1.6 %–3.0 % and accuracy of 94 %–97 %. Between-day reproducibility over three days yielded %RSD of 0.2 %–7.6 % and accuracy of 88 %–99 %, meeting acceptance criteria of ±15 %.

Benefits and Practical Applications

Fully automated sample preparation eliminates manual pipetting, reduces risk of sample mix-up and exposure, and allows parallel processing with LC-MS/MS runs to increase throughput. This approach is well suited for pharmacokinetic studies, clinical trials, and routine therapeutic drug monitoring.

Future Trends and Possibilities

Advances may include integration of robotics for higher sample capacity, miniaturized flow paths to reduce solvent consumption, and AI-driven QC monitoring. The platform can be adapted for other low-molecular-weight drugs and biomarkers, supporting personalized medicine and high-throughput screening.

Conclusion

The fully automated LC-MS/MS workflow for favipiravir in human plasma demonstrated high sensitivity, precision, and accuracy. Its robust performance and reduced manual workload make it an effective solution for clinical and research laboratories requiring reliable antiviral quantification.

References

• Shimadzu Application News No. C230: Analysis of Favipiravir in Human Plasma Using Fully Automated Sample Preparation (2020)