Method for multiple COVID-19 drugs analysis by LC-MS/MS

Significance of the topic

The ability to measure multiple COVID-19 therapeutics in a single run is crucial for drug development, therapeutic drug monitoring and pharmacokinetic studies. A unified LC-MS/MS approach with stable isotope labeled standards ensures high accuracy, precision and efficiency when analyzing compounds at trace levels in biological matrices.

Objectives and study overview

The primary goal was to develop and validate a liquid chromatography tandem mass spectrometry method for the simultaneous quantification of nine antiviral and related agents. Analytes included favipiravir, GS-441524, remdesivir, hydroxychloroquine, desethylchloroquine, chloroquine, azithromycin, ritonavir and lopinavir. Stable isotope labeled internal standards were employed to correct for matrix effects and ensure reliable quantitation across a wide dynamic range.

Methodology and instrumentation

Sample preparation involved protein precipitation followed by dilution to minimize matrix interferences. Chromatographic separation was achieved on a reversed phase column using a gradient of aqueous formic acid and acetonitrile. Key MS parameters included electrospray ionization in positive mode and multiple reaction monitoring transitions specified for each analyte and corresponding labeled standard. Major instrument settings were optimized for source temperature, gas flows and collision energies.

• Chromatography: C18 column, gradient elution over 4 minutes, column temperature 40°C, flow rate 0.5 mL/min
• Mass spectrometry: triple quadrupole with ESI positive mode, optimized MRM transitions
• Internal standards: isotopically labeled analogs for each target compound

Main results and discussion

Calibration curves demonstrated linear response over 2 to 10000 ng/mL depending on the analyte, with correlation coefficients above 0.99. Quality control samples at low, medium and high levels showed accuracy within ±10% and precision (CV) below 10%. Representative chromatograms exhibited clear separation and symmetrical peak shapes for all compounds. The use of isotope dilution effectively compensated for ion suppression and instrumental variability.

Benefits and practical application

This validated method enables high-throughput and simultaneous analysis of key COVID-19 drugs in plasma or other biological samples. It supports pharmacokinetic profiling, therapeutic monitoring and research into emerging treatment regimens. The inclusion of stable isotope labeled standards enhances data reliability and reduces the need for extensive matrix validation.

Future trends and opportunities

Advances may include extension to novel antiviral agents, integration with high resolution mass spectrometry for metabolite identification and automated sample handling for increased throughput. Microflow LC and multiplexed assays could further reduce sample and solvent consumption, meeting the demand for rapid turnaround in clinical and research laboratories.

Conclusion

A robust and efficient LC-MS/MS method with stable isotope dilution was established for nine COVID-19 therapeutics. The method meets stringent performance criteria and is suitable for research use in pharmacokinetic and therapeutic monitoring studies.

Reference

No formal reference list was provided in the source document.