Comprehending COVID-19: Mixed-Mode Chromatography for Ion Pairing Free LC-MS of Remdesivir and Remdesivir Triphosphate

Significance of the Topic

The ongoing COVID-19 pandemic has intensified efforts to understand and quantify antiviral therapeutics such as remdesivir and its metabolites. Accurate and efficient analytical methods are required to support drug discovery, in vitro assays, pharmacokinetic studies, and clinical dosing evaluations. The development of chromatography techniques that avoid ion-pairing reagents addresses challenges in sensitivity, robustness, and detector compatibility.

Objectives and Study Overview

This study presents a rapid, mixed-mode liquid chromatography–mass spectrometry (LC-MS) method for simultaneous analysis of remdesivir (GS-5734), its nucleoside metabolite GS-441524, and the active triphosphate GS-443902. By employing a hybrid reversed-phase/anion-exchange stationary phase and volatile buffers, the goal was to achieve sharp peak shapes, adequate retention, and flexibility for optical or MS detection without ion-pair reagents.

Methodology and Instrumentation

This application note utilized the Waters ACQUITY PREMIER UPLC system with quaternary pumping, an ACQUITY PREMIER PDA detector, and an ACQUITY QDa mass detector. The column was an Atlantis PREMIER BEH C18 AX (1.7 µm, 2.1 × 50 mm) maintained at 50 °C. Samples were held at 12 °C and injected in 1 µL volumes at a 0.5 mL/min flow rate.

• Mobile phases: A = acetonitrile; B = IonHance CX-MS (ammonium citrate pH 5); C = water; D = IonHance ammonium acetate pH 6.8.
• Buffers prepared by 1:5 dilution to yield 100–200 mM ammonium acetate (4% acetonitrile).
• Gradient: linear from 5 mM NH4OAc in 0% ACN to 20 mM NH4OAc in 60% ACN over 4 min, followed by re-equilibration in 0.5 min.

Main Results and Discussion

Retention factor studies showed remdesivir required at least 40–60% acetonitrile to achieve k′ between 1 and 10. Mixed-mode interactions allowed resolution of the acidic triphosphate without ion-pair reagents. pH 6.8 provided sharper peaks for GS-443902 than pH 4.8, while minor resolution adjustments between diphosphate and triphosphate forms remain possible.

• Two standard mixtures (RMD:RTP at 10:1 and 1:10) demonstrated consistent peak shapes and retention comparable to published bioanalysis conditions.
• Mass detection confirmed analyte identity through extracted m/z values, and overlay with GS-441524 illustrated no co-elution.

Benefits and Practical Applications

This method avoids strong ion-pairing reagents that can hamper ionization or require extended column equilibration. Mixed-mode columns on hybrid silica permit operation at elevated pH, expanding selectivity for both hydrophobic and ionic analytes. Fast mobile phase preparation via buffer concentrates streamlines workflow. The approach suits both UV/PDA and MS detection, supporting diverse laboratory needs in ADME, PK, and in vitro assays.

Future Trends and Opportunities

Further optimization may target peak symmetry for triphosphate analytes by adjusting injection mass or gradient slope. The methodology can be extended to other nucleotide analogs, polymerase inhibitors, or highly polar metabolites. Anticipated trends include high-throughput screening in antiviral discovery, automated sample preparation for clinical monitoring, and integration with data analytics for quantitative bioanalysis.

Conclusion

The mixed-mode Atlantis PREMIER BEH C18 AX column with ammonium acetate buffer delivers a rapid, robust LC-MS method for remdesivir and its metabolites without ion-pair reagents. The technique achieves strong retention, sharp peak shapes, and dual detection compatibility, positioning it as a versatile tool for antiviral research and clinical studies.

References

1. Williamson BN et al. Clinical Benefit of Remdesivir in Rhesus Macaques Infected with SARS-CoV-2. Nature. 2020;585:273–286.
2. Warren T et al. Therapeutic Efficacy of GS-5734 against Ebola Virus in Rhesus Monkeys. Nature. 2016;531:381–385.
3. Walter TH et al. A New Mixed-Mode Reversed-Phase/Anion-Exchange Stationary Phase Based on Hybrid Particles. Waters Application Note 720006742EN. 2020.
4. Liu C et al. Research and Development on Therapeutic Agents and Vaccines for COVID-19. ACS Cent Sci. 2020;6(3):315–331.
5. Amirian ES, Levyb JK. Current Knowledge about Remdesivir (GS-5734) and GS441524. One Health. 2020;9:100128.
6. Smith K et al. Utilization of MaxPeak High Performance Surfaces and the Atlantis PREMIER BEH C18 AX Column. Waters Tech Brief 720006745EN. 2020.