Simultaneous Determination of Major Molnupiravir Metabolite (β-D-N4-hydroxycytidine) and Favipiravir in Human Plasma by HPLC-MS/MS

Introduction. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus (severe acute respiratory syndrome-related coronavirus 2). COVID-19 is now expected to stay with us for many years as a recurring disease. Molnupiravir and favipiravir are oral antiviral drugs with anti-RNA polymerase activity. The Russian Health Ministry has approved molnupiravir and favipiravir for the treatment of COVID-19. The study describes development and validation of high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of β-D-N4-Hydroxycytidine and favipiravir in human blood plasma. The method could be applied in pharmacokinetic study of molnupiravir and favipiravir.

Aim. The aim of this study is to develop and validate a HPLC-MS/MS bioanalytical method for the determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma.

Materials and methods. The determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma by HPLC-MS/MS. The samples were processed by 0.1 % formic acid in acetonitrile. Internal standard: promethazine. Mobile phase: 0.01 mol/L Ammonium formate buffer solution (Eluent A), 0.1 % formic acid and 0.08 % aqueous ammonia in water/acetonitrile 10 : 90 (Eluent B). Column: Shim-pack GWS C18, 150 × 4.6 mm, 5 μm. Analytical range: 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. Ionization source: electrospray ionization. Detection conditions: 260.00 m/z → 82.10 m/z, 260.00 m/z → 111.00 m/z, 260.00 m/z → 127.95 m/z (β-D-N4-Hydroxycytidine); 156.15 m/z → 65.95 m/z, 156.15 m/z → 85.00 m/z, 156.15 m/z → 113.10 m/z (favipiravir); 285.05 m/z → 198.05 m/z (promethazine).

Results and discussion. This method was validated by selectivity, suitability of reference standard, matrix effect, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability.

Conclusion. The HPLC-MS/MS method for quantitative determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma was developed and validated. The analytical range was 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. This method was applied to investigate the pharmacokinetics of molnupiravir and favipiravir.

1. Li C. X., Noreen S., Zhang L. X., Saeed M., Wu P. F., Ijaz M., Dai D. F., Maqbool I., Madni A., Akram F., Naveed M. A critical analysis of SARS-CoV-2 (COVID-19) complexities, emerging variants, and therapeutic interventions and vaccination strategies. Biomedicine & Pharmacotherapy. 2022;146:112550. DOI: 10.1016/j.biopha.2021.112550.

2. Popovic M. XBB.1.5 Kraken Cracked: Gibbs Energies of Binding and Biosynthesis of the XBB.1.5 Variant of SARS-Cov-2. Preprints. 2023;2023010404. DOI: 10.20944/preprints202301.0404.v1.

3. Parums D. V. The XBB. 1.5 (‘Kraken’) Subvariant of Omicron SARS-CoV-2 and its Rapid Global Spread. Medical Science Monitor: International Medical. Journal of Experimental and Clinical Research. 2023;29:e939580. DOI: 10.12659/MSM.939580.

4. Lee C. C., Hsieh C. C., Ko W. C. Molnupiravir — A Novel Oral Anti-SARS-CoV-2 Agent. Antibiotics. 2021;10:1294. DOI: 10.3390/antibiotics10111294.

5. Pourkarim F., Pourtaghi-Anvarian S., Rezaee H. Molnupiravir: A new candidate for COVID-19 treatment. Pharmacology research & perspectives. 2022;10(1):e00909. DOI: 10.1002/prp2.909.

6. Gordon C. J., Tchesnokov E. P., Schinazi R. F., Götte M. Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template. Journal of Biological Chemistry. 2021;297(1):100770. DOI: 10.1016/j.jbc.2021.100770.

7. Sharaf Y. A., El Deeb S., Ibrahim A. E., Al-Harrasi A., Sayed R. A. Two green micellar HPLC and mathematically assisted UV spectroscopic methods for the simultaneous determination of molnupiravir and favipiravir as a novel combined COVID-19 antiviral regimen. Molecules. 2022;27(7):2330. DOI: 10.3390/molecules27072330.

8. Balakin K. V., Storozhenko R. V., Jakubova E. V. Favipiravir for the treatment of COVID-19. Nauchnyj bjulleten’ HimRar. 2023;1. (In Russ.)

9. Eloy P., Le Grand R., Malvy D., Guedj J. Combined treatment of molnupiravir and favipiravir against SARS-CoV-2 infection: One+ zero equals two? EBioMedicine. 2021;74:103663. DOI: 10.1016/j.ebiom.2021.103663.

10. Gouda A. S., Marzouk H. M., Rezk M. R., Salem A. M., Morsi M. I., Nouman E. G., Abdallah Y. M., Hassan A. Y., Abdel-Megied A. M. A validated LC-MS/MS method for determination of antiviral prodrug molnupiravir in human plasma and its application for a pharmacokinetic modeling study in healthy Egyptian volunteers. Journal of Chromatography B. 2022;1206:123363. DOI: 10.1016/j.jchromb.2022.123363.

11. Amara A., Penchala S. D., Else L., Hale C., FitzGerald R., Walker L., Lyons R., Fletcher T., Khoo S. The development and validation of a novel LC-MS/MS method for the simultaneous quantification of Molnupiravir and its metabolite ß-d-N4-hydroxycytidine in human plasma and saliva. Journal of pharmaceutical and biomedical analysis. 2021;206:114356. DOI: 10.1016/j.jpba.2021.114356.

12. Parsons T. L., Kryszak L. A., Marzinke M. A. Development and validation of assays for the quantification of β-D-N4-hydroxycytidine in human plasma and β-D-N4-hydroxycytidine-triphosphate in peripheral blood mononuclear cell lysates. Journal of Chromatography B. 2021;1182:122921. DOI: 10.1016/j.jchromb.2021.122921.

13. Hailat M., Al-Ani I., Hamad M., Zakareia Z., Abu Dayyih W. Development and Validation of a Method for Quantification of Favipiravir as COVID-19 Management in Spiked Human Plasma. Molecules. 2021;26(13):3789. DOI: 10.3390/molecules26133789.

14. Abdallah I. A., Hammad S. F., Bedair A., Mansour F. R. Menthol-assisted homogenous liquid-liquid microextraction for HPLC/UV determination of favipiravir as an antiviral for COVID-19 in human plasma. Journal of Chromatography B. 2022;1189:123087. DOI: 10.1016/j.jchromb.2021.123087.

15. Komarov T. N., Karnakova P. K., Archakova O. A., Shchelgacheva D. S., Bagaeva N. S., Shohin I. E., Zaslavskaya K. Y., Bely P. A. Development and Validation of HPLC-UV Method for the Determination of Favipiravir in Human Plasma. Drug development & registration. 2022;11(3):220–229. (In Russ.) DOI: 10.33380/2305-2066-2022-11-3-220-229.

16. Megahed S., Habib A., Hammad S., Kamal A. Chemometric Approach Based on Factorial and Box-Behnken Designs for Determination of Anti Coronavirus Drug; Favipiravir in Bulk and Spiked Human Plasma by Green HPLC Method. Turkish Journal of Analytical Chemistry. 2021;3(2):70–78. DOI: 10.51435/turkjac.963652.

17. Morsy M. I., Nouman E. G., Abdallah Y. M., Zainelabdeen M. A., Darwish M. M., Hassan A. Y., Gouda A. S., Rezk M. R., Abdel-Megied A. M., Marzouk H. M. A Novel LC-MS/MS Method for Determination of the Potential Antiviral Candidate Favipiravir for the Emergency Treatment of SARS-CoV-2 Virus in Human Plasma: Application to a Bioequivalence Study in Egyptian Human Volunteers. Journal of Pharmaceutical and Biomedical Analysis. 2021;199:114057. DOI: 10.1016/j.jpba.2021.114057.

18. Rezk M. R., Badr K. A., Abdel-Naby N. S., Ayyad M. M. A Novel, Rapid and Simple UPLC–MS/MS Method for Quantification of Favipiravir in Human Plasma: Application to a Bioequivalence Study. Biomedical Chromatography. 2021;35(7):e5098. DOI: 10.1002/bmc.5098.