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

J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Oct 1:1182:122921. doi: 10.1016/j.jchromb.2021.122921. Epub 2021 Sep 2.


The novel antiviral prodrug molnupiravir is under evaluation for the treatment of SARS-CoV-2. Molnupiravir is converted to β-D-N4-hydroxycytidine (NHC), which is the primary form found in systemic circulation. β-D-N4-hydroxycytidine-triphosphate (NHCtp) is the bioactive anabolite produced intracellularly. Sensitive and accurate bioanalytical methods are required to characterize NHC and NHCtp pharmacokinetics in clinical trials. Human K2EDTA plasma or peripheral blood mononuclear cell (PBMC) lysates were spiked with NHC (plasma) or NHCtp (PBMC), respectively. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or lysate dilution, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations. NHC can be quantified in plasma with a lower limit of quantification (LLOQ) of 1 ng/mL; the primary linearity of the assay is 1-5000 ng/mL. Assay precision and accuracy were ≤ 6.40% and ≤ ± 6.37%, respectively. NHC is unstable in whole blood and has limited stability in plasma at room temperature. The calibration range for NHCtp in PBMC lysates is 1-1500 pmol/sample, and the assay has an LLOQ of 1 pmol/sample. Assay precision and accuracy were ≤ 11.8% and ≤± 11.2%. Ion suppression was observed for both analytes; isotopically-labeled internal standards showed comparable ion suppression, resulting in negligible (<5%) relative matrix effects. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the quantification of NHC in plasma and NHCtp in PBMC lysates. The described methods are appropriate for use in clinical trials.

Keywords: Antiviral; COVID-19; LC-MS/MS; Molnupiravir; SARS Cov-2; β-D-N(4)-hydroxycytidine.

MeSH terms

  • Cytidine / analogs & derivatives*
  • Cytidine / blood
  • Cytidine / chemistry
  • Humans
  • Reproducibility of Results


  • Cytidine
  • N(4)-hydroxycytidine