Measurement of intracellular ribavirin mono-, di- and triphosphate using solid phase extraction and LC-MS/MS quantification

J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Jan 26;978-979:163-72. doi: 10.1016/j.jchromb.2014.11.032. Epub 2014 Dec 17.


Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-(13)C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection.

Keywords: Analytical methods; Clinical pharmacology; Dried blood spots; Intracellular pharmacology; LC–MS/MS; Ribavirin triphosphate.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatography, Liquid / methods
  • Dried Blood Spot Testing
  • Erythrocytes / chemistry
  • Humans
  • Intracellular Space / chemistry*
  • Leukocytes, Mononuclear / chemistry
  • Linear Models
  • Nucleotides / analysis*
  • Nucleotides / chemistry
  • Nucleotides / isolation & purification
  • Reproducibility of Results
  • Ribavirin / analogs & derivatives*
  • Ribavirin / analysis
  • Ribavirin / chemistry
  • Ribavirin / isolation & purification
  • Sensitivity and Specificity
  • Solid Phase Extraction / methods*
  • Tandem Mass Spectrometry / methods


  • Nucleotides
  • ribavirin-5'-phosphate
  • Ribavirin
  • ribavirin 5'-diphosphate
  • ribavirin 5'-triphosphate