Multiplex liquid chromatography-tandem mass spectrometry assay for simultaneous therapeutic drug monitoring of ribavirin, boceprevir, and telaprevir

Antimicrob Agents Chemother. 2013 Jul;57(7):3147-58. doi: 10.1128/AAC.00281-13. Epub 2013 Apr 29.


New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / blood*
  • Chromatography, Liquid
  • Drug Monitoring / methods*
  • Hepacivirus / drug effects
  • Hepatitis / drug therapy
  • Hepatitis / prevention & control
  • Hepatitis / virology
  • Humans
  • Liver Cirrhosis / drug therapy
  • Liver Cirrhosis / prevention & control
  • Liver Cirrhosis / virology
  • Oligopeptides / blood*
  • Oligopeptides / therapeutic use
  • Proline / analogs & derivatives*
  • Proline / blood
  • Proline / therapeutic use
  • Ribavirin / blood*
  • Ribavirin / therapeutic use
  • Tandem Mass Spectrometry


  • Antiviral Agents
  • Oligopeptides
  • Ribavirin
  • telaprevir
  • N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide
  • Proline