Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry

Abstract For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC–MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10–5000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC–MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm × 2.1 mm, 5 μm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid ( A ) and 90% acetonitrile (containing 0.5% formic acid ( B ). The flow rate was 0.4–0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were