LC–MS/MS quantitation of ribavirin in serum and identification of endogenous isobaric interferences

• Published in: Clinica chimica acta 2011-11, Vol.412 (23-24), p.2332-2335
• Main Authors: Danso, Darlington, Langman, Loralie J., Snozek, Christine L.H.
• Format: Article
• Language: English
• Subjects: Antiviral Agents - blood; Chromatography, Liquid - methods; Hepatitis C; Humans; Interference; Limit of Detection; Mass spectrometry; Ribavirin; Ribavirin - blood; Tandem Mass Spectrometry - methods; Therapeutic drug monitoring
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2011.07.016

Ribavirin is a nucleoside analog used in treatment of chronic hepatitis C. It is associated with severe, dose-dependent toxicities, including hemolytic anemia. To facilitate therapeutic drug monitoring, a liquid chromatography–tandem mass spectrometry method was validated for quantitation of ribavirin in serum. After protein precipitation, ribavirin is quantitated using a 13C5-ribavirin internal standard, on a Hypercarb analytical column designed for retention of polar analytes. The analytical method shows excellent precision, sensitivity, and specificity. In vitro drug stability was also assessed. Interestingly, endogenous isobaric compounds were noted in both human and bovine serum; these could be chromatographically separated from the ribavirin peak. Addition of exogenous uridine and cytosine increases the size of the isobaric peaks, suggesting that these compounds are the source of the endogenous interference. This method uses mass spectrometric transitions that have been used in other published methods, but also separates ribavirin from isobaric peaks that were not described. These peaks were determined to be endogenous nucleosides. Laboratories quantitating ribavirin in biological matrices should be aware of the potential for isobaric interferences, and take steps to chromatographically separate them from the ribavirin peak for accurate quantitation. ► Validation of an LC–MS/MS method for ribavirin and its isotopically labeled internal standard. ► A common MRM transition for ribavirin, 245→113, has endogenous isobaric interferences in serum. ► Isobaric interferences can be chromatographically separated from the ribavirin peak. ► Isobaric interferences are identified as uridine and cytosine.