Development and validation of a new HPLC-DAD method for quantification of sofosbuvir in human serum and its comparison with LC-MS/MS technique: Application to a bioequivalence study.

Abstract

Although for many analyses tandem mass spectrometry (LC-MS/MS) systems have significant advantage over the high-performance liquid chromatography with diode array detection (HPLC-DAD) however, the HPLC methods are easier, cheaper and more available to perform. As no published method is available for quantitative HPLC analysis of sofosbuvir (SOF), an orally administered anti-hepatitis drug in human serum, this study was aimed to evaluate applicability of the HPLC technique to quantify sofosbuvir and comparison of the two methods for analytical performance. Following extraction of the drug and an internal standard (Hexobarbital), same chromatographic conditions were used for both the systems. After the chromatographic separation on a reverse phase C18 column using a mobile phase consisting of water (containing formic acid 0.5mL/L) and acetonitrile (57:43; v/v) at a flow rate of 0.8mL/min, the eluate was introduced into a DAD detector set at 261nm, then passed through the mass spectrometry system in single ion monitoring mode (SIM). For UV and mass spectrometry detections the calibration curves were linear over a concentration range of 25-3200 and 10-3200ng/mL, respectively and the linearity was over 0.998 for both the systems. Lower limit of quantification (LLOQ) for mass spectrometry and DAD detections were 10 and 25ng/mL, respectively. In conclusion sensitivity of DAD detection is sufficient enough to determine concentrations down to 0.5% of Cmax which achieved in bioequivalence study of sofosbuvir and meet FDA requirements for these types of studies.