Development of a Robust UPLC Method for Simultaneous Determination of a Novel Combination of Sofosbuvir and Daclatasvir in Human Plasma: Clinical Application to Therapeutic Drug Monitoring

摘要

A rapid and selective UPLC-DAD method was developed and validated for simultaneous analysis of the novel two-drug combination Darvoni® for the treatment of HCV: Sofosbuvir (SF)/Daclatasvir (DC) in human plasma using Ledipasvir as internal standard (IS) where the extraction process was conducted using automated SPE. Although the analysis of the combination after concomitant oral intake of two tablets of SF and DC individually was reported in literature, yet simultaneous analysis of this new combination in human plasma after a single oral dose was not previously reported. The adopted chromatographic separation was achieved on Waters® Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) as a stationary phase using isocratic elution using a mobile phase system of ammonium formate (pH 3.5; 5 mM) and acetonitrile (60:40 v/v) pumped at a flow rate of 0.2 mL.min⁻¹. The UV detection was carried out at 261 nm for SF and 318 nm for DC and IS. SF was eluted at 1.123 min while DC was eluted at 3.179 min. The proposed chromatographic method was validated in accordance with guidelines of FDA for bioanalytical method validation. A linear range was achieved in the range of 25-6400 and 50-12800 ng.mL⁻¹ for SF and DC, respectively. The proposed UPLC-DAD method was found to be accurate with % bias ranging between -10.0-7.2 for SF and -6.9-8.0 for DC. Also it was proved to be precise with % CV for intraday precision ranging between 3.8-9.6 for SF and 2.8-9.2 for DC whereas interday precision ranged between 5.1-9.3 for SF and 3.7-9.1 for DC. Moreover, % extraction recovery ranged between 90.0-107.2 for SF and 93.1-108.0 for DC using the suggested method. The adopted chromatographic method was successfully applied to the therapeutic drug monitoring of SF and DC in healthy volunteers after the oral intake of one Darvoni® tablet.