A new validated bioanalytical method based on LC tandem MS has been developed 
for the simultaneous extraction and determination of sofosbuvir and ledipasvir 
in human plasma using antiviral daclatasvir as an internal standard (IS). 
Liquid-liquid extraction of samples was used for the purification and 
preconcentration of the analytes from a human plasma matrix. Good and consistent 
recoveries were obtained, with average extraction recoveries of 91.61 and 88.93% 
for sofosbuvir and ledipasvir, respectively. The chromatographic separation of 
the three analytes was achieved within only 2.8 min by an isocratic mobile phase 
consisting of 10 mM ammonium acetate, which was then adjusted to pH 4.0 by 
acetic acid-acetonitrile-0.1% methanolic formic acid (12 + 25 + 63, v/v/v) 
flowing through a C18 Zorbax eclipse plus column (5 μm, 100 × 4.6 mm; Agilent). 
Multiple reaction monitoring transitions were measured in positive ion mode for 
sofosbuvir, ledipasvir, and daclatasvir (IS). A detailed validation of the 
method was performed and the standard curves were found to be linear in the 
range of 0.5 to 2500 and 5 to 2100 ng/mL for sofosbuvir and ledipasvir, 
respectively, applying weighted (1/X(2)) linear regression. The developed method 
was applied to the analysis of the two drugs after a single oral administration 
of Harvoni 400/90 mg film-coated tablets containing 400 mg sofosbuvir and 90 mg 
ledipasvir to four healthy volunteers.