Simultaneous determination of ledipasvir, sofosbuvir and its metabolite in rat plasma by UPLC–MS/MS and its application to a pharmacokinetic study
Introduction
Patients with cirrhosis resulting from chronic infection with the hepatitis C virus (HCV) are at risk of developing life-threatening complications, such as decompensated liver disease and hepatocellular carcinoma [1], [2]. Treatment improves long-term outcomes for patients with cirrhosis, and sustained virological response (SVR) is associated with histological improvement and reduced risk of hepatocellular carcinoma, decompensation, and liver-related mortality [3], [4], [5]. In clinical trials and real-world settings, however, patients with cirrhosis, especially those for whom previous therapy has failed, achieve consistently lower rates of SVR than patients without cirrhosis [6], [7], [8]. Moreover, interferon-based regimens for the treatment of HCV genotype 1 are poorly tolerated by patients with cirrhosis and cause high rates of severe and serious adverse events, leading many patients to discontinue treatment [9]. Therefore, interferon-free regimens for patients with cirrhosis are needed [10].
Sofosbuvir (Fig. 1) is a nucleotide analog inhibitor of the HCV NS5B polymerase that has been extensively studied in combination with ledipasvir (Fig. 1), as well as with other direct-acting antiviral agents in treatment-naive patients with genotype-1HCV infection [11], [12], [13], [14]. Ledipasvir is a novel HCV NS5A inhibitor that has shown potent antiviral activity against genotypes 1a and 1b HCV infection, and is active against HCV with the S282T mutation, the only variant known to reduce susceptibility to sofosbuvir [15]. A fixed-dose combination of sofosbuvir–ledipasvir was approved in 2014 for treatment of patients chronically infected with genotype 1HCV [11], [16], [17].
To best of our knowledge, no published LC–MS/MS and HPLC based methods are reported for the quantification of ledipasvir in plasma. Method for the determination of sofosbuvir and its active metabolite GS-331007 in biological fluids by UPLC–MS/MS have been reported [18]. However, reports describing an UPLC–MS/MS-based method for simultaneous determination of ledipasvir, sofosbuvir and its active metabolite GS-331007 in plasma are not available. Simultaneous detection of ledipasvir, sofosbuvir and its active metabolite GS-331007 in plasma would help establishing a pharmacokinetic and pharmacodynamic co-relation in animal models that require administration of both drugs to achieve maximal efficacy.
UPLC–MS/MS has been evaluated as a faster and more efficient analytical tool compared with current chromatography [19], [20], [21]. Therefore, in this study, a quick, simple and sensitive method for simultaneous quantifying ledipasvir, sofosbuvir and GS-331007 base on UPLC–MS/MS was developed, validated, and successfully applied to the pharmacokinetic study in rats after oral administration.
Section snippets
Chemicals materials
Sofosbuvir, GS-331007, ledipasvir and diazepam (internal standard, IS) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were HPLC grade and purchased from Merck Company (Darmstadt, Germany). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA).
UPLC–MS/MS conditions
Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA, USA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm) and inline 0.2 μm
Method development and optimization
The purpose of sample extraction optimization is mainly to achieve high extraction recovery and low matrix effects in order to improve sensitivity and reliability of LC–MS/MS analysis. Strategy usually includes all possible common sample extraction options including solid phase extraction, liquid extraction and protein precipitation. With the emergence of UPLC technology combined with high sensitive tandem mass spectroscopy, it is possible to quantify analytes at a low concentration without
Conclusions
A sensitive and simple UPLC–MS/MS method for simultaneous measurement of ledipasvir, sofosbuvir and its metabolite GS-331007 in rat plasma has been developed and validated. The method was found to be accurate, precise and specific, and was successfully applied to the pharmacokinetic investigations of ledipasvir, sofosbuvir and GS-331007 in rats.
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Eco friendly stability indicating HPTLC method for simultaneous determination of sofosbuvir and ledipasvir in pharmaceutical tablets and HPTLC-MS characterization of their degradation products
2023, Microchemical JournalCitation Excerpt :LED (Fig. S2) is an inhibitor of the (HCV) NS5A protein required for viral RNA replication and assembly of HCV virions, with the chemical formula C49H54F2N8O6 and the chemical name methyl[(2S)-1-[8]-1H-benzo[d]imidazol-6-yl]-9H-fluoren-2-yl}-4,5-dihydro-1H-imidazol-2-yl]-5-azaspiro[2.4]heptan-5-yl}-3-methyl-1-oxobutan-2-yl]carbamate [8]. Since this combination is newly formulated as an antiviral, there are few methods for simultaneous determination of SOF and LED in their dosage forms, including RP-HPLC [9], LC-MS/MS [10], UPLC-MS/MS [11,12], spectrophotometric techniques [13–15] and TLC [16]. These reported methods are not eco-friendly due to their use of large quantities of organic solvents which have hazardous effects on health and the environment.
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