Development and validation for bioanalysis of VK2809, its active metabolite VK2809A and glutathione-conjugated metabolite MB06588 in rat liver using LC-MS/MS.

VK2809 is a promising drug candidate in Phase II clinical trials for the treatment of non-alcoholic steatohepatitis (NASH). It is a prodrug with a HepDirect strategy, which can achieve selective hepatic metabolic activation, generating an active metabolite VK2809A as a potent and selective agonist for thyroid hormone receptor beta (TRβ), a concomitant reactive metabolite VK2809B, and a glutathione (GSH) conjugate MB06588. Currently, there is no convenient and sensitive bioanalytical method for the simultaneous determination of the above three metabolites. Herein, we established an LC-MS/MS method to separate VK2809 and its metabolites on the XSelect HSS T3 column and quantified them in negative electrospray ionization mode. Subsequently, several factors were investigated such as the use of 60% acetonitrile for homogenization to stabilize the analytes, the addition of 20 mM glutathione for the derivation of VK2809B, and the protein precipitation with methanol containing Sobetirome as the internal standard (IS). The method exhibited good linearity for all compounds (19.4-388.4 nM for VK2809; 27.4-2744.4 nM for VK2809A and 10.6-211.0 nM for MB06588) with great correlation coefficients (r > 0.996). The method validation also demonstrated acceptable precision (RSD < 13.0% for VK2809, RSD < 7.9% for VK2809A, RSD < 14.4% for MB06588) and accuracy (92.7%-103% for VK2809, 91.2%-107.3% for VK2809A, 96%-106.7% for MB06588). The matrix effect, recovery, and stability were also suitable to determine all the analytes. This method is suitable for the bioanalysis of VK2809 and its metabolites and has been successfully applied to the study of intrahepatic exposure in rats. It is expected to be further practiced in drug design, optimization, and metabolism study in the following research.