[Show abstract][Hide abstract] ABSTRACT: Long acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease-resistant were a series of novel decapeptides structurally similar to LHRH. In the present work, a high-throughput method based on a LC-MS/MS has been developed for the simultaneous determination of pharmacokinetics of five LHRH antagonists in rat via cassette dosing. The method was performed under selected reaction monitoring (SRM) in positive ion mode. The analytes were extracted from rat plasma by liquid-liquid extraction with acetonitrile. Chromatographic separation of the analytes was successfully achieved on a Hypersil Gold (100mm×2.1mm, 3μm) using a mobile phase composed of acetonitrile-water (30:70) containing 0.05% (v/v) formic acid. The result showed good linearity and selectivity were obtained for all antagonists. The limits of quantification of the five LHRH antagonists were from 5 to 10ng/mL. The average extract recoveries in the rat plasma were all over 72%. The intra-day and inter-day precisions (R.S.D. %) were all within 10% and the accuracy was ranged from 92.54 to 109.05%. This method has been successfully applied to the pharmacokinetic studies of the five LHRH antagonists. The results indicated that the plasma drug concentrations versus time curves after intravenous injection of five antagonists via cassette dosing were all fitted to a two-compartment model. The pharmacokinetic parameters of five LHRH antagonists suggested that LY616 could be the more stable candidate drugs and optimized as the candidate drug for further study. Our studies enabled high-throughput rapid screening for pharmacokinetic assessment of new peptide candidates, and provided abundant information on the metabolic properties of these LHRH antagonists.
No preview · Article · May 2014 · Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
[Show abstract][Hide abstract] ABSTRACT: Long-acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease resistant consisted of a series of novel decapeptides structurally similar to LHRH. The aim of this study was to evaluate the in vitro metabolic stability of the LHRH decapeptides using pancreatin and homogenates models and identify the metabolites in rat liver homogenate for the purpose of illustrating the metabolic features of the decapeptides. The major metabolites in rat liver homogenate were identified by LC-ESI-MS(n). The half-lives of the 11 LHRH decapeptides were from 44 to 330 min in the pancreatin model. The half-lives of the five decapeptides in rat liver, kidney and lung homogenates were between 8 and 462 min. The most stable decapeptides were the LY616 and LY608 peptides with half-lives of 36 min in liver homogenate. Two major cleavage sites were found by analysing the metabolites of the LY618 peptide in rat liver homogenate, between the Pal(3)-Ser(4) and the Leu(7)-Ilys(8) peptide bonds. The major metabolites were produced via cleavages of peptide bonds at these sites, and further metabolic reactions such as hydroxylation, oxidative dechlorination, alcohol dehydration and isopropyl dealkylation were also observed.