Metabolite characterization of ambrisentan, in in vitro and in vivo matrices by UHPLC/QTOF/MS/MS: Detection of glutathione conjugate of epoxide metabolite evidenced by in vitro GSH trapping assay.

The focus of the present study is on in vitro and in vivo metabolite identification of ambrisentan (AMBR) a selective endothelin type - A (ETA) receptor antagonist using quadruple time-of-flight mass spectrometry (QTOF/MS). in vitro metabolism study was conducted by incubating AMBR in rat liver microsomes (RLM), rat and human liver S9 fractions. In vivo study was carried out through the collection of urine, faeces and plasma samples at various time points after oral administration of AMBR in suspension form at a dose of 25 mg/kg to six male Sprague - Dawley (SD) rats. The samples were prepared using an optimized sample preparation techniques involving protein precipitation (PP), freeze liquid extraction (FLE) and solid phase extraction (SPE). The extracted samples were further concentrated and analyzed by developing a sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method. A total of seventeen metabolites were identified in in vivo samples which includes hydroxyl, demethylated, demethoxylated, hydrolytic, decarboxylated, epoxide and glucuronide metabolites. Most of the metabolites were observed in faeces and urine matrices and few were observed in the plasma matrix. Only ten metabolites were identified in in vitro study which was commonly observed in in vivo study. The detailed structural elucidation of all the metabolites was done using UHPLC/QTOF/MS/MS in combination with accurate mass measurements. The toxicity profile of AMBR and its metabolites were predicted using TOPKAT software. In addition, a mass spectrometric method was developed for the detection and characterization of GSH-trapped reactive epoxide metabolitein human liver S9 fraction supplemented with glutathione (GSH) as trapping agent.

LC-ESI-MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer activity evaluation of silodosin and major degradation products.

Silodosin (SLD) a novel α1-adrenoceptor antagonist was subjected to forced degradation involving hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH specified conditions. The drug underwent significant degradation under hydrolytic (acidic, alkaline and neutral) and oxidative stress conditions whereas, it was found to be stable under other stress conditions. A rapid, precise, accurate and robust chromatographic method for the separation of the drug and its degradation products (DPs) was developed on a Fortis C18 analytical column (150×4.6mm, 5µm) using 0.1% formic acid and acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0mL/min. A total of 5 (DP 1 to DP 5) hitherto unknown DPs were identified by LC-ESI-TOF-MS/MS experiments and accurate mass measurements. The most probable mechanisms for the formation of DPs have been proposed based on a comparison of the fragmentation of the [M+H]+ ions of silodosin and its DPs. The major DPs (DP 1 and DP 2) were isolated and evaluated for anticancer activity using PC3 (human prostate cancer) cell lines by MTT assay. The results revealed that silodosin, DP 1 and DP 2 have potential anticancer activity with IC50 values (µM) 72.74 (±4.51), 25.21 (±2.36), and, 114.07 (±11.90) respectively.

In vivo metabolic investigation of silodosin using UHPLC-QTOF-MS/MS and in silico toxicological screening of its metabolites.

Silodosin (SLD) is a novel α1-adrenoceptor antagonist which has shown promising clinical efficacy and safety in patients with benign prostatic hyperplasia (BPH). However, lack of information about metabolism of SLD prompted us to investigate metabolic fate of SLD in rats. To identify in vivo metabolites of SLD, urine, feces and plasma were collected from Sprague-Dawley rats after its oral administration. The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid-phase extraction and then subjected to LC/HR-MS/MS analysis. A total of 13 phase I and six phase II metabolites of SLD have been identified in rat urine which includes hydroxylated, N-dealkylated, dehydrogenated, oxidative, glucosylated, glucuronide and N-sulphated metabolites, which are also observed in feces. In plasma, only dehydrogenated, N-dealkylated and unchanged SLD are observed. The structure elucidation of metabolites was done by fragmentation in MS/MS in combination with HRMS data. The potential toxicity profile of SLD and its metabolites were predicted using TOPKAT software and most of the metabolites were proposed to show a certain degree of skin sensitization and occular irritancy. Copyright © 2016 John Wiley & Sons, Ltd.