In the present study, comprehensive stress testing of amlodipine (AM) was carried out according to International Conference on Harmonization (ICH) Q1A(R2) guideline. AM was subjected to acidic, neutral and alkaline hydrolysis, oxidation, photolysis and thermal stress conditions. The drug showed instability in acidic and alkaline conditions, while it remained stable to neutral, oxidative, light and thermal stress. A total of nine degradation products (DPs) were formed from AM, which could be separated by the developed gradient LC method on a C18 column. The products formed under various stress conditions were investigated by LC–MS/MS analysis. The previously developed LC method was suitably modified for LC–MS/MS studies by replacing phosphate buffer with ammonium acetate buffer of the same concentration (pH 5.0). A complete fragmentation pathway of the drug was first established to characterize all the degradation products using LC–MS/MS and multi-stage mass (MSn) fragmentation studies. The obtained mass values were used to study elemental compositions, and the total information helped with the identification of DPs, along with its degradation pathway.

The aim of the present investigation was to demonstrate an approach involving use of liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC–MS) to separate, identify and characterize very small quantities of degradation products (DPs) of acebutolol without their isolation from the reaction mixtures. The drug was subjected to oxidative, hydrolytic, thermal and photolytic stress conditions as per International Conference on Harmonization (ICH) guideline Q1A(R2). Among all the stress conditions the drug was found to be labile in hydrolytic (acidic & basic) and photolytic stress conditions, while it was stable in water-induced hydrolysis, oxidative and thermal stress conditions. A total of four degradation products were formed. A C18 column was employed for the separation of all the DPs on a gradient mode by using high-performance liquid chromatography (HPLC). All the DPs were characterized with the help of their fragmentation pattern and the masses obtained upon LC–MS/MS and MSn analysis. All the hitherto unknown degradation products were identified as 1-(2-(2-hydroxy-3- (isopropylamino)propoxy)-5-(amino)phenyl)ethanone (DP-I), N-(4-(2-hydroxy-3-(isopropylamino) propoxy)-3-acetylphenyl)acrylamide (DP-II), 1-(2-(2-hydroxy-3-(isopropylamino)propoxy)-5-(hydroxymethylamino) phenyl)ethanone (DP-III) and 1-(6-(2-hydroxy-3-(isopropylamino)propoxy)-2,3-dihydro- 2-propylbenzo[d]oxazol-5-yl)ethanone (DP-IV). Finally the in-silico carcinogenicity and hepatotoxicity predictions of the drug and all the DPs were performed by using toxicity prediction softwares viz., TOPKAT, LAZAR and Discovery Studio ADMET. The results of in-silico toxicity studies revealed that acebutolol (0.967) and DP-I (0.986) were found to be carcinogenic, while acebutolol (0.490) and DP-IV (0.437) were found to be hepatotoxic.