A novel hydrophilic interaction liquid chromatography method for the analysis of arterolane; Isolation and structural elucidation of its major degradation product by LC-HRMS and NMR

Abstract

Adequate retention of arterolane (ART) has not been published in the literature till date; the hydrophilic interaction liquid chromatographic (HILIC) method with improved retention and separation of arterolane from its degradation products are reporting here for the first time. The present study discusses the comparative retention of the drug-using Reverse Phase C18 and HILIC columns, indicating the effective method (Syncronis HILIC-150 x 4.6 mm, 5μ). It was observed that the buffer concentration (ammonium acetate) in the mobile phase plays a crucial role in the retention of ART. Forced degradation studies of ART were conducted as per ICH Q1 (R2) prescribed conditions. The drug is not stable in hydrolytic (acid, base and neutral), oxidative and photolytic conditions and observed four unique degradation products (DPs). The optimized method for the analysis of degraded samples comprises of Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) technique having Agilent HILIC plus (100 × 4.6 mm, 3.5μ) column and acetonitrile (ACN) and 10 mM ammonium acetate 75:25 (% v/v) mobile phase with 0.4 mL/minute flow. Initially, the structure of all four DPs was proposed on the basis of accurate mass and their fragmentation pattern. The major degradation product (DP4) was isolated, and its structure was confirmed by HRMS and 1H NMR, 13C NMR, HMBC, DEPT 135 and Deuteriated NMR spectroscopy. The formation of DPs might be due to the breakdown of (1 r,3r,5 r,7r)-2-methoxyadamantan-2-ol from ART (DP4), and subsequent diol formation at 1,2,4-trioxolane moiety (DP3).