Separation and identification of degradation products in eprinomectin formulation using LC, LTQ FT-MS, H/D exchange, and NMR

Abstract

The aim of this study was to evaluate the suitability of the compendial active pharmaceutical ingredient (API) method for the analysis of finished products and characterization of degradation products in eprinomectin (EPM) samples. Heat stressed sample tests revealed a limitation of the API method in distinguishing an impurity merging with the principal analyte peak. A new selective, specific and sensitive method was therefore developed for the determination of EPM in formulations that separates its degradation products currently undetectable with the official method. The determination was carried out by reversed-phase HPLC using an isocratic solvent elution. The method was validated and found to be precise, accurate and specific; the detector response was linear over 50–150μg/ml (EPM) and 0.1–3μg/ml (degradation product) range of concentrations.Two major degradation products detected with the new method were isolated from sample matrices and characterized using LC-PDA, high resolution FT-ICR MS, NMR and hydrogen/deuterium exchange (HX-MS) studies. FTMS analysis showed accurate mass of molecular ion peaks for EPM and its two degradation products at m/z 914.52505 (mass error≤1ppm) with almost identical fragmentation patterns. Given the isomeric nature of the compounds, all three were further evaluated by 1H, 13C, 1D NOESY and 2D (COSY) NMR experiments. The interpretation of experimental data positively identified Unknown 1 as the 2-epimer of EPM and Unknown 2 as the structural isomer Δ2,3-EPM containing a conjugated enoate. The new HPLC method and identification exercise is useful for analysis of EPM and its degradation products.