Profiling and Qualitative Assessment of Enzymatically and Thermally Oxidized Egg Yolk Phospholipids using a Two-Step High-Performance Liquid Chromatography Protocol

A two-step high-performance liquid chromatography (HPLC) method for the profiling and qualitative assessment of oxidized phospholipids (oxPL) present in foods was developed. The applicability of the investigated two-step HPLC protocol was verified for separation of enzymatically and thermally oxidized hen egg yolk phospholipids (PL) as a relevant food model. In the first step, seven individual PL classes were separated using hydrophilic interaction liquid chromatography (HILIC). The collected fractions of two main egg yolk PL classes-phosphatidylethanolamines and phosphatidylcholines-were further directed to the second step of separation aimed at profiling and qualitative assessment of their oxidized species. For this purpose, the reverse phase (RP) chromatography coupled to charged aerosol, ultraviolet detection (UV) and mass spectrometry detection were employed. A database of potential oxPL including primary (hydroperoxides) and long-chain secondary PL oxidation products (epoxides, alcohols, and ketones) as well as some of their possible combinations was created. Additionally, the results were compared with the profiles of PL hydroperoxides obtained using thin layer chromatography (TLC) with N,N-dimethyl-p-phenylenediamine visualization.