Characterization and complete separation of major cyclolinopeptides in flaxseed oil by reversed-phase chromatography

Organoleptic properties of flaxseed oil deteriorate during storage due to methionine oxidation in its major cyclolinopeptides. Cyclolinopeptide E was previously identified as being responsible for the manifestation of bitter taste with flaxseed oil ageing. We developed a chromatographic procedure to monitor the oxidation of major cyclic peptides in flaxseed oil. We also used liquid chromatography with mass spectrometry and high-efficiency core-shell reversed-phase sorbents to study the separation of cyclolinopeptides in detail. The Kinetex(TM) family of stationary phases (C8, C18, phenyl-hexyl) was tested, along with the standard porous Luna(TM) C18(2) media. We found that only the phenyl-hexyl stationary phase allows for complete resolution of major cyclolinopeptides, thus permitting direct UV monitoring of degree of conversion for cyclolinopeptide B into C and L into E. We also report, for the first time, a significant effect of peak splitting for some methionine S-oxide (Mso) containing cyclolinopeptides, which most likely appear due to diastereomerization. This results in poor separation efficiency for cyclolinopeptides F, G, and E, and gives baseline resolution of diastereomeric pairs for cyclolinopeptides I and P. Thus, a single oxidation of cyclolinopeptide N yields three distinct chromatographic peaks corresponding to cyclolinopeptide T (cyclo-MsoLMPFFWV, reported for the first time) and pair of cyclolinopeptide I (cyclo-MLMsoPFFWV) diastereomers.