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

Abstract

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.

Commercial Cold Pressed Flaxseed Oils Quality and Oxidative Stability at the Beginning and the End of Their Shelf Life

“…The taste may also derive from the presence of oxidized forms of cyclolinopeptides that occur naturally in flaxseeds. The amount and type of these compounds in oil depends on flax variety, quality of seeds, methods of extraction and purification of the oil, and its storage time 28,29 .…”

Simulated moving bed purification of flaxseed oil orbitides: Unprecedented separation of cyclolinopeptides C and E

“…However, the length of their HPLC gradient was 45 min. Lao et al [10] demonstrated the separation of 1 and 7 using a Kinetex TM Phenyl-Hexyl column with an HPLC run time of at least 25 min (actual run time is not stated). The novelty of our latest HLPC method developed is in its ability to resolve nine naturally occurring flax orbitides (CLs 2, 3, 5, 6, 8, 10, 13, 14, and 17) in 2.5 min.…”

“…1). To date, twenty CLs, each possessing eight or nine amino acid residues, have been reported [2][3][4][5][6][7][8][9][10]. The biological activity of some CLs and their analogs is summarized in a recent review [11].…”

Characteristics of Specialty Natural Micronutrients in Certain Oilseeds and Oils: Plastochromanol‐8, Resveratrol, 5‐Hydroxytryptamine Phenylpropanoid Amides, Lanosterol, Ergosterol and Cyclolinopeptides

“…CL usually consist of eight or nine amino acid residues with molecular weight 960-1100 Da (Fig. 5) [97,99]. The nonoxidized CL are divided into three groups based on their number of methionine residues: 0 (e.g., CL1 and CL20), 1 (e.g., CL2, CL5, CL7 and CL18), or 2 (e.g., CL10 and CL14).…”

“…6). Traditionally, CL1 is usually considered the most stable CL due to its lack of methionine residues in its primary structure; moreover, other CL that containing methionines may produce more oxidized forms depending on the extent of oxidation [99,100].…”