A practical and fast isolation of 12 cyclolinopeptides (linusorbs) from flaxseed oil via preparative HPLC with phenyl-hexyl column.

Linusorbs, known as cyclolinopeptides, are a group of cyclic hydrophobic peptides derived from flaxseed oil with various health benefits. However, the current research efforts on both the biological activities and antioxidant capacities of linusorbs are limited because of existing issues with their purification and characterization. A practical method based on preparative HPLC for isolating 12 linusorbs simultaneously was developed and factors such as the solvent selection, gradient elution program, flow rate, loaded mass, and loading concentration, were optimized. The optimum conditions were an initial acetonitrile (ACN) to water ratio of 40%, final ACN ratio of 80%, eluting time of 21 min, a flow rate of 16 mL/min, sample load of 12.5 mg, and concentration of 80 mg/mL (in methanol). The 12 linusorbs obtained were verified using off-line MS/MS, recording purities of above 95.5%. The method could serve as a practical and fast isolation method enabling further investigation of minor linusorbs.