Direct nuclear magnetic resonance identification and quantification of geometric isomers of conjugated linoleic acid in milk lipid fraction without derivatization steps: Overcoming sensitivity and resolution barriers

Abstract

We report the first successful direct and unequivocal identification and quantification of four minor geometric (9-cis, 11-trans) 18:2, (9-trans, 11-cis) 18:2, (9-cis, 11-cis) 18:2 and (9-trans, 11-trans) 18:2 conjugated linoleic acid (CLA) isomers in lipid fractions of lyophilized milk samples with the combined use of 1D 1H-NMR, 2D 1Hsingle bond1H TOCSY and 2D 1Hsingle bond13C HSQC NMR. The significant sensitivity barrier has been successfully overcome under selective suppression of the major resonances, with over 104 greater equilibrium magnetization of the single bond(CH2)nsingle bond1H spins compared to that of the 1H spins of the conjugated bonds of the CLA isomers. The resolution barrier has been significantly increased using reduced 13C spectral width in the 2D 1Hsingle bond13C HSQC experiment. The assignment was confirmed with spiking experiments with CLA standard compounds and the method does not require any derivatization steps for the lipid fraction. The proposed method is selective, sensitive and compares favorably with the GS-MS method of analysis.