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.
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 (1)H-NMR, 2D (1)H-(1)H TOCSY and 2D (1)H-(13)C HSQC NMR. The significant sensitivity barrier has been successfully overcome under selective suppression of the major resonances, with over 10(4) greater equilibrium magnetization of the -(CH2)n-(1)H spins compared to that of the (1)H spins of the conjugated bonds of the CLA isomers. The resolution barrier has been significantly increased using reduced (13)C spectral width in the 2D (1)H-(13)C 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.
NMR Center, University of Ioannina, Ioannina GR-45 110, Greece.
Constantinos G
CG
Tsiafoulis
Department of Chemistry, University of Ioannina, Ioannina GR-45 110, Greece.
Theodore
T
Skarlas
Department of Agricultural Sciences, Biotechnology and Food Sciences, Cyprus University of Technology, P.O Box 50329, Limassol 3603, Cyprus.
Ouranios
O
Tzamaloukas
Department of Agricultural Sciences, Biotechnology and Food Sciences, Cyprus University of Technology, P.O Box 50329, Limassol 3603, Cyprus.
Despoina
D
Miltiadou
Department of Chemistry, University of Ioannina, Ioannina GR-45 110, Greece. Electronic address: igeroth@uoi.gr.
Ioannis P
IP
Gerothanassis
D000818:Animals; D057230:Limit of Detection; D044243:Linoleic Acids, Conjugated / Q000032:analysis*; D008055:Lipids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008892:Milk / Q000737:chemistry*
D044243:Linoleic Acids, Conjugated; D008055:Lipids
(1)H(1)H total correlated spectroscopy; (1)H(13)C heteronuclear single quantum coherence; Conjugated linoleic acid; Geometric cis/trans isomers