Relative retention order of all isomers of cis/trans conjugated linoleic acid FAME from the 6,8‐ to 13,15‐positions using silver ion HPLC with two elution systems

Abstract

CLA, defined as one or more octadecadienoic acids (18:2) with conjugated double bonds, has been reported to be active in a number of biological systems. GC and silver ion HPLC (Ag(+)-HPLC) have been the primary techniques for identifying specific CLA isomers in both foods and biological extracts. Recently, GC relative retention times were reported for all c,c, c/t (c,t and tc), and t,t CLA FAME from the 6,8- to the 13,15-positions in octadecadienoic acid (18:2). Presented here is the relative retention order of the same CLA FAME using Ag(+)-HPLC with two different elution systems. The first elution system, consisting of 0.1% acetonitrile/0.5% diethyl ether (DE)/hexane, has been used previously to monitor CLA composition in foods. Also presented here is the retention order of CLA FAME using 2% acetic acid/hexane elution solvent, which has advantages of more stable retention volumes and a complementary elution order of CLA FAME isomers. The data are reported using retention volumes (RV) adjusted for toluene, an estimator for dead volume, and relative to c9,t11-18:2. Measurement of relative RV in the analysis of 88 samples of cow plasma, milk, and rumen fluids using Ag(+)-HPLC is also presented here. The % CV ranged from 1.04 to 1.62 for t,t isomers and from 0 to 0.48 for c/t isomers.

A Practical Guide to the Isolation, Analysis and Identification of Conjugated Linoleic Acid

“…Several peaks (e.g., 21:0) that are not CLA isomers may occur in the CLA region of the gas chromatogram of natural CLA samples, and in samples extracted from tissue derived from animals fed commercial CLA. The 21:0 is often at similar concentrations to the minor CLA isomers and can elute anywhere between 11c,13t-(as in Fig.…”

“…3. Utilizing three Ag-HPLC columns connected in series and an isocratic solvent system of 0.1% acetonitrile in hexane, Eulitz and co-workers [12] were able to separate the very difficult 9c,11t-and 9t,11c-18:2 isomer pair as FAMEs, and, by utilizing two elution systems, others were able to determine the relative retention orders of all 32 cis/trans 6,8-to 13,15-CLA isomers [21]. Two to six columns connected in series were required to separate such isomer pairs as 8t,10c-from 7t,9c-and 11c,13t-from 9t,13c-18:2.…”

Theory and Practice of UHPLC and UHPLC–MS

“…The series of positional isomers of C18:1 and C18:2 (NikolovaDamyanova et al, 1992) or conjugated C18:2 (Delmonte et al, 2005) has been studied and their relative retention factors (k) were plotted against DB positions. The retention behavior of all series of isomers is similar, giving sinusoidal curves of k values of individual isomers.…”

Advances in Silver Ion Chromatography for the Analysis of Fatty Acids and Triacylglycerols—2001 to 2011

“…33 Introducing 2-propanol was certainly a good idea, since this solvent allowed for varying the acetonitrile content in rather broad limits. As an alternative, 2% acetic acid in hexane was used for the resolution of CLA ME, 34 and 0.15% acetonitrile in 2-octane was found to be appropriate for the fractionation of trans and cis monoenoic ME. 35 Propionitrile and butyronitrile were also tested as modifiers for both CLA 36,37 and TAG 38 separations.…”