Isolation of α-linolenic acid biohydrogenation products by combined silver ion solid phase extraction and semi-preparative high performance liquid chromatography
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C and E Trail, Lacombe, AB, Canada; Thompson Rivers University, 900 McGill Road, Kamloops, BC, Canada; Department of Animal Sciences, Faculty of AgriSciences, Stellenbosch University, P. Bag X1, Matieland, South Africa; Livestock Gentec, 1400 College Plaza 8215 112 Street, Edmonton, AB, Canada
Polyunsaturated fatty acids typically found in cattle feed include linoleic (LA) and α-linolenic acid (ALA). In the rumen, microbes metabolize these resulting in the formation of biohydrogenation products (BHP), which can be incorporated into meat and milk. Bioactivities of LA-BHP, including conjugated linoleic acid (cis (c) 9,trans (t) 11-18:2 and t10,c12-18:2) and trans fatty acid isomers (t9-, t10- and t11-18:1) have been investigated, but effects of several BHP unique to ALA have not been extensively studied, and most ALA-BHP are not commercially available. The objective of the present research was to develop methods to purify and collect ALA-BHP using silver ion (Ag+) chromatography in sufficient quantities to allow for convenient bioactivity testing in cell culture. Fatty acid methyl esters (FAME) were prepared from perirenal adipose tissue from a cow enriched with ALA-BHP by feeding flaxseed. These were applied to Ag+-solid phase extraction, and eluted with hexane with increasing quantities of acetone (1, 2, 10, 20%) or acetonitrile (2%) to pre-fractionate FAME based on degree of unsaturation and double bond configuration. Fractions were collected, concentrated and applied to semi-preparative Ag+-high performance liquid chromatography (HPLC) for the isolation and collection of purified isomers, which was accomplished using isocratic elutions with hexane containing differing amounts of acetonitrile (from 0.015 to 0.075%). Purified trans-18:1 isomers collected ranged in purity from 88 to 99%. Purity of the ALA-BHP dienes collected, including c9,t13-18:2, t11,c15-18:2 and t10,c15-18:2, exceeded 90%, while purification of other dienes may require the use of other complementary procedures (e.g. reverse phase HPLC). © 2014.
Acetone; Acetonitrile; Bioactivity; Cell culture; Chromatography; Extraction; Hexane; High performance liquid chromatography; Isomers; Linoleic acid; Liquid chromatography; Liquids; Metal ions; Olefins; Phase separation; Polyunsaturated fatty acids; Purification; Alpha linolenic acids; Biohydrogenation; Conjugated linoleic acid; Degree of unsaturations; Fatty acid methyl ester; Linolenic acids; Semi-preparative high-performance liquid chromatographies; Solid-phase extraction; Fatty acids; acetone; acetonitrile; conjugated linoleic acid; fatty acid ester; hexane; linolenic acid; silver; trans fatty acid; linolenic acid; adipose tissue; animal cell; animal tissue; Article; biological activity; concentration (parameters); controlled study; cow; fractionation; high performance liquid chromatography; hydrogenation; isomer; linseed; nonhuman; priority journal; solid phase extraction; high performance liquid chromatography; isolation and purification; procedures; solid phase extraction; Bos; alpha-Linolenic Acid; Chromatography, High Pressure Liquid; Linoleic Acids, Conjugated; Solid Phase Extraction