Journal of Chromatography A, 1216 (2009) 2757–2761 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Short communication Chemometric deconvolution of gas chromatographic unresolved conjugated linoleic acid isomers triplet in milk samples Jaroslav Blaˇ sko a , Róbert Kubinec a,∗ , Ivan Ostrovsk ´ y a , Eva Pavlíková a , Ján Krupˇ cík b , Ladislav Soják a a Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, 842 15 Bratislava, Slovakia b Institute of Analytical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 39 Bratislava, Slovakia article info Article history: Available online 14 November 2008 Keywords: Milk fat CLA isomers GC–MS Chemometric deconvolution abstract A generally known problem of GC separation of trans-7;cis-9; cis-9,trans-11; and trans-8,cis-10 CLA (con- jugated linoleic acid) isomers was studied by GC–MS on 100 m capillary column coated with cyanopropyl silicone phase at isothermal column temperatures in a range of 140–170 ◦ C. The resolution of these CLA isomers obtained at given conditions was not high enough for direct quantitative analysis, but it was, however, sufficient for the determination of their peak areas by commercial deconvolution software. Res- olution factors of overlapped CLA isomers determined by the separation of a model CLA mixture prepared by mixing of a commercial CLA mixture and CLA isomer fraction obtained by the HPLC semi-preparative separation of milk fatty acids methyl esters were used to validate the deconvolution procedure. Devel- oped deconvolution procedure allowed the determination of the content of studied CLA isomers in ewes’ and cows’ milk samples, where dominant isomer cis-9,trans-11 is eluted between two small isomers trans-7,cis-9 and trans-8,cis-10 (in the ratio up to 1:100). © 2008 Elsevier B.V. All rights reserved. 1. Introduction Conjugated linoleic acid (CLA) isomers are reported to have anti- carcinogenic, anti-atherogenic, anti-diabetic properties and they also improve the immune system, bone metabolism and body com- position [1]. Recent reports suggest that each conjugated fatty acid (FA) isomer has different physiological functions [2]. The under- standing of the biological role of these acids relies on their proper separation, identification and quantitation in complex biological extracts which contain many unsaturated and saturated FAs with a number of carbon atoms mostly from C 12 to C 22 , where the carbon number of 16–20 prevails [1]. There are 14 possible CLA positional isomers counting from carbons 2,4 to carbons 15,17–18:2. Each positional isomer has 4 geometric isomers cis,trans; trans,cis; cis,cis; trans,trans giving 56 possible isomers. The double bond positions of CLA isomers actually identified in ruminant fat range from 6,8- to 12,14-18:2 in most of the possible geometrical configurations for a total of 20 isomers [1]. The problem of determination of the isomeric CLA composi- tion of the milk fat products is a challenging analytical task [1,3]. Ag + HPLC can provide separations of CLA isomers not attainable by other means. However, to obtain reproducible results the potential ∗ Corresponding author. Tel.: +421 2 60296 330. E-mail address: kubinec@fns.uniba.sk (R. Kubinec). sources of errors should be addressed. These were summarized by Collomb et al. [4] and include: batch-to-batch variations in silver loadings of the columns; differences in instrument configuration (number of solvent pumps, mixing chambers, valves); changes in elution volumes and elution orders with sample size, solvent com- position and even storage times; lack of internal standards; control of column temperature. Further, the HPLC separation of small peak of trans-8,cis-10 isomer from large peak of cis-9,trans-11 isomer is very poor in milk fat samples and some cis,trans from trans,cis iso- mers are not distinguished. Analysis of the CLA isomers separated by Ag + HPLC requires the combination with GC. The CLA peaks are quantified by GC analyses of total fatty acids methyl esters (FAMEs). It is accepted that the 100 m capillary GC column coated with cyanopropyl polysiloxane CP-Sil 88 as highly polar stationary phase is mandatory for the analysis of milk lipids. This col- umn resolves five distinguishing peaks in the CLA region on the chromatogram of milk fat: cis-9,trans-11 + trans-7,cis-9 + trans-8,cis- 10; trans-11,cis-13 + cis-9,cis-11; trans-10,cis-12; trans-11,trans-13; trans-9,cis-11 CLA isomers. The important cis,trans isomers of CLA usually elute in a region of the chromatogram that is free from other fatty acids. However, C21:0 and C20:2 elute in the range of the cis,cis- and trans,trans-CLA isomers. Although the information on CLA iso- meric composition provided by GC is incomplete, GC is often the only method used in the analysis of FAs for CLA. In this concern, the most important analytical task is resolution of GC unresolved triplet of cis-9,trans-11; trans-7,cis-9; and trans- 8,cis-10 CLA isomers. The major cis-9,trans-11 isomer comprises 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.11.019