Journal of Chromatography B, 855 (2007) 152–158 Quantitative gas chromatographic method for the analysis of cis-9, trans-11 and trans-10, cis-12 isomers of the conjugated linoleic acid in liver A. Zabala a , M.P. Portillo a , V. Navarro a , M.T. Macarulla a , L.J.R. Barron b , A. Fern´ andez-Quintela a,∗ a Department of Nutrition and Food Science, University of the Basque Country, Paseo de la Universidad 7, 01006 Vitoria, Spain b Department of Food Technology, University of the Basque Country, Paseo de la Universidad 7, 01006 Vitoria, Spain Received 1 February 2007; accepted 25 April 2007 Available online 10 May 2007 Abstract A quantitative GC method for conjugated linoleic acid (CLA) isomers of physiological significance (cis-9, trans-11 CLA and trans-10, cis-12 CLA) as non-esterified fatty acids (NEFA) or triacylglycerols (TAG) was developed. Furthermore, the effect of the internal standard addition point (sample or fat extract) was studied. Response linearity, recovery and precision assays, detection and quantification limits were determined. Linearity was demonstrated over a range from 0.1 to 10 g/mL. When CLA isomers were present as NEFA, the recovery significantly decreased (P ≤ 0.05) from 76% to 27.1% (cis-9, trans-11 CLA) and 28.5% (trans-10, cis-12 CLA) when the standards were added to the fat extract or to the initial tissue, respectively. As an application, liver samples from hamsters fed a diet supplemented with both CLA isomers were analyzed. The CLA isomers in liver samples were detected with reasonable reproducibility. © 2007 Elsevier B.V. All rights reserved. Keywords: Quantitative method; GC; CLA analysis; Liver 1. Introduction Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of octadecadienoic acid first described by Dr Pariza’s group in the 1980s [1]. These isomers possess conjugated double bonds (from 2, 4 – 18:2 to 15, 17 – 18:2) instead of the typical methylene interrupted configuration. The position of double bonds and the isomeric configuration (cis or trans) make numerous CLA isomers possible [2]. However, only a few are naturally found in foods such as ruminant meats, dairy products and processed cheeses, the cis-9, trans-11 CLA being the predominant isomer [3], and even fewer are commercially available. In order to establish the complete isomeric distribution it is essential to use a combination of analytical methods ∗ Corresponding author at: Nutrici´ on y Bromatolog´ ıa, Facultad de Farmacia, Universidad del Pa´ ıs Vasco, Paseo de la Universidad 7, 01006 Vitoria, Spain. Tel.: +34 945 013066; fax: +34 945 013014. E-mail address: alfredo.fernandez@ehu.es (A. Fern´ andez-Quintela). (Ag + -HPLC, GC/MS) [4–6]. However, in physiological and mechanistic studies only cis-9, trans-11 and trans-10, cis- 12 CLA isomers are important at present, since beneficial effects including anticarcinogenic, antiatherogenic, antidiabeto- genic, antiobesity and immunomodulatory activities have been attributed to them [7,8]. Therefore, Kramer et al. [9] suggested that a good GC analysis might be sufficient for their quantitative analysis when the study is applied to monogastric animals or humans. Thus, considerable efforts have been made to supply data related to CLA isomer content in biological samples, most of them, however, being related as percentage of total fat [10–12] usually making it difficult to reach metabolic conclusions. This is because these isomers are always in limited amounts, which are linked to changes in the profile of the major fatty acids when they are expressed as normalized area. Nevertheless, when quan- titative data are provided another doubt is raised, since a review of the literature shows that there is a lack of consistency concern- ing the addition point of the internal standard, it being applied either to the fat extract [13,14] or to the initial tissue [15,16]. 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.04.041