Stable carbon isotope ratios of methyl-branched fatty acids are different to those of straight-chain fatty acids in dairy products.

Methyl-branched fatty acids (MBFAs) are the dominant form of fatty acid found in many bacteria. They are also found at low levels in a range of foodstuffs, where their presence has been linked to bacterial sources. In this study we evaluated the potential of compound-specific isotope analysis to obtain insights into the stable carbon isotope ratios (delta(13)C values in per thousand) of individual MBFAs and to compare them to the stable carbon isotope ratios of straight-chain fatty acids in food. Due to their low abundance in foodstuffs, the MBFAs were enriched prior to gas chromatography coupled to isotope ratio mass spectrometric (GC-IRMS) analysis. After transesterification, urea complexation was used to suppress the 16:0 and 18:0 methyl esters that were dominant in the samples. Following that, silver-ion high performance liquid chromatography was used to separate the saturated from the unsaturated fatty acids. The resulting solutions of saturated fatty acids obtained from suet, goat's milk, butter, and human milk were studied by GC-IRMS. The delta(13)C values of fatty acids with 12-17 carbons ranged from -25.4 per thousand to -37.6 per thousand. In all samples, MBFAs were most depleted in carbon-13, followed by the odd-chain fatty acids 15:0 and 17:0. 14:0 and 16:0 contained the highest proportions of carbon-13. The results from this study illustrate that MBFAs have distinctive delta(13)C values and must originate from other sources and/or from very different substrates. These measurements support the initial hypothesis that delta(13)C values can be used to attribute MBFAs to particular sources.

Enantioselective determination of anteiso fatty acids in food samples.

Anteiso fatty acids (aFAs)-long-chain carboxylic acids with a methyl branch on the (n - 2)-carbon-are among the most simple fatty acids that are chiral. The most frequently occurring aFAs in food are 12-methyltetradecanoic acid (a15:0) and 14-methylhexadecanoic acid (a17:0), structures where the asymmetric carbon is more than 10 carbons separated from the polar head group. Previously, only enantioseparation of 4-methyl-substituted carboxylic fatty acids has been reported by gas chromatography. Here we present the first direct partial enantioresolution of synthesized racemic a15:0-a17:0 on a capillary column coated with 50% heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-beta-cyclodextrin diluted in OV1701. Synthesized (S)-(+)-enantiomers were used to demonstrate that the elution order was (R)- prior to (S)-enantiomers. Using this system, food samples (butter, goat's milk fat, suet, human milk, seal oil, cod liver oil) known to contain aFAs were analyzed. Prior to the enantioselective gas chromatography, unsaturated fatty acids were preseparated by urea complexation, silver ion high performance liquid chromatography (Ag+-HPLC), or both from food samples. The fractions of the food samples enriched with methyl-branched fatty acids were then analyzed by GC/MS in the SIM mode. The measurements confirmed that the (S)-enantiomer of a15:0 (ee >96%), a16:0, and a17:0 (ee >90%, respectively) dominated in all samples. While the (R)-enantiomers could not be identified in samples from ruminants and human milk, their presence could be established in cod liver and seal oil (ee <86%).

Impact on lipid membrane organization by free branched-chain fatty acids.

Here, we exploit the non-invasive techniques of solid-state NMR (nuclear magnetic resonance) and differential scanning calorimetry (DSC) to study the effect of free iso and ante-iso branched chain fatty acids (BCFAs) on the physicochemical properties of lipid membranes. Free fatty acids are present in biological membranes at low abundance, but can influence the cellular function by modulating the membrane organization. Solid state NMR spectra of dimyristoylphosphatidylcholine (DMPC) lipid membranes containing either free 12-methyltetradecanoic acid (a15:0) or free 13-methyltetradecanoic acid (i15:0), show significant differences in their impact on the lipid bilayer. Chain order profiles obtained by deuterium NMR on fully deuterated DMPC-d(67) bilayers revealed an ordering effect induced by both fatty acids on the hydrophobic membrane core. This behavior was also visible in the corresponding DSC thermograms where the main phase transition of DMPC bilayers-indicative of the hydrophobic membrane region-was shifted to higher temperatures, with the iso isomer triggering more pronounced changes as compared to the ante-iso isomer. This is probably due to a higher packing density in the core of the lipid bilayer, which causes reduced diffusion across membranes. By utilizing the naturally occurring spin reporters nitrogen-14 and phosphorus-31 present in the hydrophilic DMPC headgroup region, even fatty acid induced changes at the membrane interface could be detected, an observation reflecting changes in the lipid headgroup dynamics.

Application of ethyl esters and d3-methyl esters as internal standards for the gas chromatographic quantification of transesterified fatty acid methyl esters in food.

Ethyl esters (FAEE) and trideuterium-labeled methyl esters (d3-FAME) of fatty acids were prepared and investigated regarding their suitability as internal standards (IS) for the determination of fatty acids as methyl esters (FAME). On CP-Sil 88, ethyl esters of odd-numbered fatty acids eluted approximately 0.5 min after the respective FAME, and only coelutions with minor FAME were observed. Depending on the problem, one or even many FAEE can be added as IS for the quantification of FAME by both GC-FID and GC-MS. By contrast, d3-FAME coeluted with FAME on the polar GC column, and the use of the former as IS requires application of GC-MS. In the SIM mode, m/z 77 and 90 are suggested for d3-methyl esters of saturated fatty acids, whereas m/z 88 and 101 are recommended for ethyl esters of saturated fatty acids. These m/z values give either no or very low response for FAME and can thus be used for the analysis of FAME in food by GC-MS in the SIM mode. Fatty acids in sunflower oil and mozzarella cheese were quantified using five saturated FAEE as IS. Gravimetric studies showed that the transesterification procedure could be carried out without of loss of fatty acids. GC-EI/MS full scan analysis was suitable for the quantitative determination of all unsaturated fatty acids in both food samples, whereas GC-EI/MS in the SIM mode was particularly valuable for quantifying minor fatty acids. The novel GC-EI/MS/SIM method using fatty acid ethyl esters as internal standards can be used to quantify individual fatty acids only, that is, without determination of all fatty acids (the common 100% method), although this is present. This was demonstrated by the exclusive quantification of selected fatty acids including methyl-branched fatty acids, erucic acid (18:1n-9trans), and polyunsaturated fatty acids in cod liver oil and goat's milk fat.

A gas chromatography/electron ionization-mass spectrometry-selected ion monitoring method for determining the fatty acid pattern in food after formation of fatty acid methyl esters.

A method using gas chromatography/electron ionization-mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode was developed for the analysis of fatty acids as methyl esters (FAMEs) in order to determine their percentage contribution to the fatty acid profile in food. In the GC/EI-MS-SIM mode, saturated fatty acids were determined with m/z 87, monoenoic fatty acids were determined with m/z 74, and polyenoic fatty acids were determined via the sum of m/z 79 and m/z 81. The ratios of these fragment ions and the GC retention data provided additional information for tentative structural assignments. The 28 FAME standards tested provided similar results for the novel GC/EI-MS-SIM method and GC/EI-MS in the full scan mode, both of which were slightly worse than GC/flame ionization detection (FID). Analysis of sunflower oil, suet, and cod liver oil verified that both major and minor fatty acids (20-60% and down to 0.001% contribution to the fatty acid pattern) were determined with sufficient quality that justifies application of the GC/EI-MS-SIM method for the analysis of food samples. Furthermore, the method was approximately 20- or approximately 10-fold more sensitive than GC/EI-MS in the full scan mode or GC/FID, respectively. The method is suited for both quantitative purposes and fatty acid identification in samples where only low amounts of lipids are available.