Comparison of available analytical methods to measure trans-octadecenoic acid isomeric profile and content by gas-liquid chromatography in milk fat.

Accurate quantification of trans-fatty acids (TFAs) could be achieved by infrared spectroscopy or by gas-liquid chromatography (GLC). Accurate quantification by GLC should be achieved using specific highly polar capillary columns such as 100 m CP-Sil 88 or equivalent. A pre-fractionation of cis and trans-fatty acids could be performed by silver-ion thin-layer chromatography (Ag-TLC), silver-ion solid-phase extraction (Ag-SPE), or by high-performance liquid-chromatography (HPLC). A pre-fractionation step allows accurate determination of the isomeric profile but it is not essential to achieve quantification of total trans-18:1 isomers nor to determine the level of vaccenic (trans-11 18:1) acid in dairy fat. TFA content could also be calculated in milk fat based on the TAG profile determined by GLC. In this paper, different GLC methods suitable to measure the total of trans-18:1 isomers, vaccenic acid and trans-18:1 acid isomeric distribution in milk fat were compared. Pre-separation of cis- and trans-18:1 isomers by Ag-TLC followed by GLC analysis under optimal conditions was selected as the reference method. Results obtained using alternative methods including pre-separation by HPLC followed by GLC analysis, direct quantification by GLC or calculation from the triacylglycerol (TAG) profile were compared to data acquired using the reference method. Results showed that accurate quantification of total trans-18:1 isomers and vaccenic acid could be achieved by direct quantification by GLC under optimal chromatographic conditions. This method represents a very good alternative to Ag-TLC followed by GLC analysis. On the other hand, we showed that pre-fractionation of fatty acid methyl esters (FAMEs) by HPLC represents a good alternative to Ag-TLC, even if some minor isomers are not selectively purified using this procedure.

Putting the 'Ome' in lipid metabolism.

The recognition that altered lipid metabolism underlies many metabolic disorders challenging Western society highlights the importance of this metabolomic subset, herein referred to as the lipidome. Although comprehensive lipid analyses are not a recent concept, the novelty of a lipidomic approach lies with the application of robust statistical algorithms to highlight subtle, yet significant, changes in a population of lipid molecules. First-generation lipidomic studies have demonstrated the sensitivity of interpreting quantitative datasets with computational software; however, the innate power of comprehensive lipid profiling is often not exploited, as robust statistical models are not routinely utilized. Therefore, the current review aims to briefly describe the current technologies suitable for comprehensive lipid analysis, outline innovative mathematical models that have the ability to reveal subtle changes in metabolism, which will ameliorate our understanding of lipid biochemistry, and demonstrate the biological revelations found through lipidomic approaches and their potential implications for health management.

Authenticity of milk fat by fast analysis of triacylglycerols. Application to the detection of partially hydrogenated vegetable oils.

Detection of foreign fat in milk fat can be performed by analyzing triacylglycerols (TAGs) by gas-liquid chromatography (GLC) using the standardized methodology. The standard methodology recommends the use of a packed column, which allows the separation of milk TAGs according to their chain length (total carbon number). This procedure is not widely applied because these columns are not commercially available. This study describes a fast methodology by using a short apolar open-tubular capillary column. The developed experimental conditions can be used to obtain the chromatographic resolution required in the standardized procedure, and the separation of milk fat TAGs (C24 to C54) is achieved in less than 4 min. As indicated by the standardized method, the quantification was performed by calibration using the certified reference material CRM-519 butterfat as standard substance. The methodology was fully validated and relative repeatability values were compared with the values provided in the standardized procedure. The developed method was applied to detect adulteration of milk fat with partially hydrogenated vegetable oils (PHVOs). PHVOs contain variable amount of trans-18:1 acids and two different PHVOs having different trans-18:1 acid levels (13 and 38%) were added to milk fat at levels ranging from 5 to 30%. The obtained mixtures were analyzed by GLC and formulas established by the European Union were applied. Calculated S values indicated that PHVOs in milk fat could be analyzed at these levels. Approximate amounts of PHVOs added to the composite samples could be calculated using the standardized formula. The impact of adulteration of milk fat with PHVOs, which contains an important amount of trans-9 and trans-10 18:1 acid isomers, was investigated as a complementary analytical criteria. We showed in composite samples, that the trans-18:1 acid isomeric distributions are distinct when referenced to the original milk fat profile and that trans-9 18:1 acid isomer is a good indicator of the occurrence of PHVOs in milk fat. Our results showed clearly that a short apolar capillary column can be used instead of a packed-column and that the mathematical model developed for the detection of foreign fat was suitable to detect adulteration of milk fat with PHVOs.

Chemical synthesis and NMR characterization of structured polyunsaturated triacylglycerols.

The chemical synthesis of pure triacylglycerol (TAG) regioisomers, that contain long chain polyunsaturated fatty acids, such as arachidonic acid (AA) or docosahexaenoic acid (DHA), and saturated fatty acids, such as lauric acid (La) or palmitic acid (P), at defined positions, is described. A single step methodology using (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), an activator of carboxyl group commonly used in peptide synthesis and occasionally used in carboxylic acid esterification, has been developed for structured TAG synthesis. Identification of the fatty acyl chains for each TAG species was confirmed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and fatty acid positional distribution was determined by (1)H and (13)C NMR spectra. The generic described procedures can be applied to a large variety of substrates and was used for the production of specific triacylglycerols of defined molecular structures, with high regioisomeric purity. Combination of MS and NMR was shown to be an efficient tool for structural analysis of TAG. In particular, some NMR signals were demonstrated to be regioisomer specific, allowing rapid positional analysis of LC-PUFA containing TAG.

Quantitative analysis of triacylglycerol regioisomers in fats and oils using reversed-phase high-performance liquid chromatography and atmospheric pressure chemical ionization mass spectrometry.

Positional distribution of fatty acyl chains of triacylglycerols (TGs) in vegetable oils and fats (palm oil, cocoa butter) and animal fats (beef, pork and chicken fats) was examined by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to atmospheric pressure chemical ionization using a quadrupole mass spectrometer. Quantification of regioisomers was achieved for TGs containing two different fatty acyl chains (palmitic (P), stearic (S), oleic (O), and/or linoleic (L)). For seven pairs of 'AAB/ABA'-type TGs, namely PPS/PSP, PPO/POP, SSO/SOS, POO/OPO, SOO/OSO, PPL/PLP and LLS/LSL, calibration curves were established on the basis of the difference in relative abundances of the fragment ions produced by preferred losses of the fatty acid from the 1/3-position compared to the 2-position. In practice the positional isomers AAB and ABA yield mass spectra showing a significant difference in relative abundance ratios of the ions AA(+) to AB(+). Statistical analysis of the validation data obtained from analysis of TG standards and spiked oils showed that, under repeatability conditions, least-squares regression can be used to establish calibration curves for all pairs. The regression models show linear behavior that allow the determination of the proportion of each regioisomer in an AAB/ABA pair, within a working range from 10 to 1000 microg/mL and a 95% confidence interval of +/-3% for three replicates.