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.

Quantification of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization by gas-liquid chromatography.

Long-chain polyunsaturated fatty acids (LC-PUFAs) of the n-3 series and especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) have important biological properties. The main dietary sources of LC-PUFAs are fish and fish oil. Geometrical isomerization is one of the main reactions happening during the thermal treatment of polyunsaturated fatty acids. Refined fish oils are used to supplement food products in LC-PUFAs and the quality of these nutritional ingredients have to be controlled. In the present study, a suitable method for the quantification of EPA and DHA geometrical isomers in fish oils by gas-liquid chromatography (GC) is presented. A highly polar capillary column (CP-Sil 88, 100 m) operating under optimal conditions was used. Method selectivity was studied by GC-mass spectrometry. The performance characteristics of the quantification method were studied using samples of fish oil deodorized at 220 degrees C for 3 h. The linearity of the method was assessed by analyzing composite samples obtained by mixing fish oil deodorized at 220 degrees C with semi-refined fish oil (control). Precision was evaluated by analyzing the same samples in triplicate. Results showed that the validated method is suitable to quantify low amounts of geometrical (trans) isomers of EPA and DHA in refined fish oils. The limits of quantification of the EPA and DHA geometrical isomers are 0.16 and 0.56 g/100 g of fish oil, for EPA and DHA, respectively. Commercially available LC-PUFA oil samples were evaluated by using the validated method. The results show that the oils analyzed contain low amounts (<1% of total fatty acids) of geometrical isomers of EPA and DHA.

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.

Development of a gas-liquid chromatographic method for the analysis of fatty acid tryptamides in cocoa products.

The determination of the occurrence and level of cocoa shells in cocoa products and chocolate is an important analytical issue. The recent European Union directive on cocoa and chocolate products (2000/36/EC) has not retained the former limit of a maximum amount of 5% of cocoa shells in cocoa nibs (based on fat-free dry matter), previously authorized for the elaboration of cocoa products such as cocoa mass. In the present study, we report a reliable gas-liquid chromatography procedure suitable for the determination of the occurrence of cocoa shells in cocoa products by detection of fatty acid tryptamides (FATs). The precision of the method was evaluated by analyzing nine different samples (cocoa liquors with different ranges of shells) six times (replicate repeatability). The variations of the robust coefficient of variation of the repeatability demonstrated that FAT(C22), FAT(C24), and total FATs are good markers for the detection of shells in cocoa products. The trueness of the method was evaluated by determining the FAT content in two spiked matrices (cocoa liquors and cocoa shells) at different levels (from 1 to 50 mg/100 g). A good relation was found between the results obtained and the spiking (recovery varied between 90 and 130%), and the linearity range was established between 1 and 50 mg/100 g in cocoa products. For total FAT contents of cocoa liquor containing 5% shells, the measurement uncertainty allows us to conclude that FAT is equal to 4.01 +/- 0.8 mg/100 g. This validated method is perfectly suitable to determine shell contents in cocoa products using FAT(C22), FAT(C24), and total FATs as markers. The results also confirmed that cocoa shells contain FAT(C24) and FAT(C22) in a constant ratio of nearly 2:1.

Triacylglycerol analysis for the quantification of cocoa butter equivalents (CBE) in chocolate: feasibility study and validation.

A new European legislation (2000/36/CE) has allowed the use of vegetable fats other than cocoa butter (CB) in chocolate up to a maximum value of 5% in the product. The vegetable fats used in chocolate are designated as cocoa butter replacements and are called cocoa butter equivalents (CBE). The feasibility of CBE quantification in chocolate using triacylglycerol (TAG) profiles was conducted by analyzing 55 samples of CBs and 31 samples of CBEs using a liquid chromatograph equipped with an evaporative light scattering detector (HPLC-ELSD). Statistical evaluation of the data obtained has been performed, and a simulation study has been carried out to assess the viability to use this method for quantifying the amount of CBE in real mixtures and in chocolates. The TAGs POP, POS, PLS, and the ratios POP/PLS, POS/PLP (P, palmityl; O, oleyl; S, stearyl; L, linoleyl) are particularly significant to discriminate between CB and CBE. Analysis of 50 mixtures between 5 different CBEs and 10 different CBs at 2 different concentration levels is presented. The data are visualized and interpreted. A mathematical model has been developed to assess the amount of CBE in real mixtures. This predictive model has been successfully applied and validated on dark chocolates including authorized CBE. The results are affected by +/-2.1% absolute average error. In particular, estimations between 10 and 20% of CBE show a very good match. On the other hand, values equal to or smaller than 5% show a larger prediction error (detection limit of the method). For the main purpose of this method (i.e., quantification of CBE at 5% max in chocolate, which represents about 15% of the total fat) this model shows very good results. For milk chocolate, the mathematical model can also be used if TAG are integrated from partition number (PN) 46 to 54. Consequently, the model proposed provides sufficient information to verify the real application of the European legislation.