Determination of trans unsaturation by infrared spectrophotometry and determination of fatty acid composition of partially hydrogenated vegetable oils and animal fats by gas chromatography/infrared spectrophotometry: collaborative study

An infrared spectrophotometric (IR) method for the determination of total trans unsaturated fatty acid (trans) content and a combined gas-liquid chromatographic/infrared spectrophotometric (GC/IR) method for determination of fatty acid composition of partially hydrogenated vegetable oils (PHVO) were studied collaboratively in 12 laboratories using 7 PHVO samples, including 1 pair of blind duplicates. The test samples were methylated and analyzed for total trans content by IR and for fatty acid composition by GC/IR using a capillary column coated with SP-2560 or another suitable cyanoalkylsiloxane stationary phase. From the measured IR absorption, the isolated trans content was calculated using a calibration curve of absorption versus trans content developed with 2-component calibration standard mixtures of methyl elaidate and oleate. The GC provided the levels of mono-transoctadecadienoates (18:2t), di-trans-octadecadienoates (18:2tt) and mono-trans-octadecatrienoates (18:3t). The trans-octadecenoate (18:1t) content was calculated with the formula: 18:1t=IR trans-0.84 x (18:2t + 18:3t)-1.74 x 18:2tt. The cis-octadecenoate (18:1c) content was obtained as the difference between total octadecenoates (18:1) and 18:1t. Reproducibility relative standard deviations (RSDR) for 15 to 35% trans content determined by IR were in the range of 8.8-11.7%, whereas RSDR for the test sample with 5% trans content was 34.6% RSDR values for 18:1t by the GC/IR followed the same pattern as that of IR trans values: 36.4% for the test sample with 4.9% 18:1t versus 7.8-12.5% for test samples with 14.9 to 32.6% 18:1t. The content of 18:1c in the test samples varied from 24.7 to 34.5% and their RSDR values ranged from 3.8 to 10.5%. The mean values for 18:1t and 18:1c compared favorably with the absolute levels determined by a silver nitrate-thin layer chromatography/GC procedure. The IR and GC/IR methods are recommended for determination of trans content and fatty acid composition, respectively, of partially hydrogenated fats derived from vegetable oils, terrestrial animal fats or such oils and fats isolated from food products containing >5% trans fatty acids. For samples containing <5% trans fatty acids, a direct GC method (American Oil Chemists' Society Official Method Ce 1c-89) is available for determination of both trans content and fatty acid composition, because at lower trans levels, overlap of 18:1 cis and trans isomers on GC with very polar capillary columns is negligible. The IR method for determination of isolated trans unsaturated fatty acid content in partially hydrogenated fats and the capillary GC/IR method for determination of total cis- and trans-octadecenoic isomers and general fatty acid composition in hydrogenated vegetable oils and animal fats have been adopted first action by AOAC INTERNATIONAL.