| As nutrition enhancers, ω-6 and ω-3 polyunsaturated fatty acids(PUFAs) are used widely in food industry. Results of study show that when total intake and the ratio of ω-6 and ω-3 PUFAs are in fitting range, it will be helpful to prevent cardiovascular disease, tumor and improve the intelligence development of infant and so on. In order to judge the quality of the products that rich in ω-6 and ω-3 PUFAs and provide basic data for people to select products reasonably, it is necessary to strengthen determination of content and ratio of ω-6 and ω-3 PUFAs.GC is widely used in methods of determination of fatty acids, including GB and literatures. But problems of double-bond isomerization and rupture when unsaturated fatty acids, especially polyunsaturated fatty acids are analysed under high temperature are found, as a result, the accuracy of determination is affected. Howere HPLC can avoid the problems above, but several problems found are as follows:(1) the underivative metods detect the polyunsaturated fatty acids in the wavelength of 200~210 nm, in which there are many chemical compositions affecting the accuracy of determination results.(2) Pretreatment methods are tedious and time consuming. Besides, reaction condition is strict.(3) The pre-column derivatization conditions of fatty acids using high performance liquid chromatography are only screened by peak area or peak height, in which derivatization yield is absent and affect the accuracy of determination results.For the drawbacks of determination methods above, MBH was obtained; The pre-column derivatization conditions of fatty acids was optimized; A HPLC method of fatty acids was demonstrated; The saponification conditions of fatty acids was optimized; Six ω-6 and ω-3 PUFAs(α-Linolenic acid ALA, Eicosapentaenoic Acid EPA, Docosahexaenoic Acid DHA, Linoleic acid LA, Gamma linolenic acid GLA, Arachidonic acid AA) in edible oil(olive oil, camellia oil etc.), health food(fish oil, cod-liver oil etc.) and milk products(infant formula milk powder etc.) was detected. Specific research results are as follows:(1) MBH was obtained. Myristic acid(MA) was selected to represent all of fatty acids(FAs) in the experiment, MBH was produced, its purity was 99.81%.(2) This study demonstrated pre-column derivatization method for the determination of six ω-6 and ω-3 PUFAs by HPLC. Through single factor test, The best conditions for the pre-column derivatization were as follows: the concentration and solvent amount of 2-NPH·HCl was 0.04 mol/L and 200 μL, the concentration of 1-EDC·HCl was 0.45 mol/L the concentration of pyridine was 3%, the solvent amount of 1-EDC·HCl working solution 200 μL, the temperature of the derivatization reaction was 60 ℃, the time of the derivatization reaction was 15 min, the optimal range of p H was 4.0~6.0.(3) This study demonstrated a HPLC method for the simultaneous determination of 6 kinds of ω-6 and ω-3 polyunsaturated fatty acids. Through screening mobile organic phase, composition of mobile phase, the p H of the mobile phase and column temperature, the best chromatographic condition was set: the analytes were separated by a Sunfire C18(250 mm×4.6 mm, 5μm), acetonitrile-aqueous solution(88:12) was used as mobile phase with a isocratic solution(88:12) under the temperature of 35 ℃, at the rate of 1 m L/min. The detection wavelength was 400 nm. the injection volume was 10 μL.(4) Saponification method was developed. Through single factor test, The best saponification conditions were as follows: the concentrationof KOH-ethanol solution(V(ethanol):V(water)=4:1) was 0.2 mol/L, the temperature of the saponification reaction was 80 ℃, the time of the derivatization reaction was 40 min.The analytes were saponified using the saponification method above and the average recoveries among different kinds of food were 93.57%~104.99% and the intro-day and inter-day prcession were lower than 1.84% and 2.05% respectively.The limits of detection ranged from 0.20 μg/m L to 0.25 μg/m L and the limit of quantification from0. 5 mg/kg to 0.6 mg/kg.(5) A large scope of samples was detected such as edible oil, health food and milk products. It was found that determination results of actual samples agreed with packaging label. Besides, the ratio of ω-6 and ω-3 polyunsaturated fatty acids in olive oil, canola oil soybean oil and cod-liver oil were in accord with recommended value.The ratio of ω-6 and ω-3 polyunsaturated fatty acids in Camellia oil, corn oil and peanut oil was far above the maximum of recommended value. A problem that ω-6 polyunsaturated fatty acids is excess in diet of our native residents has been found, so fish oil in which the ratio of ω-3 and ω-6 polyunsaturated fatty acids was 13.96 was suitable for them. AA/DHA in infant milk powder was 2.03:1, which was basically recommended value. LA/ALA was 11:1, which was not in accord with recommended value in GB 10765-2010. |
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