气相色谱同位素比质谱测定不同产地鱼油中5种不饱和脂肪酸碳同位素比值

Study on carbon isotope ratios of 5 unsaturated fatty acids in fish oil from different areas by gas chromatography-isotope ratio mass spectrometry

以IAEA-600咖啡因（δ^13C-27.771‰）作为溯源标准,建立了气相色谱同位素质谱技术测定鱼油中功能因子亚油酸（LIA）、亚麻酸（LNA）、花生四烯酸（ARA）、二十碳五烯酸（EPA）、二十二碳六烯酸（DHA）等5种不饱和脂肪酸碳稳定同位素比值δ^13C的分析方法。鱼样品先经HCl水解,乙醚液液萃取脂肪,提取的脂肪在2 mol/L KOH-甲醇溶液中反应生成脂肪酸甲酯,采用强极性毛细管气相色谱柱（Sil-88 100 m×0.25 mm×0.2μm）分离,稳定同位素比质谱测定。方法经日内、日间和人员比对验证,表明测定结果稳定,标准偏差小于0.82%。收集了不同产地的241个淡水鱼和深海鱼,对于提取的天然鱼油进行5种不饱和脂肪酸同位素比值分析,测得天然鱼油中5种不饱和脂肪酸同位素比值（δ^13C）在-32.87‰~27.07‰之间,经计算,鱼油中不饱和脂肪酸δ^13C值与硬脂酸δ^13C值之比在-5.79~1.88之间,同时测得了相同的产地、品种鱼油中不饱和脂肪酸δ^13C的分布范围,构建了天然鱼油的同位素指纹特征数据库,用于鉴定鱼油真伪。将不同浓度玉米油添加至天然金枪鱼油中进行测定,证明δ^13C值的变化与掺入C4植物油量呈良好的线性关系,方法可根据δ13C值的变化鉴别掺假的鱼油。

The IAEA-600 caffeine（ δ^13C is-27. 771‰） was used as a traceable standard. A method was established for determining carbon isotope ratio of 5 unsaturated fatty acids（ UFAs） including linoleic acid（ LIA）,linolenic acid（ LNA）,arachidonic acid（ ARA）,eicosapentaenoic acid（ EPA）,and docosahexaenoic acid（ DHA） in natural fish oil by gas chromatography- isotope ratio mass spectrometry（ GC-IRMS）. Fish samples were first hydrolyzed by hydrochloric acid and ethyl ether was used for extracting fat during liquid-liquid extraction（ LLE） procedure. The extracted fat was methyl esterified by 2 mol/L KOH methanol solution. The separation of 5 UFAs was carried out on a capillary gas chromatographic column（ Sil-88 100 m × 0. 25 mm × 0. 2μm） and the detection was finished with stable isotope ratio mass spectrometry. The method was validated with experiments of within-day,inter-day,and inter-comparison and the stable results were obtained with standard deviation less than 0. 82%. The extracted natural fish oils were analyzed for 5 UFAs which were collected from different origins including 241 fresh water fishes and sea fishes. The measured δ^13C results of 5 UFAs ranged from-32. 87‰ to ~ 27. 07‰ in natural fish oil. The ratios of unsaturated fatty acids δ^13C and stearic acid δ^13C changed from-5. 79 to 1. 88. The distribution ranges of 5 UFAs were determined in fish oil from the same area and species. The data can be characterized as isotopic fingerprint database of the natural fish oil and used for the identification of adulteration. The tuna oil was spiked with different levels corn oil and then the good linearity was obtained between δ^13C changes and added C4 plant oil. Based on δ13C changes,the new GC-IRMS method can identify adulteration of fish oil.