DOI 10.3969/j.issn.1005-6521.2013.08.019 Food Research And Development 2013 3 34 6 69 1 2 1 1 * 1 1 1 1. 361005 2. 361005 : 采用超高压液相方法检测鱼油中的二十碳五烯酸乙酯 () 和二十二碳六烯酸乙酯 (), 采用 UPLC BEH C18(2.1 mm 50 mm,1.7 μm) 色谱柱, 考察了流动相有机相比例 流速及柱温对鱼油中 和 DHA- EE 分离的影响, 确定了最佳色谱条件 : 等度洗脱, 流动相为乙腈 - 水 (80 20, 体积比 ), 流速 0.5 ml/min, 柱温 30, 检测波长 220 nm 该方法下, 和 分别在 10.4 μg/ml~209 μg/ml 10.3 μg/ml~207 μg/ml 范围内浓度和 面积呈现良好的线性关系, 相关系数 r 分别为 0.999 4 和 0.999 7; 和 的方法的检测限为 4 ng 和 6 ng, 定量限分别为 10 ng 和 15 ng, 精密度试验 RSD(n=6) 分别为 1.09 % 和 0.34 %, 重复性试验 RSD(n=6) 分别为 1.54 % 和 1.25 %, 平均回收率分别为 102.26 %(RSD=1.33 %) 和 96.64 %(RSD=1.05 %) 本方法快速 简单 可靠 灵敏 重复性 好, 可用于鱼油中 和 的快速检测 : 超高压高效液相色谱 ; 二十碳五烯酸乙酯 ; 二十二碳六烯酸乙酯 ; 鱼油 Determination of Eicosapentaenoic Acid Ethyl and Docosahexaenoic Acid Ethyl Ester in Fish Oil by Ultra Performance Liquid Chromatography CHEN Wei-zhu 1,2, JIN Wen-hui 1, HONG Zhuan 1,*, ZHANG Yi-ping 1, FANG Hua 1, YI Rui-zao 1 (1.The Third Institute of Oceanography State Oceanic Administration, Xiamen 361005, Fujian, China; 2. Department of Chemistry, College of Chemistry and Chemical Engineering, and The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, Fujian, China) Abstract The article is researching determination of eicosapentaenoic acid ethyl ester (EPA -EE) and docosahexaenoic acid ethyl ester (DHA- EE) in Fish Oil by Ultra performance liquid chromatography(uplc). The analysis was performed on an Acquity UPLC BEH C18 column (2.1 mm 50 mm, 1.7 μm). The influence of flow rate, the proportion of organic phase in mobile phase, and column temperature on the separation of and in fish oil was comprehensively studied. The optimal separation condition was as follows: a mobile phase consisting of acetonitrile and water (80 20,V/V) with a isocratic elution profile, UV detection wavelength at 220 nm, 0.5 ml/min of flow rate for mobile phase, 30 of column temperature. On the above condition, good linearity between response (peak area) and concentration was found over a concentration range of 10.4 μg/ml~209 μg/ml for, and 10.3 μg/ml~207 μg/ml for respectively. And the correlation coefficient of the calibration curve was 0.999 4 for, and 0.999 7 for respectively. The limits of detection (S/N>3) were 4ng for and 6ng for respectively. The limits of quantification (S/N>10) were 10ng for and 15ng for respectively. The average recovery rates of and were 102.26 % (RSD 1.33 %) and 2009Y0035 200905022 1981 * 1970
70 陈伟珠, 等 : 超高压液相色谱法测定鱼油中的 和 DHP-EE 96.64 % (RSD 1.05 %) respectively. The RSD of repetition was 1.54 %(n=6) for and 1.25 %(n=6) for. This method was simple, accurate and sensitive with a good reproducibility. It is suitable for fast detection of and in fish oil. Key words:ultra performance liquid chromatography ( UPLC ) ; eicosapentaenoic acid methyl ester ; docosahexaenoic acid ethyl ester; fish oil cis -5 8 11 14 17 -eicosapentaenoic acid EPA cis-4 7 10 13 1.2 16 19-docosahexaenoic acid DHA ω-3 EPA DHA EPA DHA Milli-Q 1.3 [1-2] EPA DHA 1.3.1 EPA -EE 13.041 mg EPA DHA 12.914 mg 25 ml DHA- EE EPA DHA EPA 1.2 DHA EPA DHA [3] 1.7 μm - 80 20 0.5 ml/min 220 nm 3 μl 30 [4-5] [6] 2 [7] 10 min 2.1 2 μm UPLC - - UPLC - <2 μm >10 5 kpa 75 % 80 % 85 % 1 [8-13] 1 0.5 ml/min - 75 25-80 20-85 15 3-75 25-80 20 1 1.1 2.2 TUV ACQUITY - 80 20 Waters ACQUITYUPLC TM BEHC18 2.1 mm 50 mm Sigma-Aldrich ACQUITY UPLC TM BEHC18 2.1mm 50 mm 0.3 0.4 0.5 0.6 ml/min 1.7 μm Waters Milii-Q Millipore
陈伟珠, 等 : 超高压液相色谱法测定鱼油中的 和 DHP-EE 71 3.265 4.191 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.598 0.15 0.05 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 4.468 a - 75 25 a 0.3 ml/min 1 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 b - 80 20 0.20 0.15 0.05 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 1.364 1.611 c - 85 25 Fig.1 Chromatograms of and in different mobile phases with the same flow rate and the same column temperature 2 2 0.5 ml/min 2.3 [4] 220 nm 30 35 40 3 y= 49 273.19x+ 0.60 y x 30 r = 0. 999 4-80 20 y= 14 288x-466.1 y x 0.5 ml/min 30 220 nm 2.4 EPA -EE 100 200 400 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 r = 0. 999 7 S/ N 800 1 000 1 500 2 000 μl 7 5 ml 3 4 ng 6 ng 7 S/ N 10 10 ng 15 ng 7 2.5 3 μl 1 2.656 3.289 b 0.4 ml/min 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 c 0.5 ml/min 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 1.719 2.117 d 0.6 ml/min Fig.2 Chromatograms of and in the same mobile phases and the same column temperature with different flow rate
3 72 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 a 30 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 1.950 2.394 b 35 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 1.821 2.221 c 40 Fig.3 Chromatograms of and in the same mobile phases and the same flow rate with different column temperature Table 1 1 Calibration curve for and / μg/ml 10.4 254 04 20.9 585 62 41.7 121 865 83.5 268 127 104 323 939 156 482 287 209 645 371 10.3 144 753 20.7 278 362 41.3 578 352 82.6 118 458 8 103 149 794 5 155 221 647 5 207 291 605 1 陈伟珠, 等 : 超高压液相色谱法测定鱼油中的 和 DHP-EE 104 μg/ml 103 μg/ml 3 μl 6 RSD 1.09 % 0.34 % 2.6 104 μg/ml 103 μg/ml 6 3 μl RSD 1.54 % 1.29 % 2.7 1 EPA- EE 80 100 120 μg 3 102.26 % RSD 1.33 % 96.64 % RSD 1.05 % 2 2 Table 2 The result of recovery for / / μg/ml μg/ml % /% /% 78.2 80.5 102.94 101.31 1.27 83.5 84.5 101.20 88.7 90.8 102.37 99.1 98.2 99.09 104 107 102.88 110 111 100.91 120 121 100.83 125 125 10 130 132 101.54 77.5 72.6 93.68 96.70 1.21 82.6 78.9 95.52 87.8 84.0 95.67 98.1 94.2 96.02 103 101 98.06 108 103 95.37 119 113 94.96 124 119 95.97 129 124 96.12 2 2.8 EPA- EE DHA -EE GB/T 17377-2008 [20] 3 Table 3 3 The content of and in Sample by two methods / mg/ml / mg/ml 1 31.061 38.213 2 123.383 7.976 1 30.921 38.158 2 123.021 7.904
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