色谱

色谱 ›› 2013, Vol. 31 ›› Issue (3): 275-280.DOI: 10.3724/SP.J.1123.2012.11011

• 技术与应用 • 上一篇    下一篇

柱前衍生-超高效液相色谱法测定鱼卵中的17种氨基酸

孙言春1,2, 许宪祝1*, 徐衍岭1, 谭志军3, 牟振波2, 杜宁宁2   

  1. 1. 哈尔滨工业大学基础与交叉科学研究院, 黑龙江 哈尔滨 150001; 2. 中国水产科学研究院黑龙江水产研究所, 黑龙江 哈尔滨 150070; 3. 中国水产科学研究院黄海水产研究所, 山东 青岛 266071
  • 收稿日期:2012-11-20 修回日期:2012-12-24 出版日期:2013-03-28 发布日期:2013-03-22
  • 通讯作者: 谭志军
  • 基金资助:

    中央级非营利科研机构财政专项基金项目(2012A1004,201106,2007 HSYZX-ZH-26).

Determination of 17 amino acids in fish eggs by ultra performance liquid chromatography coupled with precolumn derivatization

SUN Yanchun1,2, XU Xianzhu1*, XU Yanling1, TAN Zhijun3, MOU Zhenbo2, DU Ningning2   

  1. 1. Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150001, China; 2. Heilongjiang River Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China; 3. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
  • Received:2012-11-20 Revised:2012-12-24 Online:2013-03-28 Published:2013-03-22

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565

被引次数

55

摘要： 建立了一种快速、灵敏的柱前衍生-超高效液相色谱-光电二极管阵列检测器(UPLC-PDA)测定史氏鲟(Acipenser schrenckii)、达氏鳇(Huso dauricus)和小体鲟(Acipenser ruthenus)鱼卵中17种氨基酸含量的方法。采用6.0 mol/L的盐酸水解鱼卵,提取液经低压浓缩、碱性中和,然后以6-氨基喹啉-N-羟基琥珀酰亚胺基氨基甲酸酯(AQC)为衍生试剂在pH 8.8硼酸盐缓冲溶液中衍生化。采用的色谱分离柱为Waters BEH C18柱(100 mm×2.1 mm, 1.7 μm),流动相为30 mmol/L乙酸铵水溶液(pH 3.5)和乙腈(含0.15%(v/v)甲酸及30 mmol/L乙酸铵),梯度洗脱,流速为0.7 mL/min,在260 nm波长下检测。17种氨基酸在5.0～1000 μmol/L浓度范围内,峰面积与浓度之间的线性关系良好(r2≥0.9950)。以标准加入法测定回收率和相对标准偏差(RSD),在100、500、750 μmol/L的添加水平下,17种氨基酸的平均回收率为75.4%～107.3%, RSD为2.19%～12.3%。以3倍信噪比(S/N＞3)计方法的检出限,17种氨基酸的检出限为0.94～4.04 μmol/L。应用该方法检测了3种鲟鳇鱼鱼卵中的17种氨基酸含量。结果表明,该方法简便、准确、快速、可靠。

关键词: 氨基酸, 超高效液相色谱, 鱼卵, 柱前衍生

Abstract: A rapid quantitative method of ultra performance liquid chromatography (UPLC) has been developed for the analysis of 17 amino acids in fish eggs from Acipenser schrenckii, Huso dauricus and Acipenser ruthenus. The analytes were hydrolyzed with 6.0 mol/L HCl. The extraction solution was concentrated under low pressure and neutralized with NaOH solution before derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC) as derivatization agent in borate buffer solution (pH 8.8). Gradient UPLC separation was performed on a C18 column (BEH C18, 100 mm×2.1 mm, 1.7 μm) with 30 mmol/L ammonium acetate (pH 3.5 adjusted with formic acid) and acetonitrile (containing 0.15%(v/v) formic acid and 30 mmol/L ammonium acetate) as the mobile phases at a flow rate of 0.7 mL/min. The detection was carried out with a photo-diode array (PDA) detector and the wavelength was set at 260 nm. The linear ranges were from 5.0 to 1000 μmol/L with the correlation coefficients (r2) ≥ 0.9950. The method was validated by the analysis of seven replicates. The 17 amino acid standards were spiked in fish eggs at three levels of 100, 500 and 750 μmol/L, and the average recoveries were 75.4%-107.3% with the relative standard deviations of 2.19%-12.3%. The limits of detection (LOD) for the analytes were from 0.94 μmol/L to 4.04 μmol/L. The method was successfully applied to the analysis of the 17 amino acids in fish eggs from Acipenser schrenckii, Huso dauricus and Acipenser ruthenus. The method is simple, rapid, precise and reliable.

Key words: amino acids, fish eggs, ultra performance liquid chromatography (UPLC), precolumn derivatization