色谱

色谱 ›› 2019, Vol. 37 ›› Issue (10): 1118-1123.DOI: 10.3724/SP.J.1123.2019.03033

• 研究论文 • 上一篇    下一篇

高效液相色谱法测定生物转化反应液中N,N'-乙二胺二琥珀酸

吴智超1, 吴恩国2, 杨仲毅1, 陶宇翔3, 陈潭1, 钟永军1   

  1. 1.台州学院, 浙江 台州 318000;
    2.浙江海正药业股份有限公司, 浙江 台州 318000;
    3.台州市博纳化工有限公司, 浙江 台州 318000
  • 收稿日期:2019-03-26 出版日期:2019-10-08 发布日期:2020-12-11
  • 通讯作者: 杨仲毅,Tel:(0576)88660338,E-mail:yangzhyi@126.com.
  • 基金资助:
    台州市科技计划项目（162gy47，1701ny06）；台州市教科规划项目（gg19007）.

Determination of N,N'-ethylenediamine disuccinic acid in bioconversion reaction solution using high performance liquid chromatography

WU Zhichao1, WU Enguo2, YANG Zhongyi1, TAO Yuxiang3, CHEN Tan1, ZHONG Yongjun1   

  1. 1. Taizhou University, Taizhou 318000, China;
    2. Zhejiang Hisun Pharmaceutical Co., Ltd., Taizhou 318000, China;
    3. Taizhou Bona Chemical Co., Ltd., Taizhou 318000, China
  • Received:2019-03-26 Online:2019-10-08 Published:2020-12-11
  • Supported by:
    Taizhou Science and Technology Program Project (Nos. 162gy47, 1701ny06); Taizhou Education Planning Project (No. gg19007).

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摘要： 建立了高效液相色谱测定生物转化反应液中N，N'-乙二胺二琥珀酸（EDDS）含量的分析方法。采用InertSustain AQ-C18色谱柱（250 mm×4.6 mm，5 μm），以体积分数25%的甲醇水溶液（含有1.0 g/L一水乙酸铜、2.0 g/L四丁基氢氧化铵，以磷酸调节pH至2.80）为流动相，流速为1.0 mL/min，柱温为30℃，进样量为20 μL，检测波长为254 nm。该方法可在8 min内分离EDDS及其生物合成相关物质（苹果酸、柠檬酸、乙二胺四乙酸（EDTA）和富马酸），且峰形良好。EDDS在0.06~0.6 g/L范围内线性线性关系良好（相关系数（r）为0.9995），平均回收率为100.39%（n=9，RSD=1.15%）。EDDS生物合成反应液中EDDS含量为0.25 g/L，大部分底物被转化为苹果酸（36.56 g/L）；而EDDS的水解反应中富马酸产生较少，形成了3.05 g/L的苹果酸。该方法简便快速，灵敏可靠，适用于EDDS生物合成的研究。

关键词: 高效液相色谱, 反相离子对色谱, 生物转化, 螯合剂, N,N-乙二胺二琥珀酸

Abstract: A method was developed for the determination of N,N'-ethylenediamine disuccinic acid (EDDS) in bioconversion samples by high performance liquid chromatography (HPLC). An InertSustain AQ-C18 column (250 mm×4.6 mm, 5 μm) was used in the analysis. The mobile phase was 25% (v/v) methanol with 1.0 g/L Cu(CO2CH3)2·H2O, 2.0 g/L tetrabutylammonium hydroxide, and the pH was adjusted to 2.80 with phosphoric acid. The flow rate of the mobile phase was 1.0 mL/min, and the column temperature was set at 30℃. The detection wavelength was 254 nm. EDDS, fumaric acid, citric acid, malic acid and ethylenediaminetetraacetic acid (EDTA) were separated from one another within 8 min. EDDS showed good linearity in the range of 0.06-0.6 g/L. About 0.25 g/L EDDS was detected in the biosynthesis reaction solution catalyzed by recombinant EDDS-lyase, while 36.56 g/L malic acid was formed as the by-product from fumaric acid. The catalytic activity of the enzyme was confirmed in the hydrolysis of 10 g/L EDDS, which produced 3.05 g/L malic acid in 3 h. This analytical method is simple, rapid, sensitive, reliable, and suitable for the analysis in the research of EDDS bioconversion process.

Key words: high performance liquid chromatography (HPLC), reversed-phase ion pair chromatography, bioconversion, chelating agent, N,N'-ethylenediamine disuccinic acid (EDDS)

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