高效液相色谱法测定生物转化反应液中<i>N,N</i>'-乙二胺二琥珀酸

引用本文: 吴智超, 吴恩国, 杨仲毅, 陶宇翔, 陈潭, 钟永军. 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[J]. 色谱, 2019, 37(10): 1118-1123. doi: 10.3724/SP.J.1123.2019.03033 shu

Citation: WU Zhichao, WU Enguo, YANG Zhongyi, TAO Yuxiang, CHEN Tan, ZHONG Yongjun. Determination of N,N'-ethylenediamine disuccinic acid in bioconversion reaction solution using high performance liquid chromatography[J]. Chinese Journal of Chromatography, 2019, 37(10): 1118-1123. doi: 10.3724/SP.J.1123.2019.03033 shu

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.
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
基金项目:
台州市科技计划项目（162gy47，1701ny06）；台州市教科规划项目（gg19007）.

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:

English

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

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 Date: 26 March 2019
Fund Project:
Taizhou Science and Technology Program Project (Nos. 162gy47, 1701ny06)

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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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

English

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

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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

English

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

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