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

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-C-18 column (250 mmx4.6 mm, 5 mu m) was used in the analysis. The mobile phase was 25% (v/v) methanol with 1.0 g/L Cu (CO2CH3)(2)center dot 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 degrees C 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 cata. lyzed 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.