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Facile spectrophotometric assay of molar equivalents of N-hydroxysuccinimide esters of monomethoxyl poly-(ethylene glycol) derivatives

Ang Gao, Xiaolan Yang, Chun Zhang, Gaobo Long, Jun Pu, Yonghua Yuan, Hongbo Liu, Yuanli Li and Fei Liao*

Author Affiliations

Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry of China, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China

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Chemistry Central Journal 2012, 6:142  doi:10.1186/1752-153X-6-142

Published: 23 November 2012



A new method is developed to quantify molar equivalents of N-hydroxysuccinimide (NHS) esters of derivatives of monomethoxyl poly-(ethylene glycol) (mPEG) in their preparations with NHS acetate ester as the reference.


NHS ester of succinic monoester or carbonate of mPEG of 5,000 Da was synthesized and reacted with excessive ethanolamine in dimethylformamide at 25°C for 15 min. Residual ethanolamine was subsequently quantified by absorbance at 420 nm after reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS) at pH 9.2 for 15 min at 55°C followed by cooling with tap water. Reaction products of ethanolamine and NHS esters of mPEG caused no interference with TNBS assay of residual ethanolamine. Reaction between ethanolamine and NHS acetate ester follows 1:1 stoichiometry. By the new method, molar equivalents of NHS esters of carbonate and succinic monoester of mPEG in their preparations were about 90% and 60% of their theoretical values, respectively. During storage at 37°C in humid air, the new method detected spontaneous hydrolyses of the two NHS esters of mPEG more sensitively than the classical spectrophotometric method based on absorbance at 260 nm of NHS released by reaction with ammonia in aqueous solution.


The new method is favorable to quantify molar equivalents of NHS esters of mPEG derivatives and thus control quality of their preparations.

N-hydroxysuccinimide esters; Monomethyl ether of poly-(ethylene glycol); Ethanolamine; 2,4,6-trinitrobenzenesulfonic acid; Molar equivalent

Graphical abstract