Establishment and development of a CZE–UV method for rapid measurement of aprotinin potency

Abstract

A new method for the measurement of aprotinin potency by CZE-UV detector was established for the first time. The on-line mixing of substrate, trypsin and aprotinin using at-inlet technology was realized by the established method. Enzymatic reaction, separation, and detection of substrate and product can be performed simultaneously online. The aprotinin potency can be measured within 4 min. The response surface methodology was used to optimize the incubation conditions of trypsin and substrate, and the optimized conditions were obtained under 17.39 mM phosphate buffer at pH 7.6, 1.40 min of incubation time. The repeatability of proposed method was evaluated in three different systems of capillary zone electrophoresis: (i) only substrate; (ii) trypsin and substrate; (iii) aprotinin, trypsin and substrate, and the RSDs of migration times and peak areas of substrate were less than 2.7 and 3.1%, respectively. The RSDs of migration times and peak areas of product were less than 2.1 and 3.0%, respectively. A formula was also developed to calculate the aprotinin potency in this method. In a word, the established CZE-UV method was convenient, fast, and environmentally friendly for the measurement of aprotinin potency.

Overview of in‐capillary enzymatic reactions using capillary electrophoresis

“…The potency determined in this way, was similar to the result obtained by the assay method of the Chinese Pharmacopoeia (2015 version, Part II), which is performed by titration. This CZE‐UV method, thus, shows to be a fast, accurate, simple, and environmental friendly method that only consumes minute amounts of enzyme, substrate, and other reagents during the measurement of aprotinin potency [54].…”

“…(A) The injection sequence of different zones; (B) the process of mixing of aprotinin, trypsin, and substrate; (C) aprotinin, trypsin, substrate, and product are separated by an electric field. Reproduced with permission from [54].…”