A HPLC tool for process monitoring: rare sugar D- psicose and D- fructose contents during the production through an enzymatic path

Abstract

D-Psicose/allulose, a rare sugar, is an essential raw material in the pharmaceutical and food industries. It is scantly found in nature and to meet its demand in industries, D-Psicose is generated enzymatically using D-fructose as a substrate. In these conversions, it is important to monitor D-Psicose, in order to control the process impurities to optimize the reaction time and reduce the process cost. The available analytical methods have their limitations in quantifying D-psicose and D-fructose mixtures. Hence there is a need for the development of a routine, sensitive, quick and precise analytical method for D-psicose production on-line monitoring of reaction mixer. In the present work, a simplified reverse phase HPLC technique is developed and validated for the quick reaction monitoring of D-psicose from D-fructose, during enzymatic conversions procedures. The analysis is conducted at different concentrations ranging from 0.05 % to 0.5 % of the standard solutions of the D-psicose and D-fructose, by using water and Acetonitrile (at a ratio of 20:80) as eluent with a flow rate of 1.0 mL/min on isocratic HPLC-RID system with an aminopropyl silane stationary phase [ZORBAX SIL 4.6 x 150 mm, 5 µm particle size column (USP-L8)]. The applicability of this method is illustrated in reaction monitoring, where D-fructose (substrate) is converted to D-psicose (product) in the presence of the enzyme: D-Tagotose 3- epimerase. Separation of D-psicose and D-fructose is achieved within 8 minutes with a resolution ≥ 4 which is the key advantage for reaction monitoring and linearity is established with regression of ≥ 0.99. Additionally, the current method uses a simple mobile phase, without any buffers. It can be used routinely for reaction monitoring.