Determination of reduced glutathione by spectrophotometry coupled with anti-interference compensation

Abstract

The spectrophotometric method is usually used in enzymatic synthesis of glutathione (GSH) for tracking detection of the reaction process. However, –SH from cysteine (Cys) interferes significantly with the analysis of GSH results. By multiple interference experiments, data processing and principle analysis, an anti-interference compensation mechanism has been found for the elimination of interference caused by the reaction of alloxan and Cys. An anti-interference compensation mechanism was set by making standard curves of absorbance, introducing into these the interference coefficient and compensation coefficient, and building an interference model and a compensation algorithm. The absorbance and derivative of the samples were detected at 320 nm, then the test data was imported into the anti-interference compensation flow chart: the interference ratio and compensation coefficient was obtained, and the content of GSH and Cys could be calculated, respectively. Detection results with the new method showed that when the concentration of Cys was no more than 4 times that of GSH in the reaction liquid, the recoveries of GSH and Cys were controlled to be 95–100% and 78–120% respectively. The disturbance of the detection results by Cys could be eliminated. The standard equation of GSH concentration–absorbance was y = 3.0629x + 0.0001, R² = 0.9999. Compared with the usual alloxan method, the new method shows a significant increase in anti-interference characteristics and accuracy for detection of the GSH content and semi-quantitative analysis of Cys. It has practical application value for the quantitative detection of GSH, even without the separation of cysteine in the enzymatic synthesis of GSH.