Development of quantitative enzymatic method and its validation for the assay of glucose in human serum

Abstract

ObjectiveTo develop a simple, rapid, sensitive and affordable assay method for the determination of glucose in blood samples using a novel approach. Design and methodsA spectrophotometric method for glucose quantification in human serum samples based on self-coupling of activated 2,5-dimethoxyaniline (DMA) in the presence of peroxidase (POD)/glucose oxidase (GOD) and H2O2 is described. H2O2 generated in situ by catalytic reaction between GOD and glucose, activates DMA in the presence of POD to form a green-colored product, which has a strong absorption at λmax=740nm at room temperature (30°C) in a 100mmol/L acetate/acetic acid buffer of pH 4.2. ResultsThe linearity ranges for the quantification of glucose by rate and one-time detection method are 0.017–0.740 and 0.017–0.478mmol/L, respectively. Within-day and day-to-day precision were 0.98–1.4% (n=10) and 1.33–2.89% (n=15), respectively. Glucose recoveries ranged from 96.6 to 102%, indicating minimal interference by commonly present interferants in serum samples. Accuracy results were between 90 and 102%. The detection and quantification limits of glucose were 2.376 and 7.923μmol/L, respectively. The proposed method has good correlation coefficient of 0.999 with the enzymatic kit method. ConclusionsThis is a rapid and convenient method to determine serum glucose using simple spectrophotometer with excellent recovery and minimal interference by interferants in serum samples with low detection limit. Therefore, this method can be considered for adoption by the clinical diagnostic laboratories.