Abstract:

Objective To confirm that response surface method (RSM) is a method for amylase (AMY) reference determination procedure with optimum conditions. Methods The single factor analysis of variance was used to study pH, chloride ion (Cl^-) and ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside(EPS) concentrations in order to know whether they had statistical significance for AMY activity. Box-Behnken design was used to study the combined influence of pH (6.9-7.1), Cl^- (50-90 mmol/L) and EPS (4.5-5.5 mmol/L) concentrations on AMY activity. The quantitative relation model on pH, Cl^- and EPS concentrations for AMY activity was established in order to obtain their optimum combination. Results There was statistical significance on the influence of the 3 factors on AMY activity (P<0.05). The one-shot and second-order effects of pH,Cl^- and EPS had statistical significance on AMY activity (P<0.01). Cl^- and EPS interacted significantly (P<0.05).The optimum conditions for AMY were pH 7.02,Cl^- 72.79 mmol/L and EPS 5.07 mmol/L. Conclusions The optimum conditions of pH, Cl^- and EPS have no difference with those recommended from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). RSM is a method with optimum conditions to confirm the reference determination procedure.

Key words: Response surface method, Amylase, Single factor analysis of variance, pH, Chloride ion, Ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside