Electrochemical monitoring of the oxidation of ethinylestradiol by the horseradish peroxidase enzyme in water?

SCALA L, MARTÍNEZ N, PEREIRA S, RABA J, MESSINA G

CORDOBA

Congreso; III Reunión Internacional de Ciencias Farmacéuticas -Universidad Nacional de Córdoba; 2014

Ethinylestradiol (EE2) is a synthetic estrogen which is used as a contraceptive therapy. The presence of this compound in the environment has been attributed to their incomplete removal in wastewater treatment plant (WWTP) processes. Natural (estrone ? E1; 17b-estradiol ? E2; and estriol ? E3) and synthetic steroid estrogen (17a-ethinylestradiol ? EE2) are the major contributors to the estrogenic activity associated with WWTP effluents. In this study, Horseradish peroxidase (HRP) catalyzes the oxidation of EE2 in presence of H2O2 and the products formed are polymerized through a non-enzymatic process which leads to the formation of high molecular weight polymers. These low-solubility polymers can be removed from wastewater by co-precipitation, sorption to solids, sedimentation or filtration. Thus, the purpose of this research was to optimize the HRP enzyme-catalyzed process for removal of EE2 from synthetic water (purified deionized water) and the monitoring of the enzymatic process by square-wave voltammetry (SWV). Electrochemical experiments were performed in stirred solutions using a BAS 100B/W electrochemical analyzer (Bioanalytical System,West Lafayette, IN). Cyclic and square-wave voltammograms were obtained using a three electrodes system: glassy carbon working electrode, an Ag|AgCl|3M NaCl reference electrode and a Pt wire counter electrode. The reaction mixture was prepared by diluting stock solution of a EE2 to a concentration of 0.5 µM, in 10 mL of 0.01 M citrate buffer pH 5.0 containing HRP 0.02 U mL-1. The reaction was initiated by adding 1mM of H2O2. Then, after stirring for 10 min, the sample was centrifugated and EE2 was quantifiedby SWV EE2 calibration equation was ΔI(µA)= 1.436 + 2.382 CEE2 with a correlation coefficient of 0.998, over the concentration range of 0.3?50 µM with a limit of detection of 0.11µM.