Determination of recombinant human erythropoietin by square wave voltammetry

Abstract

The different variables that affect the determination of recombinant human erythropoietin (r-HuEPO) by square wave voltammetry are studied. The method is based on oxidation of mercury from the electrode surface and following the reduction wave of the mercury bound to the thiol groups from four cysteine amino acids contained in the peptide backbone. The electrochemical behaviour of this system is evaluated by cyclic voltammetric studies. A 0.01 M phosphate buffer of pH 7.0 is used as supporting electrolyte and an accumulation potential of 170 mV (vs. AgCl Ag ) is applied during 300 s with continuous stirring at 700 rpm. The potential scan, without previous equilibration time, is realized with a square wave frequency of 120 Hz, a scan increment ΔE of 4 mV and a square wave amplitude a of 50 mV. Under these conditions, the variation of i p with the r-HuEPO concentration is linear in the range from 0.040 to 0.400 μg l −1. A limit of detection (3σ) of 0.017 μg l −1 and a limit of determination (10σ) of 0.059 σg l −1 is obtained. The relative error of the proposed method is equal or less than 7.1%, and the relative standard deviation is equal or less than 5.3%. The proposed method is available to the determination of erythropoietin in urine.