Simultaneous homogeneous immunoassay of phenytoin and phenobarbital using a Nafion-loaded carbon paste electrode and two redox cationic labels

The dual-analyte homogeneous immunoassay of two antiepileptic drugs was carried out simultaneously at physiological pH by square-wave voltammetry at a Nafion-loaded carbon paste electrode. Phenobarbital (PB) and phenytoin (DPH) were labeled by a cobaltocenium salt (Cc+) and a ferroceneammonium salt (N+Fc), respectively, and the corresponding standard redox potentials were −1.05 V and 0.26 V. Detection limits of 0.25 and 0.2 μM were achieved for PB-Cc+ and DPH-N+Fc (S/N = 3) after a 5-minute accumulation step, with linear responses over the 0.25–5 and 0.2–5 μM ranges, respectively. The relative standard deviation was evaluated to be ≥ 11% for 1 μM of each labeled drug. The separate, as well as simultaneous calibration plots of PB and DPH were obtained at constant serum + antiserum contents (10%). It was possible to detect PB or DPH at concentrations as low as 0.5 μM, but the moderate reproducibility of the data allows only semi-quantitative assays (positive/negative test) for clinical serum samples to be envisaged.