Biosensor for the characterisation of hMAO B inhibitors and the quantification of selegiline

A novel human monoamine oxidase B (hMAO B) based biosensor for inhibitory measurements was developed. It allows both the characterisation of the type of enzyme inhibition and the sensitive and simple determination of inhibitors like selegiline hydrochloride. The sensor consists of a screen printed carbon working electrode modified with 20% manganese dioxide (MnO2) and the enzyme hMAO B, which was immobilised on the electrode via a dialysis membrane (regenerated cellulose, molecular weight cut-off 14000). Inhibition of hMAO B is evaluated by adding different concentrations of the inhibitor selegiline hydrochloride to the enzyme and applying a defined amount of the hMAO B substrate phenylethylamine (PEA). The enzymatically formed H2O2 is amperometrically detected at 0.4 V vs. Ag/AgCl in a flow injection analysis (FIA) system. With 100 µM PEA the sensor showed a linear correlation between peak height and inhibitor concentration in a range of 0.51–3.25 µg/mL selegiline hydrochloride. LOD and LOQ were determined to be 0.15 and 0.51 µg/mL, respectively. The sensor showed a repeatability of 3.7% and an intermediate precision of 8.1%. The inhibition-based biosensor was successfully employed to quantify selegiline hydrochloride in pharmaceutical samples. Kinetic studies via Lineweaver-Burk plot and enzyme quantity vs. current plot revealed that the inhibition is irreversible.