Enzymatic oxidation of tert-butylcatechol in the presence of sulfhydryl compounds: Application to the amperometric detection of penicillamine.

The high sensitivity that can be attained using an enzymatic system and mediated by 4-tert-butylcatechol (4-TBC) has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP, [EC 1.11.1.7], immobilized on a rotating disk, in presence of hydrogen peroxide catalyzed the oxidation of 4-TBC, whose back electrochemical reduction was detected on glassy carbon electrode surface at -150mV. Thus, when penicillamine (PA) was added to the solution, these thiol-containing compounds participate in Michael type addition reactions with 4-TBC to form the corresponding thioquinone derivatives, decreasing the peak current obtained proportionally to the increase of its concentration. The highest response for PA was obtained around pH 7. This method could be used to determine PA concentration in the range 0.02-80muM (r=0.998). The determination of PA was possible with a limit of detection of 7nM, in the processing of as many as 50 samples per hour. The HRP-rotating biosensor was successfully applied to the determination of PA in pharmaceutical formulations.