Sensitive voltammetric detection of allopurinol–based drug Milurit in clinical urine samples

Here we summarize three electrochemical approaches to improve the sensitivity and specificity of the voltammetric sensing of purine metabolites related to the xanthine oxidase (XO) pathway (hypoxanthine-Hyp, xanthine-Xan, and uric acid-UA), as well as of their analogues used as therapeutics in the inhibition of XO enzymatic activity (allopurinol-Alo and its enzymatic product oxypurinol-Oxy) on various carbon-based materials. Detection of purine metabolites, in all proposed protocols, is based on direct electrochemical measurement of oxidation peaks for each of the monitored substances in a single detection step by the same electrode system. A nanomolar detection of these purine metabolites is possible due to: (i) mechanical roughening of the surfaces of glassy carbon or edge plane-oriented pyrolytic graphite electrodes by 15-μm silicon carbide particles (with detection limits around 20 nM), (ii) electrochemical activation of polished carbon-based materials by cycling the potential in 0.1 M KNO3 between -0.1 and +1.85 V, and (iii) anodic stripping of the electrochemically accumulated purine-Cu(I) complexes from commonly used carbon/graphite materials in the presence of copper ions. Contrary to the mechanical roughening, both electrochemical anodization of carbon-based surfaces and anodic stripping of accumulated purine- Cu(I) complexes are less sensitive to detect Alo in the presence of Hyp due to partial overlapping of their oxidation peaks. However, all of the proposed protocols are operational to fast, sensitive, and inexpensive determination of UA, Xan as well as Oxy in a 10-μl volume of 1000 times diluted urine samples from patients treated with the Alo-based drug Milurit.