Rapid determination of xanthine metabolites by high-performance liquid chromatography with electrochemical and UV detection

Purine bases and their derivatives play an important role in the functioning of living systems. Purines are involved in many metabolic processes as cofactors which play key roles in fundamental biological processes. In particular, uric acid (UA), xanthine (XA) and hypoxanthine (HX) are degradation products of purine metabolism in human beings and higher primates. The determination of xanthine can be interesting not only in biological fluids, since involved in various diseases, but also in food industry for quality control of the products. Several analytical methods such as colorimetry, fluorometry, HPLC, mass spectrometry, anion exchange chromatography, thin layer chromatography and capillary column gas chromatography have been employed for measurement of purines [1, 2]. In the present study a simple and sensitive method using high-performance liquid chromatography with electrochemical detection (HPLC-ED) at several electrochemical sensors and with ultraviolet detection (HPLC-UV) has been developed for the determination of purines in fish meat for the evaluation of freshness. The experimental parameters were systematically optimized for each analyte of UA, XA and HX. For an efficient separation and detection of these analytes, a Hypersil ODS column, a mobile phase consisting of 0.004 M potassium phosphate buffer pH 5.8 with 1% (v/v) of methanol in isocratic mode were used. This system couples the suitable separation power of HPLC to an inherent sensitivity of electrochemical detection in a short run time, and is capable of measuring purine metabolites with minimal sample preparation and low injection volume. The estimation of the fish freshness by monitoring the purines content was also established with a low concentration level and very good reproducibility. [1] R. Devi, S. Yadav, C.S. Pundir, Analyst 137 (2012) 754-759. [2] T.C. Burdett, C.A. Desjardins, R.Logan, N.R. McFarland, X. Chen, M.A. Schwarzschild, Biomedical Chromatography 27 (2013) 122-129.