Enzymatic determination of hypoxanthine in fish samples as a freshness indicator using the CUPRAC colorimetric sensor

Fish consumption is essential for a healthy diet. However, all seafood including fish are susceptible to deterioration unless properly preserved. Controlling the freshness of fresh or packaged fish is a challenging issue for the food industry in terms of human health and shelf life determination. One of the main indicators showing the freshness of fish is undoubtedly the amount of hypoxanthine (Hx). As soon as the organism dies, Hx begins to be released with the cessation of ATP synthesis and shows a gradual increase over time. Therefore, Hx determination is an important indicator in the control of fish freshness. Based on this fact, a colorimetric method for the enzymatic determination of Hx using the CUPRAC (Cupric ion Reducing Antioxidant Capacity) sensor was developed. Uric acid (UA) and H₂O₂ are enzymatically produced by xanthine oxidase (XOD) from Hx, and both products respond to the CUPRAC reagent to produce the cuprous neocuproine (Cu(I)-Nc) chromophore chelate formed in situ on a Nafion anionic membrane on which the cationic Cu(II)-Nc complex was fixed. Hx was measured at different time intervals in the meat samples taken from sea bass (Dicentrarchus labrax), which was left to stand at room temperature for a time period between 0 and 24 h; the level of spoilage was determined from the coloration of the CUPRAC membrane sensor (via absorbance measurement at 450 nm). It was observed that there was a linear increase in the amount of Hx during the measurement period. The method was optimized for Hx determination, verified with interference analysis and standard additions to real samples, and validated against HPLC. The linear detection range of the developed method for Hx was 2.0-32.0 μM with an LOD of 0.79 μM, and early stages of fish degradation could be detected at several nanomoles of Hx per gram of fish meat. The proposed method was demonstrated to have distinct superiority over many recent colorimetric sensors of fish freshness in regard to its lower LOD for Hx, wider linear range, capability to cope with interferents (including biologically important antioxidants, such as cysteine, reduced glutathione, ascorbic acid, UA and α-tocopherol) and applicability to real samples.