Analytical Sciences

A Simple HPLC Method for Determining the Purine Content of Beer and Beer-like Alcoholic Beverages

Tomoko FUKUUCHI, Makoto YASUDA, Katsunori INAZAWA, Tatsuhiro OTA, Noriko YAMAOKA, Ken-ichi MAWATARI, Kazuya NAKAGOMI, and Kiyoko KANEKO

Laboratory of Biomedical and Analytical Sciences, Faculty of Pharma Sciences, Teikyo University, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan

Several methods for quantifying the purine content in food and drink have been described using high-performance liquid chromatography (HPLC). We have developed an improved HPLC method that is based on a method reported by Kaneko et al. and that is more sensitive yet simple, and suitable for determining the purine content of beer and beer-like alcoholic beverages. Quantitative HPLC separation was performed on a Shodex Asahi Pak GS-320HQ column with an isocratic elution of 150 mmol/L sodium phosphate buffer (H₃PO₄/NaH₂PO₄ = 20:100 (v/v)). The retention times for the four analytes, namely, adenine, guanine, hypoxanthine and xanthine, were 19.9, 25.0, 29.3 and 43.0 min, respectively. The resolution was good, and there was no excessive interference from the other compounds in the beverages at these retention times. Furthermore, the detection limit for all the analytes was improved to less than 0.0075 mg/L, and all the calibration curves showed good linearity (r² > 0.999) between 0.013 and 10 mg/L for adenine and guanine, and between 0.025 and 10 mg/L for hypoxanthine and xanthine. The pretreatment was simplified by removing some procedures and optimizing the perchloric acid hydrolysis and the enzymatic peak-shift assay. We reduced the sample dilution rate by almost 50%, and the time spent on pretreatment from 4 days to only 180 min. The recovery of the analytes from spiked samples was 94.8 – 103.8%. This method may be useful for evaluating quantitative and qualitative differences in the purine content of beer and beer-like alcoholic beverages.

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