Simultaneous quantifications of four purine derivatives biomarkers in cow milk by SPE HPLC-DAD

Abstract

In this study, simultaneous quantification of allantoin, uric acid, xanthine, and hypoxanthine in cow milk by solid phase extraction (SPE) and high performance liquid chromatography-diode array detection (HPLC-DAD) method was perform. Five different SPE cartridges were tested in order to evaluate the isolation of purine derivatives (PD) from cow milk. Chromatography was carried out on ODS-2 Hypersil column and 0.05 M (NH₄)₂HPO₄ buffer solution (pH = 7.76) as mobile phase. The HPLC-DAD validated method showed a linearity with regression coefficients higher than 0.999 and the limits of detection and quantification with values in the range 0.09–0.74 µg mL^–1 and 0.27–2.24 µg mL^–1, respectively. The method showed good precision with a relative standard deviation (RSD) below 4.48%, while the accuracy ranged from 95.34 to 104.47% for all analytes. The best recovery degree of PD by SPE were obtained on Strata SCX cartridge for xanthine (87.79%) and hypoxanthine (89.02%); on Strata NH₂ for allantoin (35.09%) and on Strata C8 for uric acid (101.08%). Finally, the HPLC-DAD method with SPE on SCX cartridges was applied to quantify the PD in a batch of thirty cow milk samples.