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Simultaneous Determination of Uric Acid, Xanthine and Hypoxanthine in Human Plasma and Serum by HPLC–UV: Uric Acid Metabolism Tracking

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Abstract

A simple reversed phase HPLC method with UV detection in isocratic conditions was developed and validated for the simultaneous determination of hypoxanthine, xanthine and uric acid levels in human plasma and serum. One analysis run takes 6.5 min including a short organic mobile phase gradient for column regeneration. Concentrations of uric acid, xanthine and hypoxanthine in plasma and serum samples were highly comparable. However, hypoxanthine levels were increased in serum compared to plasma samples due to a prolonged time between serum and blood elements separation. The method was validated for linearity, precision, accuracy, sensitivity and robustness in a similar manner to that for pharmacokinetic data and it is appropriate for physiological and pathophysiological levels of all analytes. The stability of stock standard solutions was verified using spectrophotometric analysis in different conditions. The method is simple and robust with a good precision for the measurement of hypoxanthine, xanthine and uric acid in human plasma and serum.

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Acknowledgements

This work was supported by Ministry of Health of the Czech Republic, grant number 16-28040A and by a student project grant of Masaryk University MUNI/A/1056/2015. All rights reserved. The authors would like to thank to Martina Hanouskova for an excellent technical assistance, to Milan Dastych for providing human serum based control for determination of uric acid and to Jan Jurica for providing standards of caffeine and its metabolites.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

    Anna Pleskacova, Stanislav Brejcha & Josef Tomandl

  2. Department of Pathophysiology, Faculty of Medicine Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

    Anna Pleskacova, Lukas Pacal, Katerina Kankova & Josef Tomandl

Authors
  1. Anna Pleskacova
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  2. Stanislav Brejcha
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  3. Lukas Pacal
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  4. Katerina Kankova
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  5. Josef Tomandl
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Corresponding author

Correspondence to Josef Tomandl.

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The authors declare that they have no conflict of interests.

Ethical approval

This study was performed in compliance with the Ethical Committee of Faculty of Medicine, Masaryk University and was conducted in accordance with Helsinki declaration.

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An informed consent was obtained from all study participants.

Additional information

Published in the topical collection Advances in Chromatography and Electrophoresis & Chiranal 2016 with guest editor Jan Petr.

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Pleskacova, A., Brejcha, S., Pacal, L. et al. Simultaneous Determination of Uric Acid, Xanthine and Hypoxanthine in Human Plasma and Serum by HPLC–UV: Uric Acid Metabolism Tracking. Chromatographia 80, 529–536 (2017). https://doi.org/10.1007/s10337-016-3208-8

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  • DOI: https://doi.org/10.1007/s10337-016-3208-8

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