Method development for targeted analysis of modified nucleosides and deoxynucleosides in urine samples

Małgorzata Patejko, Department Of Biopharmaceutics And Pharmacodynamics, Medical University of Gdańsk, Gdańsk, Poland (malgorzata.patejko@gumed.edu.pl)
Aleksandra Kwiecień, Department Of Biopharmaceutics And Pharmacodynamics, Medical University of Gdańsk, Gdańsk, Poland
Wiktoria Struck-Lewicka, Department Of Biopharmaceutics And Pharmacodynamics, Medical University of Gdańsk, Gdańsk, Poland
Danuta Siluk, Department Of Biopharmaceutics And Pharmacodynamics, Medical University of Gdańsk, Gdańsk, Poland
Marcin Markuszewski, Department of Urology, Medical University of Gdańsk, Poland
Marcin Matuszewski, Department of Urology, Medical University of Gdańsk, Poland
Michał J. Markuszewski, Department Of Biopharmaceutics And Pharmacodynamics, Medical University of Gdańsk, Gdańsk, Poland

Modified nucleosides and deoxynucleosides are endogenous metabolites, products of RNA and DNA turnover. They are not metabolized and cannot be utilized for synthesis of new RNA and DNA molecules. They are excreted in unchanged form. Extended DNA and RNA turnover is observed in various diseases, including cancer. Consequently, a correlation between elevated level of modified nucleosides and deoxynucleosides in urine and pathophysiological disorders development can be expected. Increased level of this group of compounds was observed in such diseases as: hepatocellular carcinoma, breast cancer or urogenital cancer.
The aim of the study is the targeted metabolomics analysis of 11 modified nucleosides and deoxynucleosides in urine samples collected from bladder cancer patients with the use of LC-QqQ/MS technique. Since proper sample treatment influences obtained results significantly, the first task of the research covered the development of sample preparation procedure. This included optimization of separation conditions and solid-phase extraction procedure (SPE). Different chromatographic conditions were compared, including: type of stationary phase, flow rate, column temperature and gradient programme. According to SPE, differences in the sugar moiety between nucleosides and deoxynucleosides cause differences in their extraction ability and consequently difficulties with sorbent selection that allows for the extraction of nucleosides and deoxynucleosides at once. Previously, nucleosides and deoxynucleosides were more often analyzed separately. The goal was to develop method for simultaneous extraction of nucleosides and deoxynucleosides from urine matrix. Different sorbents were evaluated by their selectivity, recovery and ability to extract modified nucleosides and deoxynucleosides. Method based on selected sorbent was further optimized.

Acknowledgement
The work has been supported by the National Centre of Science, project no 2018/29/N/NZ7/02299.

References

1. M. Patejko, W. Struck-Lewicka, D. Siluk, M.  Waszczuk-Jankowska, M. J. Markuszewski, Urinary nucleosides and deoxynucleosides,  Adv. Clin. Chem. 2018; vol. 83, s. 1-51

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Abstract Reference & Short Personal Biography of Presenting Author

Małgorzata Patejko is a PhD student at the Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Poland. She is doing her PhD under the supervision of Dr Danuta Siluk. Her scientific experience includes the untargeted and targeted metabolomics analysis. During her PhD thesis she mainly focuses on the quantitative analysis of modified nucleosides and deoxynucleosides in urine and plasma matrices in order to evalute the potential role of this group of compounds as a biomarker of bladder cancer development.