A fully validated bioanalytical method using an UHPLC–MS/MS system for quantification of DNA and RNA oxidative stress biomarkers
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A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5–8.9%, an inter-day precision of 3.4–8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer.
Keywords8-Hydroxy-2-deoxyguanosine 8-Hydroxyguanosine Creatinine Cancer UHPLC–MS/MS Human urine
This work was supported by projects SVV 260 412, MH CZ - DRO (UHHK, 00179906) and Progress Q42.
Compliance with ethical standards
This study has been approved by the University Hospital Hradec Kralove ethics committee 201606 S21P and has been performed in accordance with the ethical standards.
Conflict of interest
The authors declare that they have no conflict of interest.
- 1.Halliwell B. Why and how should we measure oxidative DNA damage in nutritional studies? How far have we come? Am J Clin Nutr. 2000;72:1082–7.Google Scholar
- 3.Nunomura A, Perry G, Pappolla MA, Wade R, Hirai K, Chiba S, et al. RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer’s disease. J Neurosci. 1999;19:1959–64.Google Scholar
- 9.Honda M, Yamada Y, Tomonaga M, Ichinose H, Kamihira S. Correlation of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, and clinical features of hematological disorders: a pilot study. Leuk Res. 2000;24:461–8. doi: 10.1016/S0145-2126(00)00006-0.CrossRefGoogle Scholar
- 16.Malayappan B, Garrett TJ, Segal M, Leeuwenburgh C. Urinary analysis of 8-oxoguanine, 8-oxoguanosine, fapy-guanine and 8-oxo-2′-deoxyguanosine by high-performance liquid chromatography–electrospray tandem mass spectrometry as a measure of oxidative stress. J Chromatogr A. 2007;1167:54–62. doi: 10.1016/j.chroma.2007.08.024.CrossRefGoogle Scholar
- 18.Rodríguez-Gonzalo E, Herrero-Herrero L, García-Gómez D. Development, validation and application of a fast analytical methodology for the simultaneous determination of DNA- and RNA-derived urinary nucleosides by liquid chromatography coupled to tandem mass spectrometry. J Chromatogr B. 2016;1019:132–9. doi: 10.1016/j.jchromb.2015.10.044.CrossRefGoogle Scholar
- 19.Ren L, Fang J, Liu G, Zhang J, Zhu Z, Liu H, et al. Simultaneous determination of urinary parabens, bisphenol A, triclosan, and 8-hydroxy-2′-deoxyguanosine by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Anal Bioanal Chem. 2016;408:2621–9. doi: 10.1007/s00216-016-9372-8.CrossRefGoogle Scholar
- 20.Wang C-J, Yang N-H, Chang C-C, Liou S-H, Lee H-L. Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2′-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC–MS/MS. J Chromatogr B. 2011;879:3538–43. doi: 10.1016/j.jchromb.2011.09.038.CrossRefGoogle Scholar
- 22.Zhang S, Song X, Zhang W, Luo N, Cai L. Determination of low urinary 8-hydroxy-2′-deoxyguanosine excretion with capillary electrophoresis and molecularly imprinted monolith solid phase microextraction. Sci Total Environ. 2013;450–451:266–70. doi: 10.1016/j.scitotenv.2013.02.025.CrossRefGoogle Scholar
- 23.Kataoka H, Mizuno K, Oda E, Saito A. Determination of the oxidative stress biomarker urinary 8-hydroxy-2′-deoxyguanosine by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry. J Chromatogr B. 2016;1019:140–6. doi: 10.1016/j.jchromb.2015.08.028.CrossRefGoogle Scholar
- 24.Hu J, Zhang W, Ma H, Cai Y, Sheng G, Fu J. Simultaneous determination of 8-hydroxy-2′-deoxyguanosine and 5-methyl-2′-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. J Chromatogr B. 2010;878:2765–9. doi: 10.1016/j.jchromb.2010.08.017.CrossRefGoogle Scholar
- 26.European Medicines Agency. Guideline on bioanalytical method validation online available from 21 July 2011 http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. Accessed 18 March 2017.
- 27.Food and Drug Administration. Guidance for industry: bioanalytical method validation online available from September 2013 https://www.fda.gov/downloads/Drugs/Guidances/ucm368107.pdf. Accessed 18 March 2017.