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Molecules 2018, 23(3), 605; https://doi.org/10.3390/molecules23030605

Sensitive Detection of 8-Nitroguanine in DNA by Chemical Derivatization Coupled with Online Solid-Phase Extraction LC-MS/MS

1
Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan
2
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan
3
School of Pharmacy, China Medical University, Taichung 404, Taiwan
4
Department of Optometry, Da-Yeh University, Changhua 515, Taiwan
5
Department of Occupational Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 9 February 2018 / Revised: 4 March 2018 / Accepted: 6 March 2018 / Published: 8 March 2018
(This article belongs to the Special Issue Solid Phase Extraction: State of the Art and Future Perspectives)
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Abstract

8-Nitroguanine (8-nitroG) is a major mutagenic nucleobase lesion generated by peroxynitrite during inflammation and has been used as a potential biomarker to evaluate inflammation-related carcinogenesis. Here, we present an online solid-phase extraction (SPE) LC-MS/MS method with 6-methoxy-2-naphthyl glyoxal hydrate (MTNG) derivatization for a sensitive and precise measurement of 8-nitroG in DNA. Derivatization optimization revealed that an excess of MTNG is required to achieve complete derivatization in DNA hydrolysates (MTNG: 8-nitroG molar ratio of 3740:1). The use of online SPE effectively avoided ion-source contamination from derivatization reagent by washing away all unreacted MTNG before column chromatography and the ionization process in mass spectrometry. With the use of isotope-labeled internal standard, the detection limit was as low as 0.015 nM. Inter- and intraday imprecision was <5.0%. This method was compared to a previous direct LC-MS/MS method without derivatization. The comparison showed an excellent fit and consistency, suggesting that the present method has satisfactory effectiveness and reliability for 8-nitroG analysis. This method was further applied to determine the 8-nitroG in human urine. 8-NitroG was not detectable using LC-MS/MS with derivatization, whereas a significant false-positive signal was detected without derivatization. It highlights the use of MTNG derivatization in 8-nitroG analysis for increasing the method specificity. View Full-Text
Keywords: online solid-phase extraction; LC-MS/MS; peroxynitrite; nitrated DNA lesion; derivatization; isotope-dilution online solid-phase extraction; LC-MS/MS; peroxynitrite; nitrated DNA lesion; derivatization; isotope-dilution
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Hu, C.-W.; Chang, Y.-J.; Chen, J.-L.; Hsu, Y.-W.; Chao, M.-R. Sensitive Detection of 8-Nitroguanine in DNA by Chemical Derivatization Coupled with Online Solid-Phase Extraction LC-MS/MS. Molecules 2018, 23, 605.

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