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.
Highlights
Chronic inflammation has been linked to heart disease, obesity, diabetes and cancer [1,2]
We describe a chemical derivatization coupled with online solid-phase extraction
[ C2, −N]-8-nitroG-methoxy-2-naphthyl glyoxal hydrate (MTNG) of a calf thymus DNA hydrolysate that had been treated with 10 μM
Summary
Chronic inflammation has been linked to heart disease, obesity, diabetes and cancer [1,2]. Exuberant NO production by activated macrophages is believed to be an important tissue-damage mediator as NO can be further converted into several highly reactive species, such as nitrous anhydride, nitrogen dioxide, nitryl chloride and peroxynitrite [3]. Molecules 2018, 23, 605 rationalized in terms of addition of low reactive NO2 to the highly oxidizing guanine radical that. Molecules 2018, 23, x FOR PEER REVIEW results from the deprotonation of guanine radical cation initially generated by one-electron oxidation of rationalized guanine [6].in8-NitroG is chemically unstable andoxidizing can spontaneously depurinate, terms of formed additioninofDNA low reactive. NO2 to the highly guanine radical that yielding with the resultant of GC-to-TA mutationby[7]. Adenine resultsapurinic from the sites deprotonation of guaninepossibility radical cation initially generated one-electron oxidation canofbe preferentially incorporated 8-nitroG during. Several research groups have focused on the role of[7]
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