Simultaneous determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA using online column-switching liquid chromatography/tandem mass spectrometry.
Sensitive and reliable methods are required for the assessment of oxidative DNA damage, which can result from reactive oxygen species that are generated endogenously from cellular metabolism and inflammatory responses, or by exposure to exogenous agents. The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) method is described, that utilises online column-switching valve technology for the simultaneous determination of two DNA adduct biomarkers of oxidative stress, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA). To allow for the accurate quantitation of both adducts the corresponding [(15)N(5)]-labelled stable isotope internal standards were synthesised and added prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides. The method required between 10 and 40 microg of hydrolysed DNA on-column for the analysis and the limit of detection for both 8-oxodG and 8-oxodA was 5 fmol. The analysis of calf thymus DNA treated in vitro with methylene blue (ranging from 5 to 200 microM) plus light showed a dose-dependent increase in the levels of both 8-oxodG and 8-oxodA. The level of 8-oxodG was on average 29.4-fold higher than that of 8-oxodA and an excellent linear correlation (r = 0.999) was observed between the two adducts. The influence of different DNA extraction procedures for 8-oxodG and 8-oxodA levels was assessed in DNA extracted from rat livers following dosing with carbon tetrachloride. The levels of 8-oxodG and 8-oxodA were on average 2.9 (p = 0.018) and 1.4 (p = 0.018) times higher, respectively, in DNA samples extracted using an anion-exchange column procedure than in samples extracted using a chaotropic procedure, implying artefactual generation of the two adducts. In conclusion, the online column-switching LC/MS/MS SRM method provides the advantages of increased sample throughput with reduced matrix effects and concomitant ionisation suppression, making the method ideally suited when used in conjunction with chaotropic DNA extraction for the determination of oxidative DNA damage.
Singh
Rajinder
R
Biocentre, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK. rs25@le.ac.uk
Teichert
Friederike
F
Verschoyle
Richard D
RD
Kaur
Balvinder
B
Vives
Montserrat
M
Sharma
Ricky A
RA
Steward
William P
WP
Gescher
Andreas J
AJ
Farmer
Peter B
PB
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002138:Calibration; D002251:Carbon Tetrachloride / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D003839:Deoxyadenosines / Q000032:analysis*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D008099:Liver / Q000737:chemistry; D008297:Male; D008751:Methylene Blue / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D013950:Thymus Gland / Q000737:chemistry
D003839:Deoxyadenosines; C078251:2'-deoxy-7,8-dihydro-8-oxoadenosine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D002251:Carbon Tetrachloride; D003849:Deoxyguanosine; D008751:Methylene Blue