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Objective To establish a fast and sensitive method for the detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in precision-cut rat liver slices by HPLC-MS/MS and to investigate isoniazid (INH) -induced oxidative DNA damage. Methods Precision-cut liver slices (300 μm) were prepared from male rats, and incubated with INH (0.018 mol/L) for 2 h after 1 h preincubation. DNA in the slices was extracted and digested into free nucleosides at 37℃ . The samples were injected into HPLC-MS/MS after the proteins were removed. The level of oxidative DNA damage was estimated using the ratio of 8-OHdG to deoxyguanosine (dG). Results The limit of detection of 8-OHdG was 1 ng/mL (S/N=3) and the intra-assay relative standard variation was 3.38% when one transition 284.3/168.4 was used as a quantifier and another two transitions 284.3/140.2, 306.1/190.2 as qualifiers. 8-OHdG and dG were well separated, as indicated by elution at 10.02 and 7.37 min, respectively. INH significantly increased the ratio of 8-OHdG to dG in rat liver slices (P<0.05). Conclusion 8-OHdG in precision-cut liver slices could be sensitively determined by HPLC-MS/MS. HPLC-MS/MS coupled with precision-cut tissue slices is a fast and reliable analytical technique to evaluate oxidative DNA damage of target tissues caused by procarcinogens and cytotoxins.
Objective To establish a fast and sensitive method for the detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in precision-cut rat liver slices by HPLC-MS / MS and to investigate isoniazid (INH) -induced oxidative DNA damage (300 μm) were prepared from male rats, and incubated with INH (0.018 mol / L) for 2 h after 1 h preincubation. DNA in the slices were extracted and digested into free nucleosides at 37 ° C. The levels of oxidative DNA damage was estimated using the ratio of 8-OHdG to deoxyguanosine (dG). Results The limit of detection of 8-OHdG was 1 ng / mL (S / N = 3) and the intra-assay relative standard variation was 3.38% when one transition 284.3 / 168.4 was used as a quantifier and another two transitions 284.3 / 140.2, 306.1 / 190.2 as qualifiers. 8-OHdG and dG were well separated, as indicated by elution at 10.02 and 7.37 min, respectively. INH significantly increased the ratio o Conclusions 8-OHdG in precision-cut liver slices could be sensitively determined by HPLC-MS / MS. HPLC-MS / MS coupled with precision-cut tissue slices is a fast and reliable analytical technique to evaluate oxidative DNA damage of target flora caused by procarcinogens and cytotoxins.