A new approach for the sensitive determination of DNA adduct of aristolochic acid II by using high-performance liquid chromatography with fluorescence detection

A novel and specific method for the determination of aristolochic acid-derived DNA adducts in exfoliated urothelial cells by using ultra performance liquid chromatography–triple quadrupole mass spectrometry

“…2. As described previously [19,21], 0.7 ml of 5 mM sodium chloride in 0.01 M Tris buffer (pH 7.5), 30 l of 1 mg/ml DNase I, 70 l of 0.01 M magnesium chloride in 0.01 M Tris buffer (pH 7.0) were added to the washed pellet that was lysed by three freeze/thaw cycles, and incubated at 37 • C for 1 h. After that, 0.8 ml of 0.2 M Tris buffer (pH 9.0) and 30 l of 1 unit/ml phosphodiesterase I were added and incubated for additional 48 h, followed by the addition of 22 l of 3.5 unit/ml alkaline phosphatase and another 24-h incubation at 37 • C.…”

“…A method suitable for the analysis of AA-DNA adducts should simultaneously tolerate the constraint of limited sample availability and the need on lower detec- tion limits. A number of methods have been developed for the detection of AA-DNA adducts, including 32 P-postlabelling analysis [14][15][16][17], high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) [18][19][20] and fluorescence detection (HPLC-FLD) [21]. The 32 P-postlabelling assay is an ultrasensitive method and capable of detecting adducts at levels as low as 1 in 10 9 nucleotides.…”

“…However, it required the use of large excess of radioactive ␥-32 P labeled orthophosphate that limited its application only in special laboratories with the control of radioactive material and that is a strong ␤-emitter. While HPLC-FLD allowed a sensitive detection of dA-AAII with a low detection limit of 18.3 fmol on column by monitoring the fluorescence intensity of dA-AAII [21], all of AA-DNA adducts contained the same fluorophore aristolactam and thus exhibited the similar fluorescence excitation and emission spectra, which made the method less specific.…”