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

A sensitive high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the determination of DNA adducts induced by nephrotoxic and carcinogenic aristolochic acid (AA) is presented. The DNA adduct of AAII (dA-AAII) was synthesized by in vitro incubation, purified by preparative HPLC, characterized using fluorescence spectroscopy and high-resolution mass spectrometry, and was used as the biomarker for AA exposure in rats. The developed HPLC-FLD method was validated and applied for the determination of dA-AAII in rat kidney tissues. The method provided a detection limit of 18.3 fmol, which allowed the detection of dA-AAII in the rat kidney tissue samples collected after a single oral dose of AA. dA-AAII was detected in the kidney DNA digestion extracts of the rats that were dosed with AA at 5 mg/kg and 30 mg/kg at concentrations of 6.2 ± 1.1 and 41.3 ± 8.0 dA-AAII per 109 normal dA, respectively.