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

Aristolochic acid nephropathy (AAN) is associated with the prolonged exposure to nephrotoxic and carcinogenic aristolochic acids (AAs). DNA adducts induced by AAs have been proven to be critical biomarkers for AAN. Therefore, accurate and specific quantification of AA-DNA adducts is important. In this study, a specific method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and applied for the determination of 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) in exfoliated urothelial cells of AA-dosed rats. After the isolation from urine samples, DNA in urothelial cells were subjected to enzymatic digestion and solid-phase extraction on a C18 Sep-Pak cartridge for the enrichment of DNA adducts. The sample extracts were analyzed by reverse-phase UPLC-MS/MS with electrospray ionization in positive ion mode. The quantification of the AA-DNA adduct was performed by using multiple reaction monitoring with reserpine as internal standard. The method provided good accuracy and precision with a detection limit of 1 ng/ml, which allowed the detection of trace of dA-AAI in exfoliated urothelial cells. After one-month oral dose of AAI at 10 mg/kg/day, 2.1 ± 0.3 dA-AAI per 109 normal dA was detected in exfoliated urothelial cells of rats. Compared to the traditional methods such as 32P-postlabelling and HPLC with fluorescence detection, the developed UPLC-MS/MS method is more specific and rapid with a retention time of 4 min. The outcome of this study may have clinical significance for diagnosing and monitoring AA-associated disease because detection of DNA adducts in exfoliated urothelial cells is non-invasive and convenient.