UPLC-MS/MS analysis of aristolochic acid I and aristolactam I in multiple matrices for exposure assessment

Abstract

Aristolochic acid I (AA-I) and its metabolite aristolactam I (AL-I) are nephrotoxic and carcinogenic compounds derived from Aristolochia species, which have been widely used in traditional herbal medicines. While previous studies have focused on their occurrence in herbs and human urine, their presence in environmental waters and associated population-level exposure remain largely unexplored. In this study, a sensitive and robust UPLC-MS/MS method was developed and validated for the simultaneous determination of AA-I and AL-I across five complex matrices: Chinese herbal decoctions, human urine, river water, and influent and effluent from a municipal wastewater treatment plant (WWTP). Solid-phase extraction (SPE) was optimized with a three-step rinsing protocol and NH₂ cartridge purification, while matrix-matched internal standard calibration was applied to minimize matrix effects. The method exhibited excellent linearity (R² > 0.99) over the concentration range of 10–100 ng mL⁻¹, recoveries (81.3–109.6%), and low limits of detection (0.2–2.5 ng mL⁻¹). Application of the method revealed an extremely high concentration of AA-I (112.6 μg mL⁻¹) in a commercially available herbal decoction, and elevated urinary AL-I levels (181.4–192.0 μg mL⁻¹) after ingestion, suggesting significant human exposure. AL-I was also consistently detected in influent wastewater (23.4–133.2 ng L⁻¹; 100% detection frequency), indicating widespread community-level intake. While AA-I and AL-I were largely absent in river water and WWTP effluent, potential environmental contamination during rainfall events warrants attention. These findings highlight the urgent need for stricter surveillance of AA-containing products and demonstrate the feasibility of wastewater-based epidemiology for assessing population exposure to toxic herbal constituents.