Determination of aristolochic acid I and its metabolites in cell culture with a hyphenated high-performance liquid chromatographic technique for cell toxicology

Aristolochic acid I (AA I), a major component of the carcinogenic plant extract aristolochic acid (AA), is known to be nephrotoxic, carcinogenic and mutagenic. A simple, rapid and sensitive high-performance liquid chromatography–diode array detection–fluorescence detection (HPLC–DAD–FLD) method was developed and validated for the analysis of AA I and its metabolites in cell culture medium for the first time. The samples were prepared with ethyl acetate liquid–liquid extraction (LLE). Good separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient solution. Linearities of about three orders of magnitude were gained with correlation coefficients exceeding 0.9990. The method appears to be a suitable tool for the cellular toxicokinetic study with acceptable precisions and recoveries. Cytotoxicity of AA I on human liver cells (L-02) was investigated with morphological observation and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay, cytotoxicity increased in AA I concentration-dependent manner. AA I and its metabolites were monitored with the proposed chromatographic analysis, and some preliminary toxicokinetics were investigated.