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 C(18) 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.