Determination of Aristolochic Acid Using Isocratic RP-HPLC Method

Abstract

Aristolochic acid (AA) is an abundant chemical substance commonly present in the genus, Aristolochia. In term of stereoisomerism, Aristolochic acid I (AA-I) is known to cause nephrotoxicity, genotoxicity, and carcinogenicity. Therefore, the determination of AA-I in the herbal products is imperative to avoid the unwanted adverse drug reaction. Hence, this study was undertaken to develop a low cost isocratic HPLC method to detect AA-I and AA-II in two selected products. RP-HPLC instrument (Cyberlab LC-100) equipped with photo-diode-array UV detector with Capcell Pak C₁₈ - column (5μm, 25 x 0.46 cm), and a mobile phase, methanol-water (75:25) added with 0.1% glacial acetic acid (pH 4.14) with a flow rate of 1.0 ml/min. at 254 nm was used. The total run time was 10 min. The results indicate that the isocratic RP-HPLC method developed in the present study is a reproducible and precise method with an optimum resolution for the separation of peaks. The presence of AA was determined in three herbal plants, A. debilis, A. fangchi, and A. manshuriensis and two different Chinese herbal products. The HPLC chromatogram of the standard was identified and the retention time of the standards AA-I and AA-II was found at 8.35 min and 7.0 min respectively, also the AA-I was identified in raw herbs, A. debilis, and A. fangchi at 8.4 and 8.5 min. respectively whereas, A. manshuriensis did not show the peak for AA-I. The HPLC analyses of methanol extract of the two selected Chinese herbal products Zhu Ling San and Wu Ling San indicated the absence of AA-I peaks at 8.4 min.