Simple and rapid analysis of aristolochic acid contained in crude drugs and Kampo formulations with solid-phase extraction and HPLC photodiode-array detection

Abstract

Simple and rapid analysis of aristolochic acid (AA) in crude drugs and Kampo extracts using a solid-phase extraction method and HPLC-PDA analysis was investigated. Extraction of AA from samples was accomplished by adding methanol containing 1% ammonia. The addition of ammonia ionized the AA of acidic substances so that they adhered to an acrylamide copolymer of a strong anion exchange resin (Sep-Pak QMA) coupled to diol silica easily. Furthermore, a mixture of acetonitrile-water-phosphoric acid (75:25:2, v/v) was effective in isolating AA from its carrier. Since almost all interfering peaks originating from contaminants in crude drugs and Kampo extract formulations could be removed, a satisfactory HPLC chromatogram of AA was obtained. A good result was also obtained when Aristolochiaceae and crude drugs containing AA were tested. Particularly in the case of the medicinal parts of Asarum, several interfering peaks and a ghost peak detected near the AA peak were eliminated. The AA contents of two Kampo extract formulations, tokishigyakukagoshuyushokyoto and ryutanshakanto, were calculated by HPLC analysis. The AA content (the sum of AA-I and AA-II) was 1.25-6.13 mg per daily dose. From an additional recovery experiment for Kampo formulations, high recovery rates of AA were obtained. Neither LC/MS nor special instrumentation was necessary. Our results suggest that this simple, quick, and sensitive analytical method to detect AA in crude drugs and Kampo extract formulations would be valuable in safety inspections of AA in crude drugs and their products.