Elsevier

Journal of Chromatography B

Volume 766, Issue 1, 5 January 2002, Pages 169-174
Journal of Chromatography B

Short communication
High-performance liquid chromatographic determination for aristolochic acid in medicinal plants and slimming products

https://doi.org/10.1016/S0378-4347(01)00416-9Get rights and content

Abstract

A HPLC procedure with a silica gel RP-18 reversed-phase column for the determination of aristolochic acids I, II in medicinal plants and slimming products was developed. The mobile system 0.3% ammonium carbonate solution–acetonitrile (75:25, v/v) with pH 7.5 was the optimal buffer to clearly separate aristolochic acids I, II within 20 min. The recovery of aristolochic acids I, II in medicinal plants and slimming products was better than 90% by extracting with methanol and purifying through a PHP-LH-20 column. The major component was aristolochic acid I in Aristolochia fangchi and the level ranged from 437 to 668 ppm. Aristolochic acid II was the major component for Aristolochia contorta and its range was <1–115 ppm. Twelve out of 16 samples of slimming pills and powders contained aristolochic acids I and/or II. The major component in most slimming products was aristolochic acid II and the level ranged from <1 to 148 ppm. It may indicate that slimming products were not mainly made of A. fangchi.

Introduction

Chinese herbs nephrophathy (CHN) is a new type of subacute interstitial fibrosis of the kidney. In Belgium, there have been approximately 100 women patients who had followed a weight-reducing treatment that usually consisted of Chinese herbs and appetite suppressants (fenfluramine and diethylpropion), as well as cascara, belladonna extract, and acetazolamide [1], [2], [3], [4], [5]. About one-half of the patients needed the treatment of renal replacement therapy [3], [6]. CHN is not limited to Belgium, but has been reported in other countries including France [7], Spain [1], UK [8] and Japan [9], [10].

The nephrotoxicity of these Chinese herbs has been traced to aristolochic acid (AA), which is the major alkaloid extracted from Aristolochia fangchi, inadvertently included in the slimming pills [11]. AA is a mixture of structurally related nitrophenanthrene carboxylic acids, AA-I and AA-II being major components. Not only is AA nephrotoxic, it is also a potent carcinogen in laboratory animals [12], [13]. In 1994, the first case of bladder cancer among the Belgian patients was reported [14]. By 1999, 40% of 19 kidney-biopsy specimens from a group of these patients showed multifocal, high-grade, flat, transitional-cell carcinoma in situ [15]. Hence, the relationship between the use of A. fangchi and the development of urothelia carcinoma is not a minor problem. According to these facts, Belgium, UK, Canada, Australia and Germany have banned the use of these herbs.

Usually, slimming regimens consist of two herbs Stephania tetrandra and Magnolia officianlis. Among them, S. tetrandra is often substituted by A. fangchi because the Chinese names sound similar. These slimming pills and powders including different species Aristolochia are commonly used in Taiwan. Meanwhile, these species of Aristolochia are also used to act as analgesic, diuretic and antiinflammatory agents in Taiwan. Although the incidence of CHN in Taiwan is not officially reported, this problem of CHN is noteworthy here.

The quantitative analysis of AA in the medicinal plants has recently developed by using high-performance liquid chromatography (HPLC) [16]. However, this method took about 40 min for a run. Hence, this study was carried out to establish a simple, rapid, sensitive and usable method for determining AA-I and AA-II. Then the levels of AA-I and AA-II in medicinal plants and slimming pills and powders collected from herbal stores and patients in Taiwan were analyzed.

Section snippets

Agents

A mixture of AA-I (61%) and AA-II (19%) was purchased from Sigma (lot 36H13111) (St. Louis, MO, USA) and further purified to obtain pure AA-I and AA-II standards by HPLC. The purified AA-I and AA-II were desalted by adding acetic acid and then evaporated. AA-I and AA-II were identified by 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy and gas chromatography–mass spectrometry (GC–MS) in this laboratory. The structural features of AA-I and AA-II were the same as those reported

Results and discussion

The effect of acetonitrile concentration on the retention time of eluting peaks of AA-I and AA-II in HPLC by using the buffer (pH 9.3) prepared from different ratios of 0.3% ammonium carbonate solution and acetonitrile is shown in Fig. 1. The optimal ratio of 0.3% ammonium carbonate solution–acetonitrile was 75:25 (v/v) for rapidly separating AA-I and AA-II with good resolution in HPLC. The respective retention times of AA-I and AA-II were 9.0 and 6.9 min, but it was 32.6 min for AA-I and 22.9

Conclusion

A HPLC system using a mobile phase of 0.3% ammonium carbonate solution–acetonitrile (75:25, v/v) with pH 7.5 in combination with a C18 column was useful for determining aristolochic acids I and II. The major component was aristolochic acid I in the medicinal plant A. fangchi, but aristolochic acid II in A. contora and slimming products. The profile of aristolochic acids was quite different depending on the plant species. The slimming products collected in Taiwan may not be mainly prepared from

Acknowledgements

This study was supported by a grant from the Department of Health, Taiwan. The species of plant samples collected from medicinal plant stores was identified by Dr. Chieh-Fu Chen, Director of National Chinese Medicinal College, Taipei, Taiwan.

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