Facile reversed-phase HPLC resolution and quantitative determination of aristolochic acid and aristolactam analogues in traditional Chinese medicine

A facile reversed-phase HPLC method for simultaneous resolution and quantitative determination of 17 analogues of aristolochic acid (AA) and aristolactam (AL) under gradient elution, with solvent mixture of sodium acetate and acetonitrile, is described. The retention scale of analytes examined in this study is found to be dependent on the pH of mobile phase and highly relevant to the presence of functional groups on analytes as well, under the same chromatographic conditions. Analytes with high polarity (i.e., many functional groups) elutes early. Increasing the pH of mobile phase decreases the retention time of AA analogues, however, it increases that of AL analogues slightly At higher pH (>5.0), the elution reversal between AA and AL is observed and believed to be from the ionization of carboxyl groups on aristolochic acid. Quantitative analysis of Chinese herbs such as Aristolochia heterophylla and Aristolochia fangchi has proven this method to be facile and reliable, which can be considered as fingerprints to identify the origin or species of plant in the genus Aristolochia.