NMR regulatory analysis: determination and characterization of Chinese-herb aristolochic acids

1H NMR methodology for the simultaneous determination and characterization of the nephrotoxic components of Aristolochia plants aristolochic acid I (AA-I) and aristolochic acid II (AA-II) was developed utilizing a 400 MHz spectrometer without the need of reference standards. The developed methodology is able to differentiate and assess chemical structures of these toxic injurious compounds. The quantity of each was calculated on the basis of the integrals for the signals of the H-7 and H-8 of the phenanthrene ring of AA-I and AA-II at delta7.38 and delta8.31, respectively, and the vinylic protons of the internal standard maleic acid at delta6.06. The accuracy of the method was established through the analysis of synthetic mixtures containing the internal standard maleic acid, with purified AA-I or combined AA-I and AA-II sodium salts. Excellent agreements were verified between the assay results and the quantities in the mixtures. The mean +/- SD recovery values for purified AA-I and combined AA-I and AA-II from two sets of 10 synthetic mixtures were 99.8 +/- 0.6% and 99.6 +/- 0.8%, respectively. The assay of 4 lots of commercial aristolochic acid by 1H NMR spectroscopy indicated AA-I and AA-II contents in the ranges 45.3-97.1% and 0-15.4%, respectively.