ABSTRACT
Objective:To establish a qualitative and quantitative method for the determination of aristolochic acids in <italic>Aristolochia cinnabarina</italic> dried root tubers. Method:The dried root tubers of <italic>A. cinnabarina </italic>was qualitative and quantitative analysis by ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry （UPLC-QTOF-MS/MS）. The analysis was performed on Waters ACQUITY UPLC-BEH C<sub>18</sub> column （ 2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-1 min， 10%B； 1-9 min， 10%-30%B； 9-11 min， 30%-50%B； 11-15 min， 50%-90%B）. The flow rate was 0.45 mL·min<sup>-1</sup>， column temperature was 35 ℃， and the detection wavelength was 250 nm. Mass spectral data was acquired in positive mode of electrospray ionization （ESI）. At the same time， the UPLC fingerprints of aristolochic acids in 21 batches of <italic>A. cinnabarina</italic> dried root tubers were established， and the contents of 5 aristolochic acids in <italic>A. cinnabarina</italic> dried root tubers from different producing areas and different harvesting periods were determined. Result:A total of 17 compounds， including 8 aristolochic acids， 7 aristololactams and 2 4，5-dioxoaporphine alkaloids， were identified from <italic>A. cinnabarina</italic> dried root tubers by mass spectrometry data and bibliographic information. Ten common peaks were identified in the UPLC fingerprint， and they were tuberosinone-<italic>N</italic>-<italic>β</italic>-<italic>D</italic>-glucoside， aristolactam Ⅰa-<italic>N</italic>-<italic>β</italic>-<italic>D</italic>-glucoside， aristolochic acid Ⅳa-<italic>O</italic>-<italic>β</italic>-<italic>D</italic>-glucoside， aristolactam Ⅲa-<italic>N</italic>-<italic>β</italic>-<italic>D</italic>-glucoside， aristolactam Ⅰ-<italic>N</italic>-<italic>β</italic>-<italic>D</italic>-glucoside， aristolochic acid Ⅲa， aristolochic acid Ⅳa， aristolochic acid Ⅱ， aristolactam Ⅰ and aristolochic acid Ⅰ. According to the quantitative analysis， the results exhibited that aristolochic acid Ⅲa， aristolochic acid Ⅳa， aristolochic acid Ⅱ， aristolactam Ⅰ and aristolochic acid Ⅰ had good linear relationships in the linear range. The relative standard deviations （RSDs） of precision， stability and reproducibility tests were all less than 3.0%， the recovery was 97.06%-101.84% （RSD<3.0%）. The contents of aristolochic acid Ⅰ， aristolochic acid Ⅱ， aristolochic acid Ⅲa， aristolochic acid Ⅳa， and aristolactam Ⅰ in 21 batches of <italic>A. cinnabarina</italic> dried root tubers were 0.938 6-3.567 5， 1.377 6-3.688 1， 0.056 3-0.527 7， 0.108 8-0.305 5， 0.021 0-0.081 7 mg·g<sup>-1</sup>， respectively. Conclusion:The content of aristolochic acids in <italic>A. cinnabarina</italic> dried root tubers has a certain difference， the contents of aristolochic acid Ⅰ and Ⅱ are higher than other aristolochic acids. The established method is rapid， simple， accurate and reliable， which can provide reference for the quality control and evaluation of <italic>A. cinnabarina</italic> dried root tubers.
ABSTRACT
The objective of this work was to explore the content and composition of aristolochic acid compounds in Chinese medicinal materials containing toxic aristolochic chemicals, so as to ensure the safety of these medicinal materials and their related products. Nine Chinese medicinal materials were selected for study, including the tuber of Aristolochia cinnabarina, the herbs of Asarum forbesii, the stems of Aristolochia manshuriensis., the fruits of Aristolochia debilis, the roots of Aristolochia debilis, the stems and leaf of Aristolochia debilis, the herbs of Aristolochia mollissima, the roots of Aristolochia fangchi, and the roots of Asarum heterotropoides var. mandshuricum. The aristolochic acid components in the nine Chinese medicinal materials were analyzed by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS) combined with high performance liquid chromatography diode-array detection. The separation was performed on an Agilent ZORBAX SB-Aq column (250 mm×4.6 mm, 5 μm) with gradient elution using a mobile phase consisting of acetonitrile and 0.2% acetic acid. ESI positive ion mode MS was used to investigate the ionization pathways of aristolochic acid Ⅰ, Ⅱ, Ⅲa, Ⅳa, Ⅶa, and aristololactam Ⅰ, Ⅱ using seven reference standards, and the structures of the components with UV spectrasimilar to those of the seven reference standards in the selected medicinal materials were qualitatively analyzed by following the investigated ionization pathways. The identified aristolochic acid components were quantified using an external standard method by HPLC-UV with detection at 254 nm. Twenty-two aristolochic acid components including 11 aristolochic acids and 11 aristololactams were identified from the nine selected medicinal materials; 15 aristolochic acids were found in the tuber of Aristolochia cinnabarina and the roots of Aristolochia debilis, followed by 14 aristolochic acids in the fruits of Aristolochia debilis and the stems of Aristolochia manshuriensis. The greatest content of aristolochia components was found in the tuber of Aristolochia cinnabarina and the stems of Aristolochia manshuriensis, ranging from 8.91 mg·g-1 to 13.40 mg·g-1, and the least amount was in the herbs of Asarum forbesii, at less than 0.10 mg·g-1 and containing only two aristolochia components. This study systematically explored the quantity and composition of aristolochic acid components in selected Chinese medicinal materials believed to contain toxic aristolochic compounds, providing a basis for follow-up studies on the toxicity of these substances that can lead to safety standards for their use.
ABSTRACT
More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.