HPLC-MS修改稿.docVIP

Analysis of Aristolochic Acid Derivates in Aristolochia debilis and Its Fermented Product by HPLC-ESI-TOF-MS LIU Xue-Xiang1, WU Xiao-Feng1, PAN Yang1,2*, JIANG Ya-Ping1 1Laboratory of Medicinal Fungi and Phyto-Biotech, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210029; 2Jiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, 210029, China [ABSTRACT] AIM: To develop high-performance liquid chromatography combined with electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) for the isolation and identification of aristolochic acid derivates (AAs) in Aristolochia debilis and its fermented product by Ganoderma lucidum (LQFP). METHOD: Chromatographic separation of AAs was carried out on a Hanbon Phecda C18 column (250 mm × 4.6 mm, 5 (m) at 30 °C. Elution was performed at a flow rate of 1.0 mL·min?1 using solvents of acetonitrile (A) and 0.2% acetic acid in water (B). Gradient was 0-25 min (15%→30%A), 25-45 min (30%→50%A), 45-55 min (50%→60%A), 55-75 min (60%→80%A), 75-80 min (80%→15%A). The structures of AAs were analyzed by chromatographic retention time, as well as ESI-TOF-MS. RESULT: Seven AAs in A. debilis and LQFP were recognized as AA I (6), AA II (4), AA III (5), AA Ia or IIIa (1), AA IVa, Va, VIIa or VIIIa (3), AA IV or VII (7), and AA VIa (2). The peak areas of the 7 compounds in LQFP that existed originally in A. debilis were all decreased. The decrease rate of 1, 2, 4, 5, 6, 7 was 93.5%, 87.2%, 94.1%, 42.2%, 86.2% and 69.5%, respectively, while 3 was not detected. There were also a lot of new peaks in the HPLC diagram of LQFP in the scale of retention time of 0-20 min that may have been polar substances formed by biotransformation of fermentation. CONCLUSION: The method is of great help to isolate and identify AAs in A. debilis and its fermented product LQFP. The contents of 7 aristolochic acids that are accepted as nephrotoxic substances in A. debilis de