Establishment of HPLC Fingerprint of Huafengdan Yaomu and Content Determination of 7 Nucleosides in Samples of Different Fermentation Time / 中国药房

ABSTRACT

OBJECTIVE：To establish fingerprint of Huafengdan yaomu ，and to determine the contents of 7 nucleosides in samples of different fermentation time. METHODS ：HPLC method was adopted. The determination was performed on Pntulips BP-C18 column with mobile phase consisted of methanol-water （gradient elution ）at the flow rate of 0.8 mL/min. The detection wavelength was set at 260 nm，and column temperature was 35 ℃. The sample size was 10 μL. Using xanthine as reference， HPLC fingerprint of 12 batches of Huafengdan yaomu was drawn. The similarity of samples were evaluated with Similarity Evaluation System of TCM Chromatographic Fingerprints （2012 edition）. Common peaks were confirmed. The contents of uracil ， hypoxanthine，xanthine，uridine，inosine，guanosine and thymidine were determined in samples of different fermentation time （0， 1，2，3，4 weeks）by the same method. RESULTS ：There were 8 common peaks in 12 batches of Huafengdan yaomu ，with similarities ranging from 0.712 to 0.954；7 components were identified ，namely uracil ，hypoxanthine，xanthine，uridine，inosine， guanosine and thymidine. The linear ranges of mass concentrations of above 7 components in samples at different fermentation time were 0.87-8.7 μ g/mL （r＝0.999 6）， 4030 1.51-15.1 μg/mL（r＝0.999 7），6.08-60.8 μg/mL（r＝0.999 5）， 号） 1.52-15.2 μg/mL（r＝0.999 6），1.82-18.2 μg/mL（r＝0.999 6）， 1.48-14.8 μg/mL（r＝0.999 6），1.63-16.3 μg/mL（r＝0.999 3）. The limits of quantification were 0.027 4，0.076 3，0.250 4，0.172 3，0.101 1，0.078 3，and 0.084 2 μ g/mL，and the detection limits were 0.008 7，0.025 5，0.007 9，0.084 1，0.035 7，0.026 9，0.027 5 μg/mL，respectively. RSDs of precision ， repeatability and stability tests （12 h）were all less than 3％. The sample recovery rates were 94.16％-100.16％（RSD＝2.24％，n＝ 6），93.87％-100.65％（RSD＝2.67％，n＝6），93.52％-99.66％（RSD＝2.30％，n＝6），93.67％-98.24％（RSD＝1.89％，n＝6）， 96.00％-102.18％（RSD＝1.96％，n＝6），94.62％-101.54％（RSD＝2.82％，n＝6），97.72％-104.56％（RSD＝2.97％，n＝6）. After fermentation for 0-4 weeks，the contents of the above 7 components and total nucleosides were 0.042-0.232，0.027-0.181， 0.039-0.651，0.026-0.225，0.034-0.111，0.009-0.124，0.079-0.099，0.647-1.292 mg/g，respectively. After fermentation for 1-4 weeks，the contents of uracil ，hypoxanthine，xanthine and total nucleosides were significantly increased ，compared with 0 week of fermentation；the contents of uridine ，inosine and guanosine were significantly lower than those in 0 weeks. CONCLUSIONS ：The established fingerprint has strong characteristics and simple to operate ，which can be used for the quality control of Huafengdan yaomu；the content determination method is accurate and reliable ，and can be used to simultaneously determine the contents of 7 active nucleosides ；the content of nucleosides in Huafengdan muyao is affected by fermentation time.