右归饮HPLC指纹图谱研究及9种成分定量分析

Study on HPLC fingerprint of Youguiyin and its multi-component quantitative analysis

目的建立右归饮的HPLC指纹图谱,并同时测定9种成分(京尼平苷酸、莫诺苷、绿原酸、栀子苷、马钱苷、松脂醇二葡萄糖苷、甘草苷、芦丁和甘草酸)的量,为控制右归饮的质量提供依据。方法采用Pursuit XRs 5 C18色谱柱(250mm×4.6 mm,5μm)进行分离,流动相为乙腈-0.1%磷酸水溶液,梯度洗脱,体积流量1.0 m L/min,检测波长230 nm,柱温30℃。建立10批右归饮样品的HPLC指纹图谱,并进行相似度评价;通过与各组方药味及对照品的保留时间和紫外光谱图比对,对共有峰进行归属和指认,并测定指认出的9个成分的量。结果 10批样品指纹图谱与对照指纹图谱的相似度均大于0.904;共标定30个共有峰,其中8个来自杜仲,8个来自甘草,6个来自山茱萸,2个来自附子,2个来自熟地黄,1个来自肉桂,山药及枸杞对共有峰贡献不明显,其中3个共有峰不能明确其来源;通过对共有峰进行指认,发现7号峰为京尼平苷酸、9号峰为莫诺苷、11号峰为绿原酸、12号峰为栀子苷、13号峰为马钱苷、14号峰为松脂醇二葡萄糖苷、18号峰为甘草苷、21号峰为芦丁及30号峰为甘草酸,并对这9种成分进行了定量测定,其定量测定结果分别为87.6~119.1μg/g、323.6~365.6μg/g、108.3~124.1μg/g、79.5~85.0μg/g、171.7~188.0μg/g、163.0~238.3μg/g、64.5~53.3μg/g、159.8~168.5μg/g、72.8~83.6μg/g。结论所建立的右归饮HPLC指纹图谱及定量测定方法稳定性、重复性好,可为右归饮质量控制和评价提供参考。

Objective To establish the HPLC fingerprint and determine nine components（geniposidic acid, morroniside, chlorogenic acid, geniposide, loganin, pinoresinol diglucoside, liquiritin, rutin, and glycyrrhizic acid） of Youguiyin, so as to provide a scientific basis for the quality control. Methods HPLC analysis was performed on Pursuit XRs 5 C18 column（250 mm × 4.6 mm, 5 μm）. The gradient elution was performed by the mobile phase consisting of acetonitrile and 0.1% formic acid aqueous with the flow rate of 1.0 m L/min, the detection wavelength was set at 230 nm, and the column temperature was 30 ℃. Fingerprints of ten batches of Youguiyin were determined, and the similarities among fingerprints were evaluated. Attributive analysis and identification of common peaks were performed by comparing the retention time and UV spectra among 10 batches of Youguiyin. The formula ingredients and reference substance, and nine components content were determined. Results The similarities of fingerprints of 10 batches of Youguiyin and reference fingerprints were all greater than 0.904. There were 30 mutual peaks marked in total, which were eight mutual peaks from Eucommia ulmoides, eight mutual peaks from Glycyrrhiza uralensis, six mutual peaks from Cornus officinalis, six mutual peaks from Aconitum carmichaeli, one mutual peaks from Cinnamomum cassia, and none of mutual peaks were originated from Dioscorea opposita and Lycium barbarum, and three of them cannot be originated. Based on the the identification of the common peaks, nine components [geniposidic acid（peaks 7）, morroniside（peaks 9）, chlorogenic acid（peaks 11）, gardenoside（peaks 12）, loganin（peaks 13）, pinoresinol diglucoside（peaks 14）, liquorice glycosides（peaks 18）, rutin（peaks 21）, and glycyrrhizic acid（peaks 30）] were identified and quantified. The quality faction of nine components were 87.6—119.1 μg/g, 323.6—365.6 μg/g, 108.3—124.1 μg/g, 79.5—85.0 μg/g, 171.7—188.0 μg/g, 163.0—238.3 μg/g, 64.5—53.3 μg/g, 159.8—168.5 μg/g, and 72.8—83.6 μg/g in raw material. Conclusion The method established in this study is simple, accurate and highly reproducible, and can provide basis for quality control of Youguiyin.