基于指纹图谱分析和多成分同时定量的玉屏风丸质量评价

目的建立玉屏风丸的高效液相色谱(HPLC)指纹图谱,测定7种主要成分(毛蕊异黄酮葡萄糖苷、芒柄花素、升麻素苷、升麻素、白术内酯Ⅲ、白术内酯Ⅱ、白术内酯Ⅰ)的含量,为玉屏风丸的质量控制提供可靠方法。方法色谱柱为Shim-pack VP-ODS柱(250 mm×4.6 mm,5μm);流动相为乙腈(A)-0.3%磷酸溶液(B),梯度洗脱(0~20 min时12%A,20~40 min时12%A→35%A,40~46 min时35%A→68%A,46~50 min时68%A→80%A,50~60 min时80%A→12%A);流速为0.85 m L/min,检测波长升麻素苷为300 nm(0~20 min),毛蕊异黄酮葡萄糖苷和芒柄花素及升麻素为254 nm(20~40 min),白术内酯Ⅲ及白术内酯Ⅱ为220 nm(40~46 min),白术内酯Ⅰ为275 nm(46~60 min);柱温为35℃;流速为0.85 m L/min;进样量为10μL。结果玉屏风丸HPLC指纹图谱有18个共有峰,通过比较,指认其中7个指标成分,分别为4号(升麻素苷)、9号(毛蕊异黄酮葡萄糖苷)、10号(升麻素)、11号(芒柄花素)、16号(白术内酯Ⅲ)、17号(白术内酯Ⅱ)和18号(白术内酯Ⅰ),利用相似度软件对14批样品的指纹图谱进行分析,各批样品相似度均大于0.97。在建立的色谱条件下,7组分分离度良好,精密度和重复性试验的RSD均小于1.5%(n=6),供试品溶液在24 h内稳定,各成分具有良好的线性关系和较宽的线性范围。14批玉屏风丸中毛蕊异黄酮葡萄糖苷、芒柄花素、升麻素苷、升麻素、白术内酯Ⅲ、白术内酯Ⅱ、白术内酯Ⅰ质量浓度范围分别为1.5490~1.5640 mg/g,0.7015~0.7029 mg/g,0.7960~0.7977 mg/g,0.5905~0.5922 mg/g,0.6745~0.6762 mg/g,0.5210~0.5229 mg/g,0.3992~0.4015 mg/g。结论该建立的玉屏风丸HPLC指纹图谱检测和定量测定分析方法快速、准确,可有效评价玉屏风丸的质量。

Quality Assessment of Yupingfeng Pills Based on Simultaneous Determination of HPLC Fingerprint and Multi-Components

Objective To establish a high performance liquid chromatography(HPLC)fingerprint method of Yupingfeng Pills and determine the contents of seven main components(calycosin7-O-β-D-Glucopyranoside,formononetin,prim-O-glucosylcimifugin,cimifugin,atractylenolideⅢ,atractylenolideⅡ,atractylenolideⅠ),so as to provide a reference for the quality control of Yupingfeng Pills.Methods The chromatographic column was shim-pack VP-ODS(250 mm×4.6 mm,5μm)with gradient elution by acetonitrile(A)-0.3%phosphoric acid solution(B)(0-20 min,12%A;20-40 min,12%A→35%A;40-46 min,35%A→68%A;46-50 min,68%A→80%A;50-60 min,80%A→12%A).The detection wavelength was set at 300 nm for prim-O-glucosylcimifugin(0-20 min),254 nm for calycosin7-O-β-D-Glucopyranoside,formononetin and cimifugin(20-40 min),220 nm for atractylenolideⅢand atractylenolideⅡ(40~46 min),275 nm for atractylenolideⅠ(46-60 min).The column temperature was 35℃,the flow rate was0.85 m L/min,and the injection volume was 10μL.Results The common mode of the fingerprint was set up with 18 common peaks,and seven of them were identified by comparison with the reference.The similar degrees of 14 batches of samples were over 0.97,they were prim-O-glucosylcimifugin(peak 4),calycosin7-O-β-D-Glucopyranoside(peak 9),cimifugin(peak 10),formononetin(peak 11),atractylenolideⅢ(peak 16),atractylenolideⅡ(peak 17)and atractylenolideⅠ(peak 18).The seven compounds were well separated under the determined chromatographic conditions.The RSDs of precision and repeatability experiment were all less than 1.5%and the sample solution was stable during 24 h(n=6).All the compounds had a good linearity and linear range.The contents of calycosin7-O-β-D-Glucopyranoside,formononetin,cimifugin,atractylenolideⅢ,atractylenolideⅡ,and atractylenolideⅠin the 14 batches of Yupingfeng Pills were 1.549-1.564 mg/g,0.7015-0.7029 mg/g,0.7960-0.7977 mg/g,0.5905-0.5922 mg/g,0.6745-0.6762 mg/g,0.5210-0.5229 mg/g,and 0.3992-0.4015 mg/g,respectively.Conclusion The established HPLC fingerprint method is fast and accurate,which can be used for the quality control of Yupingfeng Pills.