牛黄消炎片中土大黄苷的检查方法研究

Study on Detection of Rhaponticin in Niuhuang Xiaoyan Tablets

目的:建立牛黄消炎片中土大黄苷的检测方法。方法:通过使用聚酰胺薄膜进行薄层色谱快速筛查;采用Prevail^(TM) C18色谱柱(250 mm×4.6 mm,5μm)进行HPLC法验证,以甲醇-乙腈-水(30:7:63)为流动相,流速1 ml/min,检测波长320 nm;采用负离子模式电喷雾离子源进行HPLC-MS/MS法验证,对土大黄苷的准分子离子峰m/z 419[M-H]-进行二级质谱分析。结果:经薄层色谱法检测,测试样品中17批样品在与土大黄苷对照品R_(f)值一致的位置有相同颜色荧光斑点检出。经HPLC法验证,17批阳性样品在与土大黄苷保留时间相同的位置均有色谱峰检出,并且在280~400 nm波长范围的吸收光谱与土大黄苷对照品相同。17批阳性样品进一步经HPLC-MS/MS分析,均检测到土大黄苷准分子离子峰,且二级扫描质谱图与对照品一致。结论:建立的检测方法专属性强,准确可靠,可用于牛黄消炎片中土大黄苷的检测。

Objective:To develop a specific method for detection of rhaponticin in Niuhuang Xiaoyan Tablets.Methods:The polyamide film was used for TLC rapid detection.An HPLC method was applied for determination using a Prevail^(TM) C18 column(250 mm×4.6 mm,5μm)with a mixture of methanol-acetonitrile and water(30:7:63)as the mobile phase at a flow rate of 1 mL·min^(-1).The detection wavelength was set at 320 nm.Furthermore,the positive samples were verified by HPLC-MS/MS under ESI-mode.The quasi-molecular ion m/z 419[M-H]-was selected for monitoring and MS/MS analysis.Results:17 batches of the test samples showed the same color fluorescent spots at the position with the same R_(f) value consistent with that of rhaponticin through thin-layer chromatography.17 batches of positive samples presented chromatographic peaks at the same retention time with rhaponticin by HPLC analysis.Also,the absorption spectrum among the wavelength range of 280~400 nm was the same as that of rhaponticin.Furthermore,the quasi-molecular ions of rhaponticin were detected in 17 batches of positive samples by HPLC-MS/MS.They also showed the full scan MS2 spectrum consistent with the reference substance.Conclusion:The established methods were specific accurate and reliable for the detection of rhaponticin in Niuhuang Xiaoyan Tablets.