牛黄及代用品中胆汁酸类成分指纹图谱及含量测定研究

目的：建立牛黄及代用品中胆汁酸类成分的指纹图谱，并对甘氨胆酸、牛磺胆酸、胆酸和去氧胆酸进行定量研究。方法：采用HPLC-ELSD联用技术，使用Symmetryshield^TM C₁₈色谱柱（4.6 mm×250 mm，5 μm），以乙腈为流动相A，以0.2%甲酸水溶液（含10 mmol·L^-1醋酸铵）为流动相B，梯度洗脱（0~5 min，2% A→35% A；5~12 min，35% A→37% A；12~23 min，37% A；23~45 min，37% A→50% A），流速1.0 mL·min^-1，ELSD漂移管温度102℃，氮气1.9 mL·min^-1，进行指纹图谱分析和上述甘氨胆酸等4个成分的含量测定；采用电喷雾离子源进行正离子模式扫描，对牛黄指纹图谱中8个特征峰进行指认；并利用Chrompattern软件对测试所得到的色谱数据进行主成分分析，利用国家药典委员会相似度评价软件进行相似度评价。结果：建立了牛黄及代用品的HPLC-ELSD指纹图谱，并指认了甘氨胆酸、牛磺胆酸、胆酸、甘氨鹅去氧胆酸、牛磺去氧胆酸、猪去氧胆酸、鹅去氧胆酸和去氧胆酸8个特征峰；牛黄、体外培育牛黄和人工牛黄中胆汁酸类成分基本相同，但含量存在差异；指纹图谱经相似度和主成分分析，均可用于区分牛黄、体外培育牛黄和人工牛黄。结论：本法可为牛黄、体外培育牛黄和人工牛黄的质量控制提供有效的技术支持。

Objective: To establish the fingerprints of Calculus Bovis and its substitutes by HPLC-ELSD and simultaneously perform the quantitative analysis of glycocholic acid,taurocholic acid,cholic acid,and deoxycholic acid.Methods: The characteristic fingerprints and quantitative analysis were performed by HPLC-ELSD.The characteristic peaks were identified by LC-MS.The samples were separated on a Symmetryshield^TM C₁₈ column (4.6 mm×250 mm,5 μm),and eluted with acetonitrile(A)and the solution containing 10 mmol·L^-1 ammonium acetate and 0.2% formic acid(B)in a gradient elution(0-5 min,2%A→35%A;5-12 min,35%A→37%A;12-23 min,37%A;23-45 min,37%A→50%A)at a flow rate of 1.0 mL·min^-1.The temperature of drift tube was 102℃ and the gas flow rate was 1.9 mL·min^-1.Eight characteristic peaks in the fingerprints were identified by MS with electrospray ionization(ESI)source in the positive ion mode.The HPLC-ELSD fingerprints of Calculus Bovis and its substitutes were compared by principal component analysis and similarity analysis.Results: The characteristic fingerprints of Calculus Bovis and its substitutes were established.Eight characteristic peaks in the fingerprints were identified as glycocholic acid,taurocholic acid,cholic acid,glycochenodeoxycholic acid,taurodeoxycholic acid,hyodeoxycholic acid,chenodeoxycholic acid and deoxycholic acid.The fingerprint analysis result showed that Calculus Bovis and its substitutes had almost the same ingredients but with different content.Calculus Bovis and its substitutes were obviously distinguished by principal component analysis and similarity analysis.Conclusion: The established fingerprint can be served as an effective approach for quality control of Calculus Bovis and its substitutes.