UPLC-MS/MS同时测定八宝丹中15个胆酸类成分的含量

Simultaneous determination of 15 bile acids in Babaodan by UPLC-MS/MS

目的:建立超高效液相色谱串联质谱同时测定八宝丹中15个胆酸类成分含量的方法,并对不同批次的八宝丹进行质量评价。方法:采用CORTECS®UPLC®C_(18)(100 mm×2.1 mm,1.6μm)色谱柱,以乙腈-甲醇(1∶1)溶液-0.1%甲酸水溶液为流动相进行梯度洗脱,流速为0.2 mL·min^(-1),柱温为45℃;质谱采用电喷雾电离源,在正负离子切换的多反应监测模式下进行含量测定;采用SPSS软件对定量结果进行聚类分析与箱线图分析。结果:15个胆酸在各自线性范围内均呈良好的线性关系(r≥0.9964),其精密度、重复性及稳定性的RSD均小于7.3%,平均加样回收率为85.6%~105.1%(RSD≤9.7%)。11批八宝丹中熊去氧胆酸、猪去氧胆酸、鹅去氧胆酸、去氧胆酸、胆酸、石胆酸、牛磺鹅去氧胆酸、牛磺猪去氧胆酸、牛磺熊去氧胆酸、牛磺胆酸、甘氨胆酸、甘氨脱氧胆酸、甘氨鹅脱氧胆酸、牛磺石胆酸、牛磺脱氧胆酸的含量范围分别为0.0038~0.0109、0.1399~0.6952、1.9176~4.1660、2.1154~6.3462、11.0585~25.3614、0.1168~0.2945、0.0006~0.0038、0.0026~0.0936、0.0354~0.1942、2.6125~8.1872、0.1906~6.7183、0.3266~1.9153、0.0060~0.5309、0.0070~0.0400、0.5173~1.9818 mg·g^(-1)。11批样品被分为2类,样品S1~S7为Ⅰ类,样品S8~S11为Ⅱ类,不同生产批次的八宝丹存在较大的内在质量差异。结论:该方法能快速实现对八宝丹中15个胆酸类成分的定性定量分析,可用于八宝丹的质量评价。

Objective:To develop an ultra-high performance liquid chromatography tandem mass spectrometry method for the quantitative analysis of 15 bile acids in Babaodan,and different batches of Babaodan samples were evaluated.Methods:Chromatographic separation was performed on a CORTECS®UPLC®C_(18) column(100 mm×2.1 mm,1.6μm)with mobile phaseconsisting of acetonitrile-methanol solution(1∶1)and 0.1%formic acid solution at a flow rate of 0.2 mL·min^(-1)using gradient elution,and the column temperature was45℃.MS monitoring was performed by electrospray ionization in positive and negative ion switching mode using multiple reaction monitoring.SPSS software was employed to perform the hierarchical cluster analysis and box chart analysis for the content determination results.Results:The calibration curves for 15 bile acids had good linearity(r≥0.9964)in their linear range.The RSDs of precision,repeatability,and stability were all less than 7.3%,the average recoveries were in the range of 85.6%-105.1%(RSD≤9.7%).The content ranges of ursodeoxycholic acid,hyodeoxycholic acid,chenodeoxycholic acid,deoxycholic acid,cholic acid,lithocholic acid,taurochenodeoxycholic acid,taurohyodeoxycholic acid,tauroursodeoxycholic acid,taurocholic acid,glycocholic acid,glycodeoxycholic acid,glycochenodeoxycholic acid,taurolithocholic acid and taurodexycholic acid in 11 batches of Babaodan were 0.0038-0.0109,0.1399-0.6952,1.9176-4.1660,2.1154-6.3462,11.0585-25.3614,0.1168-0.2945,0.0006-0.0038,0.0026-0.0936,0.0354-0.1942,2.6125-8.1872,0.1906-6.7183,0.3266-1.9153,0.0060-0.5309,0.0070-0.040000 and 0.5173-1.9818 mg·g^(-1),respectively.Hierarchical cluster analysis classified 11 batches of Babaodan into 2 categories,sample S1-S7 in categoryⅠand sample S8-S11 in categoryⅡ.The results of box chart analysis showed that there were significant differences in the internal quality of Babaodan from different production batches.Conclusion:The method developed in this study can quickly achieve qualitative and quantitative detection of 15 bile acids in Babaodan,which can be used in the quality evaluation of Babaodan.