目的 建立超高效液相色谱串联四级杆-静电轨道阱高分辨质谱技术(UPLC-Q Exactive-Orbitrap MS/MS)同时测定牛黄解毒片(Niuhuang Jiedu Tablets,NJT)中牛磺胆酸、7-酮-3α,12-α-羟基胆烷酸、甘氨猪去氧胆酸、12-脱氢胆酸、甘氨胆酸、3-酮-7α,12α-二羟基-5β-胆烷酸、牛磺鹅去氧胆酸、熊去氧胆酸、7-酮基胆石酸、猪胆酸、牛磺脱氧胆酸钠、12-酮脱氧胆酸、猪去氧胆酸、胆酸、甘氨鹅脱氧胆酸、甘氨脱氧胆酸、牛磺石胆酸钠、甘氨石胆酸、鹅去氧胆酸、去氧胆酸、石胆酸21种胆酸类成分含量的方法,并对不同批次的NJT进行质量评价。方法 采用Thermo Bremen Hypersil Gold色谱柱(100 mm×2.1 mm,1.9 μm),以含0.1%甲酸水溶液-甲醇为流动相进行梯度洗脱,体积流量0.2 mL/min,柱温为40℃;质谱采用加热型电喷雾离子源(HESI),在负离子平行反应监测模式下进行含量测定,采用SIGMA 14.0软件对定量结果进行化学计量学分析。结果 21种胆酸成分在线性范围内呈良好的线性关系(r² ≥ 0.998 9),其精密度、重复性、稳定性RSD均小于6.7%,平均回收率为95.1%~102.5%(RSD ≤ 4.5%),在正交偏最小二乘-判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA)与层次聚类分析(hierarchical cluster analysis,HCA)中,样品在95%置信区间内被分为3类,变量重要性排序值(variable importance in projection values,VIP)显示猪胆酸、牛磺胆酸、7-酮基胆石酸、甘氨胆酸、胆酸为样品间的主要差异成分。结论 所建立方法具有高效、灵敏的特点,能够快速实现对NJT中21种胆酸类成分的含量测定,可为其质量控制提供理论依据。

Objective To develop a ultra high performance liquid chromatography in tandem with quadrupole electrostatic orbital hydrazine high resolution mass spectrometry (UPLC-Q Exactive-OrbitrapMS/MS) method for the content determination of 21 cholic acids in Niuhuang Jiedu Tablets (牛黄解毒片, NJT), including taurocholic acid, 7-ketodeoxycholic acid, glycohyodeoxycholic acid, 12-oxochenodeoxycholic acid, gycocholic acid, 3-oxocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, 7-oxolithocholic acid, hyocholic acid, taurodeoxycholic acid sodium salt hydrate, 12-ketolithocholic acid, hyodeoxycholic acid, cholic acid, gycochenodeoxycholic acid, glycodesoxycholic acid, taurolithocholic acid sodium salt, glycolithocholic acid, ghenodeoxycholic acid, deoxycholic acid, lthocolic acid, and to evaluate the quality of NJT from different batches. Methods The Thermo Bremen Hypersil Gold column (100 mm × 2.1 mm, 1.9 μm) was employed to gradient elution with mobile phase consisting of 0.1% formic acid solution-methanol at a flow rate of was 0.2 mL/min, and the column temperature was 40 ℃. The mass spectrum monitoring was performed by heating electrospray ion source, and using parallel reaction monitoring in negative ion mode for quantitative analysis. SIGMA 14.0 software was employed to perform the chemometrics analysis for the content determination results. ResultsThe calibration curves for 21 bile acids had good linearity in their linear range (r² ≥ 0.9989), the RSDs of precision, repeatability, stability were all less than 6.7%, the average recoveries were ranged from 95.1% to 102.5% (RSD ≤ 4.5%), in the orthogonal partial least squares discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA), the samples were classified into three categories within 95% confidence interval. The variable importance in projection values (VIP) showed that, hyocholic acid, taurocholic acid, 7-oxolithocholic acid, glycocholic acid and cholic acid were the main differential components among the samples. Conclusion The method developed in this study was efficient and sensitive, which can quickly achieve quantify 21 bile acids in NJT, and provides theoretical basis for the quality control of NJT.

R283.6

国家自然科学基金面上项目（81973437）；国家自然科学基金面上项目（82274080）；福厦泉国家自主创新示范区协同创新平台专项（2021FX02）