目的:建立同时测定牛黄蛇胆川贝液中22个胆酸成分(甘氨熊脱氧胆酸、甘氨猪胆酸、牛磺胆酸、7-酮-3α,12-α-二羟基胆烷酸、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、3α-羟基-7-酮基-5β-胆烷酸、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)的超高效液相色谱-串联质谱联用(UPLC-MS/MS)方法。方法:样品采用甲醇超声提取;以Thermo Fisher Scientfic Bremen HYPERSIL GOLD(100 mm×2.1 mm,1.9 μm)为色谱柱,0.1%甲酸水溶液(A)-甲醇(B)为流动相,梯度洗脱,流速为0.2 mL·min-1,柱温为40 ℃,进样量为2 μL;质谱采用加热电喷雾离子源(HESI),负离子模式和平行反应监测(PRM)模式,喷雾电压为3.0 kV (-)。结果:所建方法在一定范围内均呈现良好线性关系(r≥0.999 4);精密度、重复性良好,供试样品在48 h内稳定性良好,加样回收率在94.9%~105.2%,RSD为0.50%~5.2%。含量测定结果表明牛黄蛇胆川贝液中牛磺胆酸、胆酸、猪胆酸、甘氨胆酸、猪去氧胆酸含量较高,且不同厂家之间含量存在较大差异,聚类分析可分为3类。统计发现,Ⅱ类中的牛磺胆酸、3-酮-7α,12α-二羟基-5β-胆烷酸、3α-羟基-7-酮基-5β-胆烷酸、12-酮脱氧胆酸、胆酸含量明显高于类别Ⅱ、Ⅲ,类别Ⅲ中猪胆酸含量明显高于类别Ⅰ和Ⅱ。结论:本方法便捷、高效、准确、灵敏,可用于牛黄蛇胆川贝液中22个胆酸成分的测定。
Objective: To establish a method for simultaneous determination of 22 cholic acids(glycoursodeoxycholic acid, glycohyocholic acid, taurocholic acid, 7-oxodeoxycholic acid, 12-oxocholic acid, glycocholic acid, 3-oxocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid, 3α-hydroxy-7-oxo-5β-cholanic acid, hyocholic acid, taurodeoxycholic acid sodium salt hydrate, 12-ketodeoxycholic acid, hyodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycodehydrocholic, taurolithocholic acid sodium salt, glycolithocholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid) in Niuhuang Shedan Chuanbei liquid by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Methods: The samples were extracted by methanol ultrasonic extraction. Using Thermo Fisher Scientfic Bremen HYPERSI GOLD (100 mm×2.1 mm, 1.9 μm) as chromatographic column, 0.1% formic acid aqueous solution (A)-methanol (B) as mobile phase with gradient elution. The flow rate was 0.2 mL·min-1, the column temperature was 40 ℃, the injection volume was 2 μL. The mass spectrometry adopted heating electrospray ion source (HESI) with negative ion mode and parallel reaction monitoring (PRM) mode. The spray voltage was 3.0 kV (-). Results: The established method showed a good linear relationship in a certain range (r≥0.999 4). The precision, repeatability and stability of the tested samples were good within 48 h. The recovery rate were 94.9%-105.2% and the RSDs were 0.50%-5.2%. The results of content determination showed that the contents of taurocholic acid, cholic acid, porcine cholic acid, glycine cholic acid and porcine deoxycholic acid in Niuhuang Shedan Chuanbei liquid were higher, and there were great differences among different manufacturers. Cluster analysis could divided the samples into three categories. It was found that the contents of taurocholic acid, 3-oxocholic acid, 3α-hydroxy-7-oxo-5β-cholanic acid, 12-oxocholic acid and cholic acid in class Ⅱ were significantly higher than those in class Ⅱ and Ⅲ, and the content of porcine cholic acid in class Ⅲ was significantly higher than that in class Ⅰ and Ⅱ. Conclusion: This method is convenient, efficient, accurate and sensitive, and can be used for the determination of 22 cholic acids in Niuhuang Shedan Chuanbei liquid.
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