Snake bile is one of the most expensive traditional Chinese medicines (TCMs). However, due to the complicated constitutes of snake bile and the poor ultraviolet absorbance of some trace bile acids (BAs), effective analysis methods for snake bile acids were still unavailable, making it difficult to solve adulteration problems. In present study, ultrahigh-performance liquid chromatography with triple quadrupole linear ion trap mass spectrometry (UHPLC-QqQ-MS/MS) was applied to conduct a quantitative analysis on snake BAs. The mass spectrometer was monitored in the negative ion mode, and multiple-reaction monitoring (MRM) program was used to determine the contents of BAs in snake bile. In all, 61 snake bile from 17 commonly used species of three families (Elapidae, Colubridae and Viperidae), along with five batches of commercial snake bile from four companies, were collected and detected. Nine components, Tauro-3α,12α-dihydroxy-7-oxo-5β-cholenoic acid (T1), Tauro-3α,7α,12α,23R-tetrahydroxy-5β-cholenoic acid (T2), taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), cholic acid (CA), Tauro-3α,7α-dihydroxy-12-oxo-5β-cholenoic acid (T3), and Tauro-3α,7α,9α,16α-tetrahydroxy-5β-cholenoic acid (T4) were simultaneously and rapidly determined for the first time. In these BAs, T1 and T2, self-prepared with purity above 90%, were first reported with their quantitative determination, and the latter two (T3 and T4) were tentatively determined by quantitative analysis multi-components by single marker (QAMS) method for roughly estimating the components without reference. The developed method was validated with acceptable linearity (r² ≥ 0.995), precision (RSD ＜ 6.5%) and recovery (RSD ＜ 7.5%). It turned out that the contents of BAs among different species were also significantly different; T1 was one of the principle bile acids in some common snake bile, and also was the characteristic one in Viperidae and Elapidae; T2 was the dominant components in Enhydris chinensis. This quantitative study of BAs in snake bile is a remarkable improvement for clarifying the bile acid compositions and evaluating the quality of snake bile.

蛇胆汁是最昂贵的中药之一。然而，由于蛇胆汁的成分复杂，某些痕量胆汁酸（BAs）的紫外线吸收性差，因此仍缺乏有效的蛇胆汁酸分析方法，难以解决掺假问题。在本研究中，采用三重四极杆线性离子阱质谱（UHPLC-QqQ-MS / MS）超高效液相色谱法对蛇毒BA进行定量分析。以负离子模式监测质谱仪，并使用多反应监测（MRM）程序确定蛇胆中BA的含量。共有来自三个科的17种常用物种的61种蛇胆汁，分别为蛇形科，蛇形科和蛇形科，以及来自四家公司的五批商业蛇胆被收集和检测到。九种成分，Tauro-3α，12α-dihydroxy-7-oxo-5β -cholenoic酸（T1），牛磺3α，7α，12α，23 - [R -四5 β -cholenoic酸（T2），牛磺胆酸（TCA），甘氨胆酸（GCA），牛磺鹅去氧酸（TCDCA），牛磺脱氧胆酸（TDCA），胆酸（CA），牛磺3α，7α二羟基-12-氧代-5- β -cholenoic酸（T3），以及牛磺3α，7α，9α，16α四羟基-5- β -cholenoic酸（首次同时快速确定T4）。在这些BA中，首先报告了自行制备的纯度高于90％的T1和T2，并对其进行了定量测定，而后两种（T3和T4通过单一标记（QAMS）方法对多组分进行定量分析，初步确定了这些组分，以粗略估算组分，而无需参考。所提出的方法以可接受的线性关系（r进行了验证²  ≥0.995），精密（RSD <6.5％）和回收率（RSD <7.5％）。事实证明，不同物种之间BA的含量也存在显着差异。T1是某些常见蛇胆中主要的胆汁酸之一，也是蛇蝎科和蛇蝎科的特征性胆汁酸。T2是中华hydr中的主要成分。对蛇胆中BA的定量研究对于澄清胆汁酸成分和评估蛇胆质量具有显着改善。