Analysis of bile acids by ultra-high performance convergent chromatography (UPC 2 ) in series ESI-MS/MS
Kaori Taguchi, Eiichiro Flukusaki and Takeshi Bamba
1 Waters Corporation (Milford, MA, USA)
2 Department of Biotechnology, Osaka University, Osaka, Japan
purpose
A new method for rapid bile acid analysis and quantification was established using UltraPerformance Convergence ChromatographyTM (UPC 2 TM) ultra-high performance convergent chromatography.
background
As a signaling molecule, bile acids play an important role in regulating the metabolism of triglycerides, cholesterol and glucose. These signaling pathways have become targets for the development of drugs for the treatment of metabolic diseases. In addition, bile acids can also be used as biomarkers in serum to reveal metabolic mechanisms of liver disease and bile acid regulation. Therefore, it is important to establish an analytical method for measuring bile acids in the body.
The separation of bile acids is particularly complicated by the presence of structural analogs (like isomers) and the difference in polarity between unbound and bound bile acids. In the past, researchers used gas chromatography (GC) and liquid chromatography (LC) to analyze these compounds, but these methods have certain limitations. The GC method usually requires a cumbersome derivatization step, and a certain amount of bile acid is lost in each step of the derivatization. In addition, when the composition or concentration of the bound bile acid is determined by GC analysis, several aliquots of the sample are separately extracted.
Using an ultra-high performance convergent chromatography tandem ESI-MS/MS system, 25 bile acids can be analyzed simultaneously in 13 min. 
Figure 1. Simultaneous analysis of 25 bile acid standard mixtures in 13 min, including glycine and taurine conjugates. The names of the compounds in each MRM chromatogram are indicated in order of retention time.
Figure 2. Quantification of bile acids in rat serum using the UPC 2 /MS system (n=6).
Although LC analysis can detect both bound and unconjugated bile acids, the single needle analysis time is up to 30 min. In this application note, we will introduce a UPC 2 /MS method for rapid bile acid analysis and quantification.
solution
This experiment used the Waters® ACQUITY UPC 2 TM system to establish a method for simultaneously separating 25 different bile acids (including combinations thereof) with supercritical carbon dioxide (SCCO 2 ) as the main mobile phase. Using SCCO 2 as the mobile phase accelerates the diffusion of analytes and reduces back pressure, which reduces analysis time. To increase the sensitivity and specificity of the analysis, we used the ACQUITY UPC 2 system with the Xevo® TQ-S mass spectrometer. The results of the separation of 25 different bile acids (including glycine and taurine conjugates) are shown in Figure 1.
All bile acids were separated within 13 min, and the analysis time was reduced by a factor of about 2 compared to previous assays. The method is optimized using a combination of different stationary phases, column temperature settings, additives, and modifiers of different pH values. The Xevo TQ-S's detection mode is set to ESI negative ion mode for maximum sensitivity without the use of makeup gas.
This method was used to quantitatively analyze bile acids and their conjugates in actual biological samples (rat serum). The purchased rat serum was deproteinized using methanol at a ratio of methanol: serum 3:1 (v/v), followed by vortexing and centrifugation. The supernatant was injected into UPC 2 /MS/MS to determine the concentration of each bile acid and its conjugate in the serum sample. The results are shown in Figure 2, indicating that the method can determine the levels of bile acids and their conjugates in the biological matrix with good reproducibility.
to sum up
Ultra-high performance convergent chromatography (UPC 2 ) tandem ESI-MS/MS system can simultaneously analyze 25 bile acids and their combinations in biological matrix. Separation of all analytes was achieved within 13 min using a column with sub-2 μm packing, and the resolution was satisfactory. This application note demonstrates that this new separation method is ideal for evaluating the effects of bile acids on triglyceride, cholesterol and glucose metabolism, and for the development of novel drug targets for the treatment of metabolic diseases.
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