Parallel derivatization strategy for comprehensive profiling of unconjugated and glycine-conjugated bile acids using...

Bile acids (BAs) are steroidal compounds that play important roles in the occurrence and development of liver injury. However, comprehensive characterization of BAs was rarely reported due to the limitations of both standards access and detection sensitivity. In this study, a parallel derivatization strategy was established for the sensitive and comprehensive profiling of unconjugated and glycine-conjugated BAs by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Two structural analogues 2-hydrazinyl-4,6-dimethylpyrimidine (DMP) and 2-hydrazinylpyrimidine (DP) were used as the parallel derivatization reagents for BAs labeling, facilitating the improvements of both detection sensitivities and chromatographic performances. The derivatization reactions can be completed in 20 min at room temperature, with derivatization efficacy higher than 99 %. Through derivatization, the sensitivity of BAs increased dozens or hundreds of times compared to their non-derivatized forms. Due to the structural similarities of derivatized BAs, general MS parameters can be forged for the analysis of DMP and DP labeled BAs. In addition, the DP labeled BAs were incorporated into the DMP derivatized biological samples for both the discovery and comprehensive characterization of BAs. Retention time shift (RTS) and peak area ratio (PAR) induced by the parallel DMP and DP labeled BAs were used for the rapid identification of BAs from complex biological samples. More than 200 BAs were profiled in rat serum using this parallel derivatization strategy. Further, the new strategy was successfully implemented in BAs profiling of serum samples from tripterysium glycosides-induced liver injury rat model. The disturbance of the BA metabolism network was further interpreted.