Two Standards-Aided LC-MS Technologies Uncover 30 "Unreported" Compounds in Natural Bear Bile Powder to Add a New Pharmacopoeia Quality-Marker.

Natural bear bile powder is a precious traditional Chinese medicine that has a long history of use in clinical practice. The current study analyzed four batches of natural bear bile powder samples (B-1∼B-4) using standards-aided ultra-high-performance liquid chromatography-quadrupole-Orbitrap-mass spectrometry and ultra-performance liquid chromatography coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry. A total of 37 compounds (1-37) were accurately identified, while 30 "unreported" compounds were found in natural bear bile powder for the first time. In particular, two "unreported" isomers (2'-hydroxydaidzein and genistein) were strictly distinguished by means of ΔR.T. matching in chromatographic stick-peak and MS/MS peak matching. The quantification work indicated that taurochenodeoxycholic acid (TCDCA) and its epimer tauroursodeoxycholic acid (TUDCA) had the highest contents in natural bear bile powder. However, TCDCA showed different binding energy towards pregnane X receptor from TUDCA in the molecular docking simulation. Based on the analytical chemistry, molecular docking simulation, and documentary evidence, TCDCA was recommended as a potential Q-marker for pharmacopoeia. The new Q-marker was validated to be feasible by analysis of 11 batches of natural bear bile powder samples (B-5∼B-15), and the specific ratio of TCDCA/TUDCA (0.58∼0.98) can distinguish natural BBP from artificial BBP and other animal biles. All these findings not only lay a foundation for manufacturing technique improvement of artificial natural bear bile powder, but also promote pharmacopoeia to update the quality-control method.