Abstract
BACKGROUND: As one of the most famous natural products, salvianolic acid A (SAA) is undergoing clinical trials for the treatments of angina pectoris and coronary heart disorders. However, the in vivo metabolites of SAA have only been tentatively identified, leading to a barrier for precise therapeutical drug monitoring. METHODS: Ultra-high performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry (UPLC-Qtof-MS/MS) was firstly employed to acquire high-resolution MS(1) and MS(2) spectra for all metabolites. Through paying special attention onto the features of ester bond dissociation, metabolism sites were restricted at certain regions. To further determine the metabolism site, such as the monomethylated products (M23, M25, and M26), post collision-induced dissociation energy-resolved mass spectrometry (post-CID ER-MS) was proposed through programming progressive exciting energies to the second collision chamber of hybrid triple quadrupole-linear ion trap mass spectrometry (Qtrap-MS) device. RESULTS: After SAA oral administration, 29 metabolites (M1-M29), including five, thirteen, and sixteen ones in rat plasma, urine, and feces, respectively, were detected in rats. The metabolism route was initially determined by applying well-defined mass fragmentation pathways to those HR-m/z values of precursor and fragment ions. Metabolism site was limited to SAF- or DSS-unit based on the fragmentation patterns of ester functional group. Through matching the dissociation trajectories of concerned 1(st)-generation fragment ions with expected decomposition product anions using post-CID ER-MS strategy, M23 and M25 were unequivocally assigned as 3'-methyl-SAA and 3''-methyl-SAA, and M26 was identified as 2-methyl-SAA or 3-methyl-SAA. Hydrolysis, methylation, glucuronidation, sulfation, and oxidation were the primary metabolism channels being responsible for the metabolites' generation. CONCLUSION: Together, the metabolism regions and sites of SAA metabolites were sequentially identified based on the ester bond dissociation features and post-CID ER-MS strategy. Importantly, the present study provided a promising way to elevate the structural identification confidence of natural products and metabolites.