Systematic Analysis of Chemical Constituents in Shenzhuo Decoction Based on Iterative Ultra‐High Performance Liquid Chromatography Coupled With Quadrupole Time‐of‐Flight Tandem Mass Spectrometry and Multidimensional Data Annotation

Shenzhuo Decoction (SZT), a classical traditional Chinese medicine (TCM) formula from Synopsis of the Golden Chamber (Jinkui Yaolue), demonstrates clinical efficacy in treating ankylosing spondylitis (AS); however, its systematic chemical profiling remains insufficient. This study established an integrated strategy for chemical constituent analysis of SZT, combining iterative ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF MS/MS) with multidimensional data annotation. The iterative UHPLC-Q-TOF MS/MS technology employed 20 eV/40 eV dual collision energies, along with three iterative scans and a dynamic exclusion method. The multidimensional data annotation for SZT analysis was derived from an internal database containing 997 constituents, combined with Progenesis QI and SIRIUS. A total of 130 constituents were identified from SZT using the above methods. Compared with a conventional LC-MS identification method (single 20 eV collision energy, no iterative scanning), the iterative strategy identified an additional 33 constituents from SZT, indicating a 34% improvement in detection sensitivity. More importantly, network pharmacology validation revealed that three of the additionally identified constituents (medicarpin, licochalcone A, and acetoxy-methylisoflavone) exhibited high anti-AS pharmacological activity (ranking among the top ten in the 130-component scoring values of SZT), further demonstrating that constituents identified via the iterative strategy more comprehensively reflect the material basis of SZT's pharmacological effects. This strategy establishes a foundation for research into the chemical constituents of SZT and provides a reliable paradigm for complex TCM profiling.