Comprehensive Profiling and Identification of C21 Steroids in the Root of Marsdenia Tenacissima (Dai-Bai-jie) Using Offline Two-Dimensional Chromatography (LC × SFC) with Q-TOF/MS

Dai-Bai-Jie, the root of the plant Marsdenia tenacissima from the Asclepiadaceae family, is well-known for its therapeutic effects in clearing heat, detoxifying, reducing swelling, and relieving pain as one of the most commonly used Dai medicine. Due to numerous structurally similar C21 steroidal compounds in Dai-Bai-Jie, chemical composition profiling has been substantially challenged. In this study, an offline two-dimensional chromatographic method (LC x SFC separation system) was developed to address these issues. Using the Hypersil Gold (1stD LC column) and 2-PIC (2ndD SFC column) based on 40 reference standards, the orthogonality was as high as 83.83 %. Most profiled ion peaks were tentatively identified through quadrupole time-of-flight mass spectrometry and a self-built compound virtual library. Consequently, the integrated method effectively addressed and resolved the issues associated with co-elution, thus significantly expanding the peak capacity. This advancement identified 362 C21 steroidal components, 319 of which were speculated to be potentially novel compounds. Furthermore, 86 groups of isomeric compounds were distinguished. This method provides a comprehensive understanding of chemical composition of Dai-Bai-Jie and an integrated qualitative analysis method for the C21 steroids.