An enhanced chemical profiling characterization strategy integrating off-line two-dimensional counter-current chromatography × UHPLC-MS/MS and feature-based molecular networking: Comprehensive characterization of the fibrous root of Bletilla striata as a case study.

This study establishes a multidimensional analytical platform combining two-dimensional counter-current chromatography (CCC) × ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with feature-based molecular networking (FBMN) for systematic phytochemical profiling of Bletilla striata fibrous root extract. The orthogonal separation system employed a biphasic solvent system (n-hexane/ethyl acetate/methanol/0.1% formic acid aqueous, 9:11:10:10, v/v) in the first dimension CCC, followed by UHPLC-MS/MS analysis for the secondary separation and characterization. This configuration achieved 80.47% spatial coverage. Total peak capacity was evaluated, and approximately 1328 peaks were obtained through the comprehensive off-line 2D CCC × UHPLC-MS/MS separation. Data-dependent acquisition mode (DDA) generated high-fidelity MS2 spectra, enabled comprehensive structural annotation through visualization, through an integrated workflow combining an in-house database, an online database, and a FBMN analysis. The methodology identified 263 compounds through targeted database matching, with FBMN analysis revealing 15 additional compounds (10 potentially novel natural product and 5 previously unreported in B. striata). Systematic classification identified 142 phenanthrenes, 37 bibenzyls, 16 anthraquinones, and 68 other compounds. Providing a more extensive chemical inventory reported for the fibrous root of B. striata to date (2D CCC × UHPLC-MS/MS 278 total vs. 1D UHPLC-MS/MS 80 total). Advanced visualization tools, including molecular networking and 2D contour plotting, provided more profound insight into structural relationships and chromatographic retention behaviors. This CCC × UHPLC-MS/MS-FBMN platform establishes a new paradigm for comprehensive phytochemical analysis with a broad applicability to natural product research and quality control applications.