The current study presents a comprehensive analysis to explore the compositions of both the supernatant and naturally-occurring precipitate of Huanglian Jiedu decoction employing ultra-high-performance liquid chromatography hyphenated with quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). Totally 109 constituents (32 alkaloids, 39 flavonoids, 12 iridoids, 9 phenolic acids, and 17 other compounds) were identified from accurate-mass measurements in full-scan MS/data-dependent MS/MS mode of acquisition. Furthermore, a quantitative method was developed for determination of 14 marker compounds in Huanglian Jiedu decoction. Experimental results revealed that all of these marker compounds were present in both the supernatant and naturally-occurring precipitate. Most notably, the contents of baicalin and berberine were significantly higher in the naturally-occurring precipitate than supernatant, presumably due to self-assembly complexation. The formation of the baicalin/berberine complex was comprehensively investigated by electrospray ionization (ESI)-MS, nuclear magnetic resonance (NMR), ultraviolet-visible (UV–vis), Fourier transform infrared (FTIR), and fluorescence spectroscopy, etc. The morphology and size distribution of the baicalin/berberine self-assembled nanoparticles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). This study provides fundamental scientific evidence of the presence of a self-assembled phytochemical complex in the naturally-occurring precipitate, enabling better understanding of Huanglian Jiedu decoction.
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