Integrated post-acquisition data processing strategy for rapid steroid sulfate characterization in Toad gall-bladder

Abstract

LC-MS serves as a workhorse for chemical profile characterization of Chinese medicinal materials (CMMs) attributing to the ability of measuring fruitful MS/MS spectral information. However, it is laborious to extract the information belonging to the compounds-of-interest from the massive data matrixes even employing those well-defined post-acquisition data processing strategies. Here, efforts were devoted to propose an integrated strategy allowing rapid chemical homologs-focused data filtering through integrating the fit-for-purpose existing strategies, such as molecular weight imprinting (MWI), diagnostic fragment ion filtering (DFIF), neutral loss filtering (NLF), and isotope pattern filtering (IPF). Homologs-focused chemical characterization of a precious CMM namely Toad gall-bladder (Chinese name: Chandan) that is rich of diverse effective steroid sulfates, particularly bufogenin sulfates, bile acid sulfates and bilichol sulfates, was employed as a proof-of-concept. Recombinant human SULT2A1-catalyzed in vitro metabolism was undertaken to generate eight bufogenin sulfates to facilitate summarizing MS/MS spectral behaviors. After in-house data library construction and MS¹ and MS² spectral acquisition, data filtering was conducted as follows: 1) MWI and IPF was utilized in combination to capture deprotonated molecular ions and the ³⁴S isotopic ions for the sulfates of those reported steroids; 2) m/z 79.9568 (SO₃^-·) and 96.9596 (HSO₄^-) were applied to DFIF; and 3) SO₃ (79.9568 Da) served as the feature to achieve NLF. Those captured MS/MS information subsequently participated in tentatively structural annotation through applying those empirical mass fragmentation rules. As a result, 71 compounds including 7 bufogenin sulfates, 17 bile acid sulfates, 13 bilichol sulfates and a C-23 steroid sulfate were detected from Toad gall-bladder and thereof, 39 ones received plausible identities assignment. Above all, the steroid sulfates in Toad gall-bladder were profiled in depth, and more importantly, the proposed strategy should be a meaningful option for, but not limited to, submetabolome characterization in CMMs.

Keywords: (34)S isotopically molecular ion; Bilichol sulfate; Post-acquisition data processing strategy; Steroid sulfate; Toad gall-bladder.