关键词: 液相色谱-质谱联用技术, 导向分离, 角型吡喃香豆素, 代谢产物, 结构鉴定

Abstract:

Identifying the structures of metabolites is of great importance towards elucidating the therapeutic material basis of traditional Chinese medicines （TCMs）. However， current studies lack sufficient reference standards， hindering the structural identification of metabolites. This study establishes a strategy guided by the retention times and mass spectrometric information of microsomal metabolites. By coupling liquid chromatography-mass spectrometry （LC-MS） to trace and isolate target compounds from the raw materials， these compounds served as reference standards to confirm the structures of microsomal metabolites， thereby elucidating the metabolic pathways. Peucedani Radix （PR， Chinese name： Qianhu） consists of the dried roots of Peucedanum praeruptorum Dunn and contains abundant angular-type pyranocoumarins （APs）， which serve as primary chemical constituents and effective compound clusters as well. From the structural standpoint， C-3′ and C-4′ sites of APs are frequently substituted by acetoxyl， angeloxyl， isovaleroxyl and some other moieties， leading to the extensive occurrences of regio-isomers， which not only possess comparable structures but also similar chemical properties. Previous studies have preliminarily unveiled that APs primarily undergo hydrolysis and acyl migration in vivo， and these hydrolytic metabolites can be observed in both in vitro metabolism and the raw materials. In this study， a pair of regio-isomers from PR， i.e.， （+）-（3′S，4′S）-praeruptorin A （（+）-PA） and （+）-（3′R，4′R）-pteryxin （（+）-Pte）， was incubated separately with human liver microsomes （HLMs） in the presence of necessary cofactors to simulate the metabolic pathways occurring in vivo. After carefully processing LC-MS datasets and applying the well-defined mass fragmentation rules of APs， eight metabolites were detected and putatively identified for （+）-PA， including three hydrolysis products together with five oxidation products， whereas nine metabolites were observed and putatively identified for （+）-Pte， encompassing five hydrolysis products and four oxidation products. Noteworthily， phase I metabolism of （+）-Pte in HLMs was profiled for the first time. Various column chromatographic tools， such as silica gel column chromatography， C₁₈ reversed-phase chromatography （ODS）， Sephadex LH-20 and semi-preparative high performance liquid chromatography equipped with achiral or chiral columns， were employed to isolate compounds-of-interest from PR extracts under the guidance of LC-MS through taking both retention time and MS/MS information into account. A total of nine AP compounds were successfully purified from PR extracts， and notably， four pairs of AP enantiomers were involved. By matching ¹H- and ¹³C-NMR spectral information with the data archived in the literature， these compounds were identified as （3′S，4′S）-qianhucoumarin C （1a）， （3′R，4′R）-qianhucoumarin C （1b）， （3′S，4′S）-qianhucoumarin B （2a）， （3′R，4′R）-qianhucoumarin B （2b）， （3′S，4′S）-3′-angeloylkhellactone （3a）， （3′R，4′R）-3′-angeloylkhellactone （3b）， （3′S，4′S）-4′-angeloylkhellactone （4a）， （3′R，4′R）-4′-angeloylkhellactone （4b） and （3′S，4′S）-isoepoxypteryxin （5）. Thereof， compounds 1b and 2b represent new configurational structures of known planar structures. The study summarized mass fragmentation rules for APs and achieved accurate identification of two pairs of regio-isomers： qianhucoumarin B and C， and 3′- and 4′-angeloylkhellactone. Thereafter， in vitro metabolites of （+）-PA and （+）-Pte were successfully identified by comparing the retention times and MS/MS information with authentic compounds， including cis-khellactone， qianhucoumarin B， qianhucoumarin C， 3′-angeloylkhellactone， 4′-angeloylkhellactone and （3′S，4′S）-isoepoxypteryxin. Regarding the three hydrolytic metabolites， i.e.， cis-khellactone， qianhucoumarin B and qianhucoumarin C， they were shared by （+）-PA and （+）-Pte. Hydrolysis and oxidation reactions， as well as acyl migration， served as the primary metabolic pathways for APs in HLMs. In this study， （+）-PA and （+）-Pte were selected as representatives to demonstrate that it is feasible to obtain their metabolites from PR extracts through LC-MS-guided isolation and purification. After structural identification through analyzing ¹H- and ¹³C-NMR spectral information， these pure compounds were eligible authentic compounds enabling confirmative structural identification of metabolites， providing pronounced insights into the primary metabolic pathways of APs and offering an effective approach for the accurate identification of metabolite structures through isolating authentic compounds that are available in the original TCM. However， due to differences in metabolism between in vitro and in vivo， the study failed to construct a definitive structure-activity relationship for the metabolites. Future studies should be devoted to validating these findings through performing in vivo investigations.

Key words: liquid chromatography-mass spectrometry （LC-MS）, guided separation, angular-type pyranocoumarins, metabolites, structural identification