集成靶向和拟靶向策略测定健康及溃疡性结肠炎小鼠体内四逆散原型成分及代谢物

Determination of prototype components and metabolites of Si-Ni-San in healthy and ulcerative colitis mice by an integrated targeted and pseudo-targeted UPLC-QqQ-MS method

摘要

建立可同步定量四逆散原型成分并筛查其代谢物的分析方法，以阐明其在溃疡性结肠炎（UC）模型中的组织分布于代谢特征。为此，开发并验证了一种基于 UPLC-QqQ-MS 的集成分析策略，结合 12 种原型成分的靶向定量与基于离子对列表触发数据依赖采集的“拟靶向”代谢物筛查技术。样品经 80% 甲醇（100 mg/mL）提取并加内标处理后，采用 Waters Acquity UPLC HSS PFP 色谱柱，以乙腈-5 mmol/L 醋酸铵（含 0.1% 甲酸）为流动相进行梯度洗脱，在电喷雾离子源（ESI）多反应监测模式下分析。方法学验证显示各项参数符合生物样本分析要求。应用本方法发现，四逆散原型成分在 UC 小鼠结肠、肝及肾中的含量显著高于健康小鼠；且所有样本中Ⅱ相代谢物数量均明显多于Ⅰ相代谢物。结果表明，所建方法可靠，初步揭示了四逆散在 UC 状态下的组织分布特点及以Ⅱ相结合为主的代谢模式，为深入研究其效应靶组织与药效物质基础提供了方法学依据和数据支持。

Abstract

This study aimed to establish an analytical method capable of simultaneously quantifying the prototype components of Si-Ni-San and screening their metabolites to elucidate its tissue distribution and metabolic characteristics in an ulcerative colitis (UC) mouse model. To this end, an integrated analysis strategy based on UPLC-QqQ-MS was developed and validated, combining the targeted quantification of 12 prototype components with a pseudo-targeted metabolite screening technique based on ion pair list-triggered data-dependent acquisition. Samples were extracted with 80% methanol (100 mg/mL) and processed with internal standards. Separation was achieved on a Waters Acquity UPLC HSS PFP column using a gradient elution with a mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate containing 0.1% formic acid. Analysis was performed using an electrospray ionization (ESI) source in multiple reaction monitoring mode. Methodological validation confirmed that all parameters met the requirements for bio-sample analysis. Application of this method revealed that the content ofSi-Ni-San prototype components in the colon, liver, and kidneys of UC mice was significantly higher than that in healthy mice. Furthermore, the number of phase II metabolites was markedly greater than that of phase I metabolites in all tested samples. The results demonstrate the reliability of the established method and preliminarily reveal the tissue distribution characteristics of Si-Ni-San under UC conditions and its metabolism pattern dominated by phase II conjugation, which provides a methodological basis and experimental data for further in-depth research into its effective target tissues and pharmacodynamic material basis.

关键词

四逆散 / 溃疡性结肠炎 / 体内成分 / 靶向和拟靶向分析 / UPLC-QqQ-MS

Key words

Si-Ni-San / ulcerative colitis / in vivo components / targeted and pseudo-targeted analysis / UPLC-QqQ-MS

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基金资助

商丘医学高等专科学校开放课题(KFKT23002)