Optimizing Analytical Conditions and Creating a Multidimensional Reference Library for Over 300 Bile Acids

Bile acids are increasingly recognized as pivotal regulators of macronutrient metabolism (lipids, carbohydrates, proteins) and systemic inflammatory homeostasis. Their metabolic and signaling pathways are essential for maintaining health, with dysregulation contributing to cardiometabolic, inflammatory, and neoplastic disorders. Thus, the accurate and reliable identification of the many bile acid isomers is essential to understand their distinct biological functions and diagnostic potential. To date, conventional analytical workflows combining liquid chromatography and mass spectrometry (LC-MS) struggle to differentiate bile acid isomers due to frequent chromatographic coelution, identical precursor masses and often indistinct fragmentation spectra. Here, we compared different analytical parameters in a multidimensional workflow and created a library to improve the resolution and identification of over 300 bile acids, including unconjugated and conjugated versions such as the recently discovered microbially conjugated bile acids (MCBAs). Specifically, we systematically optimized and evaluated extraction protocols for the bile acids in stool, serum and plasma samples and determined advantages and caveats to different LC conditions and MS source ionization modes.

胆汁酸（bile acids）日益被认定为宏量营养素代谢（脂质、碳水化合物、蛋白质）与全身性炎症稳态的关键调控因子。其代谢与信号通路对维持机体健康至关重要，通路失调可引发心代谢紊乱、炎症性疾病及肿瘤性疾病。因此，精准可靠地鉴定众多胆汁酸异构体，是解析其独特生物学功能与诊断潜力的必要前提。迄今为止，常规采用液相色谱与质谱联用（liquid chromatography and mass spectrometry, LC-MS）的分析流程难以有效区分胆汁酸异构体，其原因包括频繁出现的色谱共洗脱现象、前体离子质量一致以及通常辨识度低下的裂解谱图。本研究通过多维度分析流程对比了多种分析参数，并构建了一套胆汁酸鉴定数据库，以提升300余种胆汁酸的分离分辨率与鉴定准确率，涵盖游离型与结合型胆汁酸，例如新近发现的微生物结合型胆汁酸（microbially conjugated bile acids, MCBAs）。具体而言，本研究系统优化并评估了粪便、血清及血浆样本中胆汁酸的提取方案，并明确了不同液相色谱条件与质谱源电离模式的优势与局限性。