Enhancing metabolite coverage using dedicated mobile phases for individual polarity modes in HILIC-MS

We have developed a new hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method with mobile phases optimized for high metabolite coverage in individual polarity modes. This dual mobile phase strategy expands the range of annotated metabolites and improves identification confidence, providing broader and more accurate metabolic profiles. The incorporation of a bioinert chromatographic system further enhances sensitivity. The bridged ethyl hybrid (BEH) amide column yields the best results for metabolomic analysis among the three chromatographic columns in this comparison. The method development involves investigating the effects of mobile phase composition, pH, and a medronic acid additive on the MS response under bioinert chromatographic conditions. The results highlight the important role of alkaline pH for the sensitive detection of polyphosphorylated metabolites, while demonstrating the redundancy of chelating additives in a fully bioinert system. Finally, the optimized method is applied to mouse plasma, pancreas, and liver samples to demonstrate its versatility and reliable performance in complex biological matrices, establishing it as a powerful tool for confident and reproducible metabolomics studies.本研究开发了一种新型亲水作用色谱-质谱联用（hydrophilic interaction liquid chromatography-mass spectrometry, HILIC-MS）方法，其流动相针对各单一极性模式分别优化，以实现高代谢物覆盖度。该双流动相策略可拓宽可注释代谢物的覆盖范围，提升代谢物鉴定的置信度，从而获得更全面且精准的代谢组学图谱。引入生物惰性色谱系统可进一步提升检测灵敏度。在本次对比的三款色谱柱中，桥联乙基杂化（BEH）酰胺色谱柱在代谢组学分析中表现最优。本方法的开发过程中，我们在生物惰性色谱条件下，考察了流动相组成、pH值以及甲膦酸（medronic acid）添加剂对质谱响应的影响。研究结果显示，碱性pH环境对多磷酸化代谢物的灵敏检测至关重要，同时证实了在完全生物惰性系统中，螯合剂添加剂并无必要。最后，我们将优化后的方法应用于小鼠血浆、胰腺及肝脏样本，验证了其在复杂生物基质中的通用性与可靠性能，使其成为可实现高置信度、可重复代谢组学研究的有力工具。