評估液相層析條件對代謝體學於塑膠微粒毒性研究的影響

Abstract

近年來，塑膠微粒汙染成為國際關注的議題，許多文獻指出生物體攝入塑膠微粒後，會導致炎症、細胞壞死等毒性影響。因此，眾多研究正在探討這些毒性背後的運作機制。液相層析-質譜聯用(LC-MS)是一種強大的工具，適用於疾病相關差異代謝物的非靶向和靶向分析。然而，不同LC條件之間缺乏系統比較，限制了分析效率。在本研究中，我們使用了Amide、Silica、Obelisc N、CN、F5和C18六種管柱，並使用甲酸、乙酸、甲酸銨、乙酸銨、氫氧化銨五種移動相添加劑，綜合出十六種層析條件，探討了不同LC條件對小鼠中受塑膠微粒毒性影響的代謝物的影響。我們將研究分為電極模式、管柱、添加劑三大部分進行討論。總體而言，RPLC在負電模式下增強了代謝物的鑑定，並檢測到更多的碳水化合物和脂類。而HILIC管柱在正離子模式下顯示出更強的檢測能力，增加了胺基酸、核苷酸和有機酸的檢測。我們也發現，使用甲酸作為添加劑比乙酸能夠得到更好的峰型。最後，我們結合了HILIC和RPLC兩種層析模式，獲得代謝物總和的最大值，找出了小鼠攝入塑膠微粒後產生的差異代謝物。本研究強調了優化LC條件的重要性，揭示了塑膠微粒在生物系統中對生化影響的顯著見解，並成功找出了其毒性影響。In recent years, plastic microbead pollution has become an issue of international concern. Studies indicate that ingesting plastic microbeads can cause toxic effects such as inflammation and cell necrosis. As a result, researchers are investigating the mechanisms behind these effects. Liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for both untargeted and targeted analysis of disease-related metabolites, but the lack of systematic comparisons between different LC conditions limits efficiency. In this study, we used six columns (Amide, Silica, Obelisc N, CN, F5, and C18) and five mobile phase additives (formic acid, acetic acid, ammonium formate, ammonium acetate, and ammonium hydroxide), creating sixteen chromatographic conditions. We examined the effects of these conditions on metabolites in mice affected by microplastic toxicity, focusing on electrode mode, column type, and additives. RPLC in negative ion mode enhanced metabolite identification and detected more carbohydrates and lipids. In positive ion mode, HILIC columns increased the detection of amino acids, nucleotides, and organic acids. Formic acid as an additive resulted in better peak shapes than acetic acid. Combining HILIC and RPLC modes maximized the identification of differential metabolites in mice after ingesting plastic microbeads. This study underscores the importance of optimizing LC conditions and provides significant insights into the biochemical impacts of microplastics.