以氰丙基液相層析質譜技術精進目標代謝體學與途徑解析

Abstract

代謝體學探討生物體內小分子代謝物的組成，透過高效能液相層析串聯式質譜儀定性與定量分析，獲取全面性的代謝途徑變化，有助於疾病早期診斷及發現有效治療策略。本研究開發並應用氰丙基層析法，透過調整環境pH值，使目標物在5分鐘內達到良好分離，並成功建立生物分子分析平台。該平台探索了51種代謝物，涵蓋廣泛的生物途徑，包括核苷和核苷酸、氧化還原代謝體、糖解途徑、戊糖磷酸途徑、嘌呤合成途徑、氨基酸以及與神經疾病相關之代謝物。此外，透過在移動相中添加檸檬酸來防止分析物吸附在金屬表面，從而改善波峰形狀並提高靈敏度。質譜系統採用正負模式電噴灑游離法與多重反應監測模式。生物分析平台良好的精密度（RSD%< 15.1%）與準確度（回收率 81.3 - 117.8%），線性範圍1.0 - 2000 ng mL⁻¹，相關係數r > 0.99，偵測極限0.1至10 ng mL⁻¹，定量極限0.1至25 ng mL⁻¹。該平台應用於細胞、腦組織、血液與蜂王漿等檢體，以評估基質效應並驗證平台的可靠性。接著應用於dSH-SY5Y細胞之代謝分析，顯示在神經毒素6-OHDA的環境下，導致細胞內粒線體損傷，進而引起細胞內氧化壓力增加以及能量ATP減少，驗證其可行性與可靠性。接著進一步探討ENERGI在SH-SY5Y細胞與小鼠腦中的神經保護效果，展現其在疾病研究中的潛在應用。該生物分子分析平台具快速、高效且符合綠色化學原則，適用於食品、細胞、組織及生物流體的代謝物分析，成為解析生物體內代謝途徑的強大工具。Metabolomics is the comprehensive study of small molecules involved in metabolic pathways, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to explore dynamic metabolite profiles. In this study, a cyanopropyl (CN) liquid chromatography method was developed and optimized for efficient metabolite separation. The CN-based LC-MS/MS platform enabled the quantification of 51 metabolites within 5 minutes through pH adjustment, covering wide biological pathways including nucleosides, nucleotides, the oxidative-redox metabolome, glycolysis, the pentose phosphate pathway, purine de novo synthesis, amino acids, and metabolites associated with neurological disorders. Additionally, citric acid was added as an anti-adsorption agent to improve peak shapes and sensitivity. The MS system used electrospray ionization (ESI) in positive and negative modes with scheduled multiple reaction monitoring (sMRM) for quantitation. The method demonstrated excellent linearity (r> 0.99) across the range of 1.0 to 2000 ng mL⁻¹, with detection limits from 0.1 to 10 ng mL⁻¹ and quantification limits from 0.1 to 25 ng mL⁻¹. Recovery ranged from 81.3% to 117.8%, with relative standard deviations (RSD) <15.1%. The bioanalytical platform was validated using various biological and food-related samples, including cells, brain tissue, blood, and royal jelly. Subsequently, analysis of dSH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) confirmed the reliability of the bioanalytical platform by revealing mitochondrial damage and alterations in key metabolic pathways. The study further investigated the neuroprotective effects of ENERGI in SH-SY5Y cells and mouse brain tissue, providing valuable insights into the application of metabolomics in disease mechanism studies and therapeutic development.