透過兩階段策略以二維液相層析-串聯式質譜儀進行血漿三酸甘油脂分子組成分析

Abstract

三酸甘油脂 (Triglycerides) 是脂質分類中甘油脂的一種，在哺乳類的代謝路徑中飾演著重要的角色，通常除了肝臟能生產三酸甘油脂以外，也能藉由攝食消化其他生物體轉化成所需的三酸甘油脂，它能幫助我們調節脂肪和血糖之間的可逆變化。 在過去有許多研究都指出血漿中的三酸甘油脂是疾病的一項高風險因子，當血漿中三酸甘油脂濃度異常時更被視為許多疾病 (如心血管疾病、第二型糖尿病、代謝症候群等) 的一個警訊，此外，也有研究指出血清中三酸甘油脂含量的改變也是肝毒性的徵兆。探討個體中三酸甘油脂的種類及濃度變化能幫助我們更清楚其與不良健康效應之間的關係，本次實驗目的就是針對三酸甘油脂建立有效的分析方法。本實驗所建立的方法主要由二維液相層析質譜儀搭配兩階段分析策略來完成，在液相層析部分，我們在C18管柱前方串聯Hilic管柱來分離不同的脂質族群以降低基質效應的影響，在第一階段定性分析策略中針對50個重要的三酸甘油脂脂肪酸鏈來進行中性分子丟失掃描分析ICR公鼠的脂質萃取物，第二階段則是利用「預定時間-多重反應監測」來進行三酸甘油脂的半定量分析。為了驗證這套分析方法，我們檢驗其的校準曲線、偵測極限及範圍、添加回收測試、再現性分析還有測試Hilic管柱串聯與否的效果差異，最後以此分析方法分析暴露萘後的小鼠樣本來測試應用性。本研究建立的分析方法能有效地針對生物樣本中的三酸甘油脂進行分析，並且可以應用在血漿或是血清的樣本分析。

Triglycerides, one of the major lipid classes, plays an important role in metabolic pathway in mammals. Plasma triglyceride levels were used as a risk factor for monitoring certain health status. Unusual levels of plasma triglyceride become a sign of cardiovascular disease, type 2 diabetes or metabolic syndrome. Moreover, changed serum triglycerides were often associated with liver dysfunction caused by toxicants. Consequently, triglyceride profiling can help to examine the roles of triglyceride species in adverse health effects more completely than before. The objectives of the present study were to optimize an analytical method for triglycerides from lipid extracts. The analytical method was conducted by a two-stage mass spectrometry experiments coupled with two-dimensional chromatography strategy. Hilic column was connected with C18 in series for separation lipid classes. 50 major fatty acids were chosen to apply neutral loss scans for composition identification at stage 1 and then scheduled-multiple reaction monitoring (sMRM) was employed for triglyceride quantitation at stage 2 for serum lipid extracts from ICR male mice. We validated the performance by linearity, detection limit, reproducibility, recovery rate, and the effects of Hilic column connected. Further, three serum samples from mice treated with low or high doses of naphthalene (100 mg/kg or 200mg/kg, ip) or the control (with olive oil only) were used to examine the practical usability of the analytical method. The principle component analysis, orthogonal projections to latent structures discriminant analysis, and ANOVA were used for statistical analysis. The results show that the analytical method can analyze triglycerides in lipid extract efficiently. It can be applied to examine triglycerides in both serum and plasma samples.