高解析質譜搭配同位素標定衍生化建立羰基體學平台：食用油分析初探

Development of Carbonylomics with Isotope-Coded Derivatization using High Resolution Mass Spectrometry: Preliminary Evaluation of Carbonyls in Cooking Oil

王苡蓁(Yi-Jhen Wang)

指導教授 ： 趙木榮

共同指導教授 ： 胡瓊文(Chiung-Wen Hu)

中山醫學大學/健康管理學院/職業安全衛生學系/碩士(2022年)

https://doi.org/10.6834/csmu202200051

摘要

本研究為科技部計畫MOST 109-2628-B-040-001及MOST 110-2628-B-040-002成果的一部分，係使用高解析度質譜 (High resolution mass spectrometry, HRMS) 四極柱/線性離子阱/軌道阱混成式質譜儀 (Q-LIT-Orbitrap MS)，透過2,4-Dinitrophenylhydrazine (DNPH) 衍生具羰基的醛酮標準品 (Carbonyl-d0/d3-DNPH) 在負電荷模式下探索碎裂機制，整合出48種羰基化合物的產物離子特徵，利用資料依賴動態訊號配對啟動產物離子掃描模式 (Dynamic peak-pairing and product ion triggering MS2 acquisition, DPP-PI-MS2) 建立新的羰基體學 (Carbonylomics) 技術。此方法基於d0-DNPH/d3-DNPH衍生化物，當MS1母離子間存在同位素的精確質量差 (3.0188 Da或6.0377 Da)，則啟動MS2碎裂並根據產物離子交 (聯) 集進行篩選，符合特徵離子的前驅母離子將重新啟動MS2以高能量碎裂獲得豐富的分子結構資訊。本研究成功將48種羰基化合物以d0-DNPH/d3-DNPH精確質量差進行配對，最終根據DNPH基團分別篩檢出單羰基 (monocarbonyls) 與雙羰基 (dicarbonyls) 化合物，此方法透過同位素配對能進一步降低方法偽陽性。
本研究運用建立完成的LC-Q-LIT-Orbitrap DPP-PI-MS2 掃描模式篩檢大豆油加熱前/後生成的羰基化合物。從羰基體學地圖 (Carbonylomic map) 呈現大豆油加熱後 (於180℃下，加熱30及120分鐘)，許多已知與未知的羰基化合物迅速生成。利用主成分分析 (Principal component analysis) 發現大豆油中羰基化合物的特徵指紋能將未加熱、加熱30及120分鐘，這3種樣本明顯分群。此外，透過統計發現trans,trans-2,4-Nonadienal為大豆油加熱裂解的良好指標。本研究所開發的羰基體學能廣篩加熱油中已知與未知的醛酮物，並為油品品質評估提供一項新的方法，未來將搭配其他技術進一步鑑定未知羰基化合物的結構。

關鍵字

羰基體學 ； 醛/酮 ； 食用油氧化 ； 四極柱/線性離子阱/軌道阱混成式質譜儀 ； 同位素標定衍生化

並列摘要

This study was part of two projects funded by the Ministry of Science and Technology, Taiwan [grant numbers: MOST 109-2628-B-040-001 and MOST 110-2628-B-040-002]. This study explored the fragmentation profile of 48 carbonyls (i.e., aldehydes and ketones) following 2,4-dinitrophenylhydrazine (DNPH) derivatization, using a liquid chromatography coupled with a quadrupole/linear ion trap/orbitrap tribrid mass spectrometer (LC-Q-LIT-Orbitrap MS) in a negative ion mode, and further established a novel Carbonylomics approach using a dynamic peak-pairing and product ion triggering MS2 acquisition (DPP-PI-MS2). DPP-PI-MS2 scan mode is based on the screening of both carbonyls- d0-DNPH and d3-DNPH derivatives. The MS2 fragmentation event will be triggered using collision energy dissociation (CID) if a pair of isotope-labeling precursor ion with an exact mass difference of 3.0188 Da (for monocarbonyl compounds) or 6.0377 Da (for dicarbonyl compounds) was detected. A second MS2 fragmentation event will be triggered using higher collision energy dissociation (HCD) upon observations of the specific product ions to obtain abundant fragments for structure analysis. Using the proposed Carbonylomics approach, this study successfully paired 48 carbonyls- d0-DNPH and d3-DNPH derivatives and identified both the monocarbonyl compounds and dicarbonyl compounds by the DNPH moieties. This “isotope peak pairing” method could further reduce the false positives.
The proposed Carbonylomics approach was applied to comprehensively detect the carbonyl compounds formed in the soybean oil after heating. The results showed that many known and unknown carbonyl compounds were rapidly formed after heating oil at 180 ℃ for 30 and 120 minutes, and was successfully visualized as the Carbonylome maps. Furthermore, principal component analysis was performed and revealed that all the characteristic carbonyl compounds formed in soybean oil could be grouped into three classes: unheated, heated for 30 and 120 minutes. The statistical analysis also demonstrated that trans,trans-2,4-Nonadienal could be a good indicator for the oxidation of edible oil. Numerous unknown carbonyl compounds were detected in this study and needed to be identified in the future study. Taken together, the present Carbonylomics approach using the LC-Q-LIT-Orbitrap MS with the DPP-PI-MS2 scan mode is able to comprehensively detect both the known and unknown carbonyls in oil, and could be a useful tool for assessing the quality of edible oil.

並列關鍵字

Carbonylomics ； aldehydes ； ketones ； oxidized edible oil ； Q-LIT-Orbitrap MS ； isotope-coded derivatization

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