運用連線固相萃取-液相層析串聯質譜儀搭配同位素稀釋法分析嚼食檳榔者唾液中的檳榔特有亞硝胺

Determination of areca nut-specific nitrosamines in saliva of areca nut chewers by isotope dilution LC-MS/MS with automated solid-phase extraction

廖啟軒(Chi-Syuan Liao)

指導教授 ： 趙木榮

中山醫學大學/健康管理學院/職業安全衛生學系/碩士(2019年)

https://doi.org/10.6834/CSMU201900038

摘要

檳榔特有亞硝胺 (areca nut-specific nitrosamines, ASNAs)是由檳榔特有生物鹼經內生性亞硝化作用產生，且過去動物文獻中顯示部分檳榔衍生亞硝胺物質確定為具有致癌性的。早期文獻中使用GC-TEA量測ASNAs，其方法的敏感度佳，但特異性差。本研究成功建立連線固相萃取液相層析串聯質譜儀搭配同位素稀釋法，可快速準確及同時分析ASNAs：3-(methylnitrosamino)propionitrile (MNPN)、3-(methylnitrosamino)propanal (MNPA)、N-nitrosoguvacine (NGCI)、N-nitrosonipecotic acid (NNIP) 以及N-nitrosoguvacoline (NGCO)。MNPN、MNPA、NGCI、NNIP、NGCO的方法偵測極限 (LOD) 分別為0.34、0.48、0.45、0.05及0.17 ng/mL。本分析方法的組內變異 (intraday) 和組間變異 (interday) 之變異係數皆小於10 %，而ASNAs在低、中、高濃度的平均回收率分別介於98-102 %、86-104 %、69-128 %。
本研究將所開法方法應用於檳榔鹼亞硝化生成ASNAs的體外試驗，分別探討各種檳榔鹼及檳榔汁在中性環境、酸性環境及唾液中培育後是否會亞硝化生成ASNAs。結果顯示guvacine和guvacoline皆生成MNPA、NGCI及NGCO；arecoline生成MNPN及MNPA；arecaidine只生成MNPA；N-methylnipecotic acid生成MNPA及NNIP；檳榔汁經亞硝化反應可驗出NGCI及NGCO。本研究所發現各種檳榔鹼亞硝化生成ASNAs的路徑和過去文獻略有差異。我們也同時分析5位嚼食檳榔者的唾液樣本分析，結果只有2位檢測到MNPN，濃度分別為0.89、0.86 ng/mL，只有1位檢測到NGCO，濃度為4.04 ng/mL。我們是第一個研究測出台灣嚼食「未成熟檳榔」者唾液中的ASNAs，期望此研究方法未來能廣泛用於探討嚼食檳榔導致癌症的分子流行病學研究。

關鍵字

檳榔特有亞硝胺 ； 檳榔鹼 ； 亞硝化作用 ； 連線固相萃取 ； 液相層析串聯質譜儀 ； 唾液

並列摘要

Areca nut-specific nitrosamines (ASNAs) are produced from the endogenous nitrosation of areca nut-specific alkaloids. ASNAs have been classified as carcinogenic to animals. In the previous studies, ASNAs were mainly analyzed by GC-TEA. This method has high sensitivity but low specificity. In this study, we developed an isotope dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) method coupled with on-line solid phase extraction (SPE) for simultaneous detection of five ASNAs, including 3-(methylnitrosamino)propionitrile (MNPN), 3-(methylnitrosamino)propanal (MNPA), N-nitrosoguvacine (NGCI), N-nitrosonipecotic acid (NNIP) and N-nitrosoguvacoline (NGCO). The detection limits of this method are 0.34 ng/mL for MNPN, 0.48 ng/mL for MNPA, 0.45 ng/mL for NGCI, 0.05 ng/mL for NNIP and 0.17 ng/mL for NGCO. Inter- and intraday imprecision is < 10 % and mean recoveries of ASNAs in low, medium and high concentration are 98-102 %, 86-104 % and 69-128 %, respectively.
This method is further applied to investigate the ASNAs formation in the incubation of various areca nut-specific alkaloids (or areca nut juice) with nitrite and thiocyanate in the test medium (pH 7.0 or pH 2.1) or in the saliva. The results showed that both guvacine and guvacoline could produce MNPN, NGCI and NGCO; arecoline could produce MNPN and MNPA; arecaidine could produce MNPA; N-methylnipecotic could produce MNPA and NNIP; areca nut juice could also produce NGCI and NGCO after treating with nitrite. Our findings on the relationship between the areca nut-specific alkaloids and the resulting/corresponding ASNAs are inconsistent with previous studies. We also determined the ASNAs in five saliva samples of current areca nut chewers. The results showed that two chewers had a detectable concentration of MNPN (0.89 and 0.86 ng/mL) and one chewer had a detectable NGCO (4.04 ng/mL) in saliva.
This is the first study to detect the ASNAs in saliva of unripe areca nut chewers in Taiwan, and this method would be useful in molecular epidemiology study on cancer associated with the habit of chewing betel quid.

並列關鍵字

Areca nut-specific nitrosamines ； areca nut-specific alkaloids ； nitrosation reaction ； on-line SPE ； LC-MS/MS ； saliva

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