開發連線固相萃取液相層析串連質譜儀搭配同位素稀釋法分析尿液中的九種亞硝胺

亞硝胺 (N-nitrosamines) 為人類可能的致癌物質，常存於食物、飲用水及環境中。人體可藉由食入或吸入的途徑暴露亞硝胺。除了外在暴露，亞硝胺也可藉由體內硝酸鹽轉換為亞硝酸鹽，並與體內的二級胺反應生成內生性亞硝胺。本研究開發利用連線固相萃取 (on-line solid- phase extraction) 液相層析串聯質譜儀 (LC-MS/MS) 搭配同位素稀釋法 (isotope-dilution) 分析尿液中的九種亞硝胺，分別為N-nitrosodimethylamine (NDMA)、N-nitrosomethylethylamine (NMEA)、N-nitrosopyrrolidine (NPyr)、N-nitrosodiethylamine (NDEA)、N-nitrosopiperidine (NPip)、N-nitrosomorpholine (NMor)、N-nitrosodi-n-propylamine (NDPA)、N-nitrosodi-n-buthylamine (NDBA) 及N-nitrosodiphenylamine (NDPhA)。同時評估並比較九種亞硝胺在電灑游離源 (electrospray ionization, ESI) 及大氣壓力化學游離源 (atmospheric-pressure chemical ionization, APCI) 下其分析敏感度。12 ml 尿液在添加同位素內標後，經活性碳管柱純化並以氮氣吹拂濃縮體積至0.1 ml，之後直接注射至on-line SPE LC-MS/MS進行分析。結果顯示 ESI 游離源下，NDMA、NMEA、NPyr、NDEA、NPip、NMor、NDPA、NDBA、NDPhA 的偵測極限分別為0.88、0.15、0.11、0.09、0.08、0.03、0.01、0.003、0.0004 fmol；而在游離源 APCI 下，NDMA、NMEA、NPyr、NDEA、NPip、NMor、NDPA、NDBA 偵測極限分別為0.04、0.03、0.04、0.04、0.09、0.002、0.02、0.008 fmol。因 NDPhA 為熱不穩定的亞硝胺，所以在 APCI 游離源下無法定量分析。本方法進一步應用於人體尿液分析。在44位健康受試者的尿液樣本，我們測得NDMA (0.7±0.52 ng/mg creatinine), NMEA (0.04±0.04 ng/mg creatinine), NDEA (0.17±0.09 ng/mg creatinine), NPyr (0.12±0.05 ng/mg creatinine), NDBA (0.024±0.007 ng/mg creatinine), NDPhA (0.09±0.13 ng/mg creatinine)，其他亞硝胺則是未檢測出(低於本方法偵測極限)。本研究建立之方法未來可應用於大量人體尿液樣本，以評估人體暴露亞硝胺的程度。

關鍵字

亞硝胺；液相層析串聯質譜儀；電灑游離源；大氣壓力化學游離源；同位素稀釋法

並列摘要

N-nitrosamines are a class of probable human carcinogens. They often present in food, drinking water and environment. Human exposed to N-nitrosamines through ingestion and inhalation. In addition to exogenous exposure, N-nitrosamines can also be formed endogenously inside the body from its precursors, nitrate or nitrite and secondary or tertiary amines. In this study, we developed an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) with on-line solid-phase extraction (on-line SPE) for a quantitative analysis of nine N-nitrosamines in human urine, namely N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPyr), N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPip), N-nitrosomorpholine (NMor), N-nitrosodi-n-propylamine (NDPA), N-nitrosodi-n-buthylamine (NDBA) and N-nitrosodiphenylamine (NDPhA). Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) ion sources were evaluated and compared for nine N-nitrosamines determination. A 12 ml of crude urine was added with isotopic internal standards, then purified by activated carbon and concentrated to 0.1 ml under a high purity nitrogen stream. A 10 μl aliquot of prepared sample was then directly injected into the on-line SPE LC-MS/MS. The results showed that in the ESI mode, limit of detection (LOD) for NDMA, NMEA, NPyr, NDEA, NPip, NMor, NDPA, NDBA, NDPhA were 0.88, 0.15, 0.11, 0.09, 0.08, 0.03, 0.01, 0.003, 0.0004 fmol, respectively, while in the APCI mode, the LOD for NDMA, NMEA, NPyr, NDEA, NPip, NMor, NDPA, NDBA were 0.04, 0.03, 0.04, 0.04, 0.09, 0.002, 0.02, 0.008 fmol, respectively. Because NDPhA is a thermally labile nitrosamine, it was unable to be quantitatively detected in the APCI mode. This method was further applied to measure urinary nine N-nitrosamines levels in 44 healthy subjects. The results showed that among nine N-nitrosamines only NDMA (0.7±0.52 ng/mg creatinine), NMEA (0.04±0.04 ng/mg creatinine), NDEA (0.17±0.09 ng/mg creatinine), NPyr (0.12±0.05 ng/mg creatinine), NDBA (0.024±0.007 ng/mg creatinine), NDPhA (0.09±0.13 ng/mg creatinine) were detectable, while the others were under LODs of our method. Overall, the present method would be useful to assess the human exposure to N-nitrosamines.

並列關鍵字

N-nitrosamines ； on-line SPE ； ESI ； APCI ； LC-MS/MS ； isotope-dilution

參考文獻

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開發連線固相萃取液相層析串連質譜儀搭配同位素稀釋法分析尿液中的九種亞硝胺

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