液相層析串聯式質譜儀於砷、苯及多環芳香烴多重生物偵測分析之應用

Multiple Biomonitoring Analysis of Arsenic, Benzene and Polycyclic Aromatic Hydrocarbons by Using Liquid Chromatography Tandem Mass Spectrometry

林佳怡(Chia-Yi Lin)

指導教授 ： 沈振峯

國立虎尾科技大學/文理學院/生物科技系碩士班/碩士(2016年)

摘要

砷化物(arsenic)、苯(benzene)及多環芳香烴（polycyclic aromatic hydro- carbons, PAHs）為工業上三種主要的致癌物質，長期的環境曝露可能會導致癌症的發生。目前台灣的工業漸漸走向複合型工業，一個工廠可能會產生數個危害物質的環境汙染及對人體的曝露。以往對於此三類化合物檢驗方法多為單一檢測，本研究即欲建立多重致癌物質之同步生物分析方法以縮短分析上的時間並提供勞工及鄰近居民了解其曝露情形的方法。本研究起初嘗試針對上述三類致癌物在尿液中之主要代謝物進行偵測，其分別為單甲基砷酸 (Monomethylarsonic acid, MMAA)、二甲基砷酸(Dimethylarsonic acid, DMMA)、苯基硫醇酸(Sphenylmercapturic acid, S-PMA)、1-羥基焦腦油(1-hydroxypyrene, 1-OHP)等四個化合物。發現因代謝物極性差異甚大，因而選用逆相層析管柱Kinetex PFP 2.6 µm 100 x 2.1 mm，以於層析上提供適當的滯留。樣品前處理部分，使用4 mL尿液引入β-glucuronidase酵素水解，將1-OHP共軛物(glucuronid、sulfate)水解還原，接著以正己烷(含10%乙酸乙酯)進行第一次萃取1-OHP，再以氯仿和碳酸氫銨水溶液進行第二次萃取/浮冰反萃取可將MMAA、DMMA、S-PMA萃出。以添加尿液樣品對其他四個指標物進行方法確效，以經空白修正後面積對添加濃度計算檢量線，可達線性範圍MMAA為2-64 ng/mL、DMAA為1-64 ng/mL、S-PMA為0.78125-100 ng/mL及1-OHP為0.05-6.4 ng/mL，其檢量線均有良好的線性r2=0.99以上，測試偵測極限(S/N ratio≧3)：MMAA為0.117 ng/mL、DMAA為0.167 ng/mL、S-PMA為0.046 ng/mL及1-OHP為0.007 ng/mL，對其做方法確效，精密度(CV %, 3濃度, 6重複)範圍2.03-30.99%，而準確度(% Error, 3濃度, 6重複)達±30%以內。分析十個臨床尿液樣品，四個指標物(MMAA、DMAA、S-PMA及1-OHP)大多可清楚測得，測得指標物濃度範圍MMAA為N.D.(沒有偵測到)或0.92(外插法)-8.38 ng/mL，DMAA為0.63(外插法)-10.71 ng/mL，S-PMA為N.D.或0.08(外插法)-2.55 ng/mL，1-OHP為0.01(外插法)-0.17 ng/mL，均在文獻報導背景值範圍。

關鍵字

液相層析串聯式質譜儀 ； 砷 ； 苯基硫醇酸 ； 苯 ； 多環芳香烴碳氫化合物 ； 1-羥基焦腦油 ； 生物偵測

並列摘要

Arsenic, benzene and polycyclic aromatic hydrocarbons (PAHs), are three major industrial carcinogens, long term environmental exposure may cause very cancer. At present, the industrial emissions are usually quite complicate, the workers or residents may be exposed to a number of hazardous substances. Thus, the biominitoring of the multiple carcinogens is highly required. Currently, the three kinds of carcinogens are usually determined by separated analytical method. The aim of this study is to develop a multiple biomonitoring method for simultaneous determination of the urinary markers of the three carcinogens. The major marker metabolites are monomethylarsonic acid (MMAA), dimethylarsonic acid (DMAA), sphenylmercapturic acid (S-PMA), 1-hydroxypyrene (1-OHP). In order to provide suitable retention of the four marker compounds, a LC column of Kinetex PFP 2.6 µm 100 x 2.1 mm was used. After enzymatic deconjugation, 4 mL urine sample was extracted by 2 steps of solvent extraction. The 1-OHP in urine was firstly extracted with n-hexane, and MMAA、DMMA、S-PMA in urine were then extracted with a 2nd extraction/ice back-extraction with the chloroform/ ammonium bicarbonate aqueous solution. The developed method was validated with spiked urine samples. The calibration curve was calculated with corrected area against the spiked concentrations. The linear ranges of the urinary markers were MMAA: 2-64 ng/mL, DMAA: 1-64 ng/mL, S-PMA: 0.78-100 ng/mL and 1-OHP: 0.05-6.4 ng/mL, the satisfid linearity was obtained for all markers ( r2>0.99), the obtained detection limits (S/N ratio≧3) were MMAA: 0.117 ng/mL, DMAA: 0.167 ng/mL, S-PMA: 0.046 ng/mL and 1-OHP: 0.007 ng/mL, the accuracy(%error) and precision (CV%) were ranged of -16.67-29.17% and 2.03-30.99 %, respectively (3 spiked levels, 6 replicates). The method was applied for 10 clinical urine samples, the presences of MMAA, DMAA, S-PMA and 1-OHP were clearly detected and no significant difference was observed between the healthy volunteer group and lung cancer group. The detected concentrations were MMAA ranged from N.D. (no detection) or 0.92(Extrapolation method) -8.38 ng/mL, DMAA ranged from 0.63(Extrapolation method)-10.71 ng/mL, S-PMA ranged from N.D. or 0.08(Extrapolation method)-2.55 ng/mL and 1-OHP ranged from 0.01(Extrapolation method)-0.17 ng/mL, which all committed with the reported background values.

並列關鍵字

LC–MS/MS ； Arsenic ； S-phenylmercapturic acid ； Benzene ； Polycyclic aromatic hydrocarbons ； 1-OHP ； Biomonitoring

參考文獻

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