分散式浮冰液相微萃取/液相層析-螢光法同步偵測尿液中多環芳香烴與甲苯曝露指標物

甲苯(toluene)與多環芳香烴(polycyclic aromatic hydrocarbons, PAHs)為工業上常見的危害物，長期曝露於甲苯可能造成中樞神經損害，而長期曝露於PAHs環境可能導致癌症發生。本研究欲建立此兩類危害物之同步生物偵測法，縮短樣品前處理時間，及降低分析成本。本研究首次針對此兩類危害物在尿液中之主要代謝物，鄰-甲酚(o-cresol)和1-羥基焦腦油(1-hydroxypyrene, 1-OHP)，以液相層析-螢光法(LC-FL)進行同步分析，樣品前處理部分則使用分散式浮冰液相微萃取(dispersive liquid-liquid microextraction based on solidification of floating organic droplet, DLLME-SFO) 技術與水解法結合。先以3 mL尿液加入β-glucuronidase進行酵素水解，將 o-cresol與1-OHP 共軛物(conjugates)水解還原，接著注入50 μL 1-十一烷醇(萃取試劑)與1 mL丙酮(分散試劑)震盪萃取(1 min)，離心後置入-20 ℃冰箱冷凍30 min至萃取相凝結成冰珠，以鑷子取出冰珠後待溶化與等體積甲醇置入1.2 mL樣品瓶(vial)混合，並注入10 μL進行LC-FL分析。以活性碳處理後空白尿樣與其他尿樣相對基質比對，相對標準差在20%以內。以空白尿樣進行方法確效，經面積計算檢量線，可達線性範圍o-cresol為2.81-180.00 ng mL-1、1-OHP為37.50-300.00 pg mL-1，其檢量線均有良好線性r2　≥ 0.99，測試偵測極限(S/N ratio≧3):o-cresol為0.35 ng mL-1、1-OHP為9.38 pg mL-1。精密度(CV %, 3濃度, n=6)範圍為3.74-12.22 %，而準確度(% Error, 3濃度, n=6)在±20 %以內。分析7個臨床尿樣，o-cresol範圍為1.87-6.00 ng mL-1、1-OHP則為50.61-104.82 pg mL-1。

關鍵字

分散式浮冰液相微萃取；液相層析-螢光法；甲苯；多環芳香烴碳烴化合物；鄰-苯酚； 1-羥基焦腦油；生物偵測

並列摘要

Toluene and polycyclic aromatic hydrocarbons(PAHs) were common hazard materials on the industry, long-term exposure to toluene and PAHs can cause central nervous system damage and cancer respectively.In this stady, simultaneously the biological detection method for hazard, shorten the sample preparation time and reduce the cost of analysis. Urinary 1-hydroxypyrene(1-OHP) and o-cresol are main metabolites of the two types of hazards, and detected with liquid chromatography-fluorescence(LC-FL).The technique of dispersive liquid-liquid microextraction based on solidification of floating organic droplet method (DLLME-SFO)is used for sample processing.At first 3 mL urine was hydrolyzed for 2 hours by enzyme of β-glucuronidase.followed by addition of 50 μL 1-undecanol(extraction solvent) and 1 mL acetone(dipsersive solvent) for DLLME-SFO(1 min).After centrifuged and frozen 30 min in-20 ℃, the solidification droplet(ice) was mixed by equal volumes methanol, transferred into 1.2 mL vial and injected to LC-FL analysis.The blank urine was stripped by charcoal powder and compared the relative standrd deviation with other source urine, the result of relative standrd deviation was less than 20 %. The developed method was calidated with spiked blank urine samples. The calibration curve was calculated with area against the spiked concentration. The linear ranges of the urinary markers were o-cresol: 2.81-180.00 ng mL-1、1-OHP:37.50-300.00 pg mL-1, the satisfid linearity was obtained for each markers (r2>0.99) , the obtained detection limits(S/N ratio≧3) were o-cresol: 0.35 ng mL-1, 1-OHP: 9.38 pg mL-1, the accuracy ( % Error) and precision(CV %) were range 3.74-12.22 %and ±20 %, respectively(3 spiked levels, 6replicates). The method was applied for 7 clinical smaples, the presence of o-cresol and 1-OHP were detected.

並列關鍵字

DLLME-SFO ； LC-FL ； Toluene ； PAHs ； o-cresol ； 1-OHP ； Biomonitoring

參考文獻

1. 楊秀宜、李聯雄. (2011). 生物偵測曝露指標建議值訂定機制研究，Strategies for setting biological exposure indices，IOSH99-A309. 行政院勞工委員會勞工安全衛生研究所.

3. Jeng, H. A.,Pan, C. H., & Chao, M. R. (2013). 1-Hydroxypyrene as a biomarker for assessing the effects of exposure to polycyclic aromatic hydrocarbons on semen quality and sperm DNA integrity. J Environ Sci Health A Tox Hazard Subst Environ Eng, 48(2), 152-158.

4. Jongeneelen, F. J. (1994). Biological monitoring of environmental exposure to polycyclic aromatic hydrocarbons; 1-hydroxypyrene in urine of people. Toxicology Letters, 72(1–3), 205-211.

5. Kamal, A.,Cincinelli, A.,Martellini, T., & Malik, R. N. (2016). Biomarkers of PAH exposure and hematologic effects in subjects exposed to combustion emission during residential (and professional) cooking practices in Pakistan. Environ Sci Pollut Res Int, 23(2), 1284-1299.

7. Jongeneelen, F. J.,Anzion, R. B. M., & Henderson, P. T. (1987). Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in urine. Journal of Chromatography B: Biomedical Sciences and Applications, 413, 227-232.

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分散式浮冰液相微萃取/液相層析-螢光法同步偵測尿液中多環芳香烴與甲苯曝露指標物

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