新型三液相萃取裝置結合液相層析串聯質譜儀偵測尿液中甲苯暴露指標成分

Analysis of Urinary Markers for Toluene Exposure by LC-MS/MS with a New Liquid-Liquid-Liquid Extraction (LLLE) Device

黃豊鈞(Li-Jun Huang)

指導教授 ： 沈振峯

國立虎尾科技大學/電機資訊學院/光電與材料科技研究所/碩士(2010年)

摘要

甲苯為國內工業界常用之溶劑，甲苯暴露過量會造成神經系統、肝和腎臟危害。因此對甲苯暴露的監控可藉由分析尿中甲苯代謝物進行對偵測。本研究主要利用三液相萃取裝置結合液相層析-串聯式質譜儀分析尿樣中甲苯曝露代謝物芐基硫醇酸(Benzylmercapturic acid,BMA) 與鄰-甲酚(o-cresol)。裝置所使用的材料皆為實驗室易取得的離心管及樣品瓶，且容易操作。因為萃取相必須使用緩衝液，所以我們選擇使用化學游離界面(APCI)來進行液相層析質譜儀分析。本報告使用三液萃取裝置的萃取條件為：萃取溶劑為toluene與diethyl ether(1:1;v/v)、萃取相體積400μL、尿液樣品pH值調整為2。經過裝置萃取後，使用液相層析串聯質譜儀進行分析，其尿液樣品萃取前後BMA訊號增加將近100倍，而o-cresol訊號增加將近8倍。此外，本方法在分析物的濃度範圍10ng/mL~1000ng/mL亦可得到良好的線性(r2>0.99)。為減低尿樣的相對基質效應，七種不同來源尿樣需經十倍稀釋後再進行三液相萃取，所得各指標物訊號峰面積之相對變異(RSD)皆小於15%。

關鍵字

液相微萃取法 ； 化學游離界面 ； 芐基硫醇酸 ； 鄰-甲酚

並列摘要

Toluene is a widely used solvent in industry. It is a human central nervous system depressant. Long-term exposure of toluene is associated with liver and kidney damage in humans. Bio-monitoring of low level toluene exposure is commonly performed by determination of urinary benzylmercapturic acid (BMA) and o-cresol. In this study, a new three phase liquid-liquid-liquid extraction (LLLE) device was developed and applied in the analysis of the urinary markers of toluene exposure. The new device was simply constructed with centrifuge tube and sample vials, and it is very easily to operate. Because of the higher buffer contents of the acceptor phase, the APCI mode was chosen for the LC-MS/MS analysis. The mainly applied parameters were urine sample mixed with phosphorate buffer (pH~2, donor layer), 1.5 mL toluene and diethyl ether (1:1, organic layer) and 400μL 0.1 M NaOH(aq) (pH~13, acceptor). After extraction and back-extraction, the urinary markers were cleaned up and enriched in the donor phase. In comparing with unprocess urine sample, after LLLE, the ion signals were increased about 100 times for BMA and about 8 times for o-cresol. Good linearity (r2 > 0.99) was also obtained in the range of 10-1000 ng/mL for both urinary BMA and o-cresol. To reduce the relative matrix effect, 10-fold dilution was needed before LLLE. The obtained RSD values of the markers of 7 lots urine samples were all less than 15% .

並列關鍵字

LLLE ； Toluene ； APCI ； BMA ； o-cresol

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