2-氯乙醇於全血中的穩定度測試

Abstract

2-氯乙醇(CAS 107-07-3)是台灣農民常用於葡萄催芽之用的試劑，雖因毒性強烈被禁用，但因比49 %氰滿素效果較佳且費用便宜，還是常被農民非法使用，因此常有誤食或工作接觸造成意外中毒的事件。民國100年南投縣發生因感情糾紛而使用2-氯乙醇毒殺，造成4人死亡的謀殺案件，從屍體中檢測出2-氯乙醇成為重要的法庭證據。 因2-氯乙醇並非一般常規篩檢毒物，目前並沒有標準方法可以檢測血中濃度，因此我們使用HS-GC/MS系統並以1-pentanol為內標準品建立全血中2-氯乙醇的檢測方法，此方法利用選擇性離子方法以定量(m/z 31)與定性離子(m/z 49, 80)測得2-氯乙醇之偵測極限為1 μg/mL，定量極限為5 μg/mL，靈敏度佳且線性關係良好(在5-200 μg/mL之間可得r2=0.999)、易於操作。 為得知其穩定度，以EDTA抗凝固劑管採取10位自願者血液，將血液檢體每組配製10 μg/mL與100 μg/mL二種濃度，分二組分別置於室溫及4℃二種不同儲存溫度的環境下儲存，觀察不同時間點對於2-氯乙醇穩定度之影響。結果顯示10 μg/mL濃度2-氯乙醇於血液檢體中從第0週到第12週不論是儲存於4℃或室溫條件下都沒有明顯下降趨勢，4℃平均為10.6 μg/mL (SD 0.86)，室溫平均為10.5 μg/mL (SD 0.62)；100 μg/mL濃度2-氯乙醇血液檢體則是在4℃儲存條件下沒有明顯下降(平均93.6 μg/mL，SD 5.37)，但儲存於室溫的檢體從第0週到第6週都還保持穩定，但第9週起就降低到78.3 μg/mL，第12週則為83.4 μg/mL。 2-氯乙醇的代謝途徑為經酒精脫氫酶氧化成為氯乙醛後，再經乙醛脫氫酶代謝為氯乙酸。為證實2-氯乙醇中毒，除2-氯乙醇外尚須檢驗其代謝物氯乙酸的濃度，參考檢測水質中氯乙酸方法，同時考慮時間與操作簡易性，挑選以液相-液相微萃取法/甲基化，在室溫下震盪三分鐘即可萃取氯乙酸，同時將氯乙酸衍生為甲基化酯類以HS-GC/MS偵測，並同時偵測2-氯乙醇。於本實驗方法中可同時於水中驗出這二種物質，但血液檢體，還有其LOD、LOQ、衍化率等均需再進一步評估。

2-chloroethanol (2-CE, ethylene chlorohydrin, CAS 107-07-3) is a chemical once widely used in hastening grape vine sprouting among Taiwanese farmers. Due to its severe toxicity upon acute exposure, such use of 2-CE is now prohibited in Taiwan. However, because of its superior potency and cheaper price compared to 49% cyanamide, 2-CE is still being illegally used by some farmers, so cases of poisoning due to accidental ingestions or occupational exposures were reported from time to time. In 2011, a man murdered his ex-girlfriend with 2-CE due to an emotional entanglement in Nantou County; four persons died in this case, and the detection of 2-CE from the corpses was an important forensic evidence. To date, there are no standardized methods for the detection and quantification of 2-CE in human blood, because it is not a routinely-screened toxin. We developed a sensitive and specific method by employing static headspace gas chromatography with mass spectrometry (HS-GC/MS) for the quantitative determination of 2-CE in whole blood sample using 1-pentanol as internal standard. It was performed using selected ion monitoring (SIM) with quantitative ion (m/z 31) and qualitative ion (m/z 49, 80). We found that the method produced results with good linearity (r2=0.999, in the concentration range of 5-200 μg/mL), is sensitive (with the limit of detection and the limit of quantitation as 1 μg/mL and 5 μg/mL, respectively), and is easy to operate. To evaluate the stability of 2-CE, whole human blood samples were taken from 10 volunteers (with EDTA added as anticoagulant), and two different concentrations of 2-CE solutions (10 μg/mL and 100 μg/mL) were prepared from each sample. They were further divided into two groups, with one group stored at 4℃ and another stored at room temperature until analyzed in triplicate by headspace gas chromatography-mass spectrometry (HS-GC/MS). After twelve weeks, the result showed that there were no significant alterations in the concentrations of the 10 μg/mL 2-CE solutions, whether stored at 4℃ (average 10.6 μg/mL with SD 0.86) or room temperature (average 10.5 μg/mL with SD 0.62). In the 100 μg/mL 2-CE solutions, no significant alterations of the concentrations were noticed when stored at 4℃ (average 93.6 μg/mL with SD 5.37); however, when stored at room temperature, it decreased significantly to 78.3 μg/mL in the 9th week and to 83.4 μg/mL in the 12th week. 2-chloroethanol has been assumed to be metabolized to chloroacetaldehyde (CAA) via alcohol dehydrogenase, and then to chloroacetae (CA) by aldehyde dehydrogenase. Both 2-CE and its metabolites should be detected in order to prove that it’s indeed 2-CE poisoning. Looking to the method of determining CA concentration in water for inspiration, we developed a simple and quick way that combines simultaneous liquid-liquid microextraction/methylation and HS-GC/MS for quantification of CA and 2-CE concentrations. Methylation and derivatization were completed in 3 minutes by shaking at room temperature. The methyl ester derivatives and the organic phase were completely volatilized by static headspace technique, and then analyzed by GC/MS. It seems that the concentrations of CA and 2-CE could be simultaneously determined in water, but further evaluations of LOD, LOQ, linearity and yield rate of the method in blood samples are still needed.