探究尸体血样中乙醇、乙基葡萄糖醛酸苷和硫酸乙酯的产生

Research of generation of ethanol, ethyl glucuronide and ethyl sulfate in post-mortem blood sample

目的探索尸体血样保存过程中乙醇的产生情况及乙基葡萄糖醛酸苷(EtG)和硫酸乙酯(EtS)的产生可能。方法对照组为7例阳性静脉血,而实验组则为7例阴性尸体血。每例血样分成3份并保存在室温(18~22℃),4℃及-20℃等3种不同的条件下,在保存天数为0、2、3、5、7、9、11、13、15、17、19、21等时间点取样。使用顶空气相色谱法(HS-GC)检测乙醇,采用固相萃取提取EtG和EtS,使用高效液相色谱-三重四级杆质谱(LCMS/MS)法检测EtG和EtS。结果保存期间,对照组各血样中的乙醇、EtG和EtS浓度均呈下降趋势;实验组中1、2、4、5、6、7号血样的室温及4℃的样本在保存第2~3天时检出乙醇,而7号-20℃的样本在第6天检出乙醇。其中,6号室温血样的乙醇峰值浓度为64.27mg/100mL。各血样中均未检出EtG,EtS。结论室温及4℃保存的尸体血可产生乙醇且产生速度较快,反复冻融可导致-20℃保存的尸体血产生乙醇,乙醇峰值浓度可超过法定酒驾标准,但实验组血样中均无EtG和EtS产生。因此,尸体血中的EtG,EtS可以作为乙醇生前入体的特异性标志物,区分乙醇生前入体和腐败产生乙醇的依据。实际工作中,乙醇原体检测的酒精认定应注意血样保存和运输条件造成的影响。为了避免假阳性结果,涉及死亡的案件进行酒精认定时有必要辅以EtG和EtS的检测。

Objective To study the generation of ethanol and the possibility of ethyl glucuronide(EtG) and ethyl sulphate(EtS) generation in post-mortem(PM) blood samples Methods There were 7 negative PM samples in experimental group, with 7 positive venous blood samples in control group. Each sample was divided into 3 aliquots and kept in ambient temperature(AT, 18~20℃), 4℃ and-20℃ respectively. On the initial day and the 2 nd, 3 rd, 5 th, 7 th, 9 th, 11 th, 13 th, 15 th, 17 th, 19 th, and the 21 st day, ethanol in blood samples was detected by Headspace-Gas chromatography(HS-GC) system. Meanwhile, after pre-treatment by solid phase extraction(SPE), samples were analyzed by Liquid chromatography-tandem mass spectrometry(LC-MS/MS) for EtG and EtS. Results During storage, ethanol, EtG and EtS in all the samples of control group decreased. On the 2 nd or 3 rd day of storage, the No.1, 2, 4, 5, 6, 7 samples kept in AT and 4℃ were detected as ethanol positive. The No.7 sample kept in-20℃ was ethanol positive on the 6 th day. Among the ethanol-positive samples, the blood alcohol concentration(BAC) of the No.6 AT sample had once been up to 64.27 mg/100 mL. None of the samples was detected as EtG or EtS positive. Conclusion Ethanol would generate fast in PM blood samples that were kept at AT and 4℃. Besides, freezing-thawing would lead to ethanol generation in-20℃ samples. However, neither EtG nor EtS was detected in experimental group. Thus, EtG and EtS in PM blood sample were thought to be specific biomarker of alcohol consumption before death, as an ideal way to differentiate blood ethanol of the consumption from ethanol of putrefaction. In practice, attention should be paid to the effects of sample storage and transportation on the determination of ethanol detection. To avoid false positives, it is necessary to conduct assay of EtG and EtS for determination alcohol consumption before death in cases involving death.