血液和尿液中酒精代谢指标物乙基葡萄糖醛酸苷的GC-MS检测方法
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摘要
目的优化并建立血液和尿液中乙基葡萄糖醛酸苷(Et G)含量的气相色谱-质谱(GC-MS)检测法。方法用丙酮沉淀血样中的蛋白质,离心取上清液,并经固相萃取小柱(Clean Screan)富集净化,采用衍生化试剂双(三甲基甲硅烷荃)三氟乙酰胺-三甲基氯硅烷[BSTFA+TMCS,BFT]对提取物进行衍生化,采用GC-MS检测,全扫描(Full Scan)模式和选择离子扫描(SIM)模式,内标法定量分析。结果 Et G在血液样品0.1～3.0μg/ml浓度范围内与色谱响应值具有良好线性关系(R~2=0.999 8),回收率范围为95.86%～101.88%,准确度标准偏差(SD)为1.52～2.91,相对标准偏差(RSD)为0.51～3.45;Et S在尿样0.1～3μg/ml浓度范围内与色谱响应值具有良好线性关系(R~2=0.999 2),回收率为98.10%～102.71%,SD值为0.81～6.65,RSD值为0.27～9.94之间。结论本方法有较高的灵敏度和特异度,分析结果准确,适合用于Et G在血液和尿液中的定性定量分析。
[Objective]To optimize and establish the determination method for ethyl glucuronide(Et G) content in human blood and urine by gas chromatography-mass spectrometry(GC-MS) method.[Methods]The acetone was used to precipitate the proteins in the bloodsamples,the supernatant was taken after centrifugation,and the extracts were enriched and purifiedby by solid-phase extraction with small columns(Clean Screan). The extract was derived from BSTFA+TMCS(BFT) to detected by GC-MS with ion scan mode(SIM)and full scan mode,and internal standard method for quantitative analysis.[Results]For the blood samples,good linearity was obtained at the mass concentration range of 0.1-3 μg/ml with chromatographic response(R~2=0.999 8),the average recovery rate was 95.86%-101.88%,the the accuracy(standard deviation,SD) was 1.52-2.91,and the relative standard deviation(RSD) was 0.51-3.45. For the urine samples,good linearity was obtained at the mass concentration range of 0.1-3 μg/ml with chromatographic response(R~2=0.999 2),the average recovery rate was 98.10%-102.71%,SD was 0.81-6.65,and RSD was 0.81-6.65.[Conclusion]The method has high sensitivity and specificity,and the analysis results are accurate. It is suitable for qualitative and quantitative analysis of Et G in blood and urine.
[Objective]To optimize and establish the determination method for ethyl glucuronide(Et G) content in human blood and urine by gas chromatography-mass spectrometry(GC-MS) method.[Methods]The acetone was used to precipitate the proteins in the bloodsamples,the supernatant was taken after centrifugation,and the extracts were enriched and purifiedby by solid-phase extraction with small columns(Clean Screan). The extract was derived from BSTFA+TMCS(BFT) to detected by GC-MS with ion scan mode(SIM)and full scan mode,and internal standard method for quantitative analysis.[Results]For the blood samples,good linearity was obtained at the mass concentration range of 0.1-3 μg/ml with chromatographic response(R~2=0.999 8),the average recovery rate was 95.86%-101.88%,the the accuracy(standard deviation,SD) was 1.52-2.91,and the relative standard deviation(RSD) was 0.51-3.45. For the urine samples,good linearity was obtained at the mass concentration range of 0.1-3 μg/ml with chromatographic response(R~2=0.999 2),the average recovery rate was 98.10%-102.71%,SD was 0.81-6.65,and RSD was 0.81-6.65.[Conclusion]The method has high sensitivity and specificity,and the analysis results are accurate. It is suitable for qualitative and quantitative analysis of Et G in blood and urine.
引文
[1]缪绿青,王华荣,莫闲,等.血液中不同浓度酒精对驾驶员脑电及心理功能的影响[J].交通医学,2016,26(2):141-144
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[9]APPENZELLER BM,AGIRMAN R,NEUBERG P,et al.Segmental determination of ethyl glucuronide in hair:A pilot study[J].Forensic Sci Int,2007,173(2-3):87-92.
[10] MORINI L,POLITI L,ZUCCHELLA A,et al. ALDO P.Ethyl glucuronide and ethyl sulphate determination in serum by liquid chromatography-electrospray tandem mass spectrometry[J].Clinica Chimica Acta,2007,376(1-2):213-219.
[11] KETEN A,TUMER AR,et al.Measurement of ethyl glucuronide in vitreous humor with liquid chromatography-mass spectrometry[J].Forensic Sci Int,2009,193(1):101-105.
[12]WURST FM,SKIPPER GE,WEINMANN W.Ethyl glucuronide on the time course of excretion in urine during detoxification[J].Addiction Biology,2003,98(2):51-59.
[13]JANDA I,AIT A. Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction[J].J Chromatogr B,2001,758(2):229-234.
[14]KEREKES I,YEGLES M,GRIMM U,et al.WENNIG R. Ethyl glucuronide determination:head hair versus non-head hair[J]. Alcohol and Alcoholism,2009,44(1):62-66.
[15]ALEKSA K,WALASEK P,FULGA N,et al. Simultaneous detection of seventeen drugs of abuse and metabolite in hair using solid phase micro extraction with GC/MS[J].Forensic Sci Int,2012,218(2):31-36.
[16]JURADO C,SORIANO T,GIMENEZ MP,et al. Diagnosis of chronic alcohol consumption-Hair analysis for ethyl glucuronide[J].Forensic Sci Int,2004,145(2-3):161-166.
[2]翟红梅,肖颖,肖霄,等.酒在人体内的代谢及酒精中毒[J].石家庄学院学报,2010,12(3):27-29.
[3]赵晖,卓先义,严慧,等.液相色谱一串联质谱法测定血液、尿液中乙基葡萄糖醛酸苷[J].法医学杂志,2010,26(4):269-272.
[4]赵晖,卓先义,沈保华.乙基葡萄糖醛酸苷的检测及其在法医毒理学中的应用[J].法医学杂志,2009,25(1):63-67.
[5]GUDRUN H,JEAN PB,RITVA K,et al. A pharmacokinetic study of ethyl glucuronide in blood and urine:Applications to forensic toxicology[J].Forensic Sci Int,2007,172(2):119-124.
[6] WOLFGANG W,PATRICK S. Confirmatory Analysis of Ethyl glucuronide in urine by Liquid-chromatography/electrospray Ionization/tandem mass spectrometry According to forensic Guidelines[J].J Am soc Mass spectrom,2004,1(5):188-193.
[7]KAUSHIK R,WR LACOURSE,B LEVINE.Determination of ethyl glucuronide in urine using reversed-phase HPLC and pulsed electrochemical detection(Part II)[J]. Analytica Chimica Acta,2006,556(2):267-274.
[8]OHYAMA K,KISHIKAWA N,NAKAGAWA H. Simultaneous determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human serum by capillary zone electrophoresis[J].Pharmaceutical and Biomedical Anal,2008,47(1):201-206.
[9]APPENZELLER BM,AGIRMAN R,NEUBERG P,et al.Segmental determination of ethyl glucuronide in hair:A pilot study[J].Forensic Sci Int,2007,173(2-3):87-92.
[10] MORINI L,POLITI L,ZUCCHELLA A,et al. ALDO P.Ethyl glucuronide and ethyl sulphate determination in serum by liquid chromatography-electrospray tandem mass spectrometry[J].Clinica Chimica Acta,2007,376(1-2):213-219.
[11] KETEN A,TUMER AR,et al.Measurement of ethyl glucuronide in vitreous humor with liquid chromatography-mass spectrometry[J].Forensic Sci Int,2009,193(1):101-105.
[12]WURST FM,SKIPPER GE,WEINMANN W.Ethyl glucuronide on the time course of excretion in urine during detoxification[J].Addiction Biology,2003,98(2):51-59.
[13]JANDA I,AIT A. Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction[J].J Chromatogr B,2001,758(2):229-234.
[14]KEREKES I,YEGLES M,GRIMM U,et al.WENNIG R. Ethyl glucuronide determination:head hair versus non-head hair[J]. Alcohol and Alcoholism,2009,44(1):62-66.
[15]ALEKSA K,WALASEK P,FULGA N,et al. Simultaneous detection of seventeen drugs of abuse and metabolite in hair using solid phase micro extraction with GC/MS[J].Forensic Sci Int,2012,218(2):31-36.
[16]JURADO C,SORIANO T,GIMENEZ MP,et al. Diagnosis of chronic alcohol consumption-Hair analysis for ethyl glucuronide[J].Forensic Sci Int,2004,145(2-3):161-166.