不同温度保存血痕中EtG、EtS的稳定性研究

目的建立血痕(DBS)中乙基葡萄糖醛酸苷(EtG)、硫酸乙酯(EtS)液相色谱-串联质谱(LC-MS/MS)的检测方法,研究不同温度保存血痕中EtG、EtS的分解规律,应用于乙醇相关案件的法医学鉴定。方法采集8名志愿者酒后静脉血并制成血痕,均分为3组,分别保存在20℃、4℃、-20℃温度下,于保存当时(0d)、5d、9d、14d、20d、23d、26d、44d、50d,采用多反应离子监测、液相色谱-串联质谱法检测血痕中EtG、EtS的含量。结果血痕中EtG、EtS在10ng/ml～1μg/ml之间线性范围良好,相关系数(r)≥0.998,最低检出限分别为40ng/ml、20ng/ml。不同温度保存血痕中EtG、EtS的含量均呈下降趋势,其中20℃分解最快,-20℃分解最慢,且EtS的检测时限较EtG长。结论在乙醇相关案件的法医学鉴定中,应注意将血痕冷冻(-20℃)保存、及时送检,同时检测其中EtG、EtS的含量,避免假阴性结果的出现。     

乙基葡萄糖醛酸苷的检测及其在法医毒理学中的应用

乙基葡萄糖醛酸苷（ethyl glucuronide,EtG）是乙醇在人体内的特异性代谢物。迄今,已有相当数量的文献针对EtG的检测方法、代谢动力学及实际应用等进行了深入探讨。本文综述了EtG的各种检测方法、EtG含量与乙醇摄入量的相互关系以及如何利用EtG含量区分是否酗酒、判断尸体中乙醇来源等,阐述EtG在法医毒理学领域的重要意义。     

保存温度对血液酒精含量检测的影响

目的为筛选最佳的保存温度,准确检测酒驾血液酒精含量,为交管部门客观判断酒驾行为提供技术支撑。方法本研究选取EDTA-2真空抗凝采血管,采取酒后人体静脉鲜血后,分别在-20℃、4℃~8℃、25℃常温、35℃~42℃高温等4个温度条件下保存,GC法按0、3d、7d、14d、21d及28d后检测血液酒精含量,并对测试结果进行比较统计分析。结果在35℃~42℃和25℃温度下存储的血液酒精含量在0~3d内基本稳定,3d后显著下降(P0.05);4℃~8℃温度下存储的血液酒精含量在0~14 d内基本稳定,14d后显著下降(P0.05);-20℃温度保存条件下血液酒精含量测试28d统计结果间无显著差异。结论建议血样采集后低温保存,-20℃温度为血样的最佳保存温度。     

尿样品中乙醇浓度变化

动物实验和实际案例分析结果表明，尸体低温保存较长时间，或室温保存数天，血，尿中可生成乙醇正丙醇等，乙醇量一般为正丙醇的20倍内。另一组实验考察了血，尿样品保存时乙醇的稳定性。在评价乙醇含量，判断是否生前饮酒时需考虑这些因素对乙醇含量的影响。     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法检测血斑中乙醇生物标志物

目的建立了超高效液相色谱-四极杆/静电场轨道阱高分辨质谱(UPLC-Q/Orbitrap HRMS)测定血液斑痕中两种乙醇生物标志物乙基葡萄糖醛酸苷(EtG)和硫酸乙酯(EtS)的检测方法。方法采用空白静脉血液加标后在载玻片上制备成血液斑痕,取样后用50%甲醇提取,通过超高效液相色谱高分辨质谱进行检测;色谱柱Synicronis C18(150mm×2.1mm×5.0μm)分离,流动相A为0.1%的甲酸水,流动相B为含0.1%甲酸的甲醇-乙腈(1∶1),梯度洗脱,离子源为加热电喷雾离子化源(HESI),扫描方式全扫描模式,负离子模式检测。结果该方法在EtG、EtS浓度为0.2~100ng/patch范围内有良好线性关系(R^(2)=0.9991、0.9994),样品回收率良好,日内与日间精密度小于15%,基质效应在85%~120%之间。结论利用该方法可有效测定血液斑痕中的乙醇生物标志物。     

血、尿中乙醇含量的测定及其评价——Ⅲ. 乙醇的死后生成及其血、尿样品中乙醇浓度变化

动物实验和实际案例分析结果表明,尸体低温保存较长时间,或室温保存数天,血尿中可生成乙醇正丙醇等,乙醇量一般为正丙醇的20倍内.另一组实验考察了血、尿样品保存时乙醇的稳定性.在评价乙醇含量,判断是否生前饮酒时需考虑这些因素对乙醇含量的影响.     

顶空气质联用法测定腐败血、肝检材中CO

目的研究CO中毒腐败血、肝组织检材中CO的HS/GC/MS检测。方法用HS/GC/MS法分析碳氧血红蛋白(COHb)血的线性范围。配制10%、30%、50%、70%浓度COHb血样,分别在室温、冷藏、冷冻条件下保存,分别在当日、第4、14、45d进行测定,比较实验结果。腐败肝组织由雄性健康家兔通CO气体致死,当天解剖,家兔肝常温隔绝空气保存并放35d至腐败,期间进行不定期顶空测定分析。结果制备的COHb血在0-100%之间有良好的线性关系Y=2.4X+2.2(r=0.9995)。以此方法测定家兔CO中毒致死的COHb新鲜血的浓度和4℃下放置45dCOHb腐败血,结果表明温度对血样中COHb%的测定影响最大。采用HS/GC/MS法检测,每次只需0.25ml血样或1g肝脏,分析一次时间只需3min,均可检测出新鲜检材和常温放置45d的腐败肝组织检材CO的含量。结论HS/GC/MS法能检出CO中毒的腐败生物检材中CO。     

头发中乙基葡萄糖醛酸苷分析的研究进展

乙基葡萄糖醛酸酐（ethy lglucuronide,EtG）为乙醇的体内代谢物,摄入的乙醇大约0.02%～0.06%以EtG的形式排出,其消除时间较乙醇缓慢,即使当乙醇在体内完全消除后,EtG仍可作为酒精滥用的生物学标志。测定头发中的EtG可延长检测时限,获取饮酒史的信息,在法庭科学和临床医学领域已成为研究热点。头发中EtG的检测主要采用以串联质谱为主的高灵敏的分析方法,且注重头发采样、去污、水解、提取、分析等各环节的质量控制,避免出现假阳性结果。头发中EtG的研究结果可广泛应用于法庭科学、临床医学、征兵、招工、驾驶能力测试等领域。     

人体组织ABO血型检测方法的研究

探讨人体组织ABO血型检测方法。对已知ABO血型尸体的不同组织,用红细胞粘连试验、吸收—抑制试验和吸收－解离试验进行ABO血型测定。12例尸体的16种组织中均检出与尸体血痕相同的ABH物质。对不同温度保存的组织块进行ABH物质检测的结果显示,4℃保存的组织ABH物质的检出时间长于室温,空腔脏器的检出时间短于实质脏器。三种方法中,红细胞粘连试验简单易行,适用于基层单位,吸收－抑制试验用于组织块的测定时优于吸收－解高试验。     

保存犬尸体中自生醇的研究

目的建立组织中自生醇直接进样毛细管柱气相色谱检测方法；建立犬尸体中自生醇研究模型．观察犬尸体中自生醇的产生情况。方法犬经CO2处死，置于室温，分别于死后0、2、4、8、12、24、48、72、96、120h采集样品，样品匀浆后（体液除外）离心去上清，内标溶液1：2（体液为1：4）稀释，直接进样，气相色谱法检测，保留时间定性，内标法定量。结果甲醇、丙酮、乙醇、正丙醇、异丁醇、异丙醇、正丁醇、异戊醇的保留时间分别为（2．77±0．01）、（3．42±0．01）、（3．87±0．01）、（5．92±0．01）、（7．54±0．01）、（7．92±0．01）、（10．33±0．01）、（13．55±0．01）min；大部分组织如心血、下腔静脉血、尿、胆汁、肝、脾、肺和肾在犬死后12h可检出乙醇，肌肉、玻璃体液、和大脑分别在死后48、72、96h检出乙醇；乙醇和正丙醇的生成存在正相关。结论死亡犬体内会产生自生醇，尿中检出乙醇不能判定为生前饮酒，在涉及乙醇的法医学检案中。特别是腐败样品．应注意自生醇对乙醇定性和定量可能造成的影响，应采集不易腐败的组织如玻璃体液、肌肉或大脑，检测乙醇及自生醇含量，并观察与正丙醇的比例关系来进行综合判定。     

A study of ethyl glucuronide in post-mortem blood as a marker of ante-mortem ingestion of alcohol

The possibility of post-mortem production of ethanol makes correct interpretation of ethanol detection in forensic autopsy samples difficult. Even though the levels of ethanol formed post-mortem are generally low, this may be highly relevant in cases where intake of alcohol was forbidden, for instance for pilots, professional drivers and countries with low legal alcohol limits for driving. Different criteria are used to determine whether a finding of ethanol is of exogenous origin, but there is no marker for alcohol ingestion that has been studied in detail. In this study, we wanted to evaluate the sensitivity and specificity of ethyl glucuronide (EtG), a direct minor metabolite of ethanol, measured in blood, as a marker of ante-mortem alcohol ingestion. Forensic autopsy cases were divided into groups with and without ante-mortem alcohol ingestion, according to strict inclusion criteria. In 93 cases with information on ante-mortem alcohol ingestion, EtG was detected in blood in all cases, even when levels of ethanol were low. In another 53 cases where there were no indications of ante-mortem alcohol intake, EtG could not be detected in blood in a single case, also in 11 cases in which ethanol was detected and considered to be most probably formed post-mortem. In conclusion, blood EtG determination seems to be a reliable marker of ante-mortem ingestion of alcohol, and it could be considered in forensic autopsy cases when post-mortem formation of ethanol is questioned.     

Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death

To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.     

Markers of chronic alcohol use in hair: comparison of ethyl glucuronide and cocaethylene in cocaine users

Two direct ethanol metabolites, namely ethyl glucuronide (EtG) and cocaethylene (CE), in the hair of cocaine (COC) users were compared in this study. Hair samples (n=68) were submitted to the determination of EtG (by liquid chromatography-electrospray-tandem mass spectrometry) and of COC and metabolites, including CE (by gas chromatography-mass spectrometry). Quantitative and qualitative results were compared. No quantitative correlation was found between EtG and CE, as well as between EtG and the cocaethylene concentration divided by the concentration of COC and its metabolites (benzoylecgonine and ecgonine methylester, as COC equivalents). Nevertheless, many factors are supposed to affect the amount of the two substances incorporated in the hair matrix, such as the subject's habits in ethanol and COC use, genetic variability in the metabolism of both substances, and the different chemical and physical properties of EtG and CE. When establishing a cut-off of 4 pg/mg for EtG and of 200 pg/mg for CE, 47 samples tested positive for EtG and 41 samples tested positive for CE; 12 samples out of the 47 EtG-positives tested negative for CE (25%), whereas 6 samples out of the 41 CE-positives tested negative for EtG (15%). According to these data, EtG appears to be a more sensitive and specific marker of non-moderate alcohol users than CE.     

Comparison of ethyl glucuronide in hair with phosphatidylethanol in whole blood as post-mortem markers of alcohol abuse

Ethyl glucuronide (EtG) is a direct metabolite of ethanol and has been used as a marker of alcohol abuse in both urine and hair. This study investigated the value of EtG testing in post-mortem hair for diagnostic improvement of alcohol abuse in forensic medicine. Material from 70 consecutive medico-legal autopsies was collected in accordance with the recommendations on ethics by the Swedish National Board of Forensic Medicine. A method for determination of EtG in hair samples was developed using ultra performance liquid chromatography/electrospray tandem mass spectrometry (UPLC/ESI-MS/MS; LOQ, 2.5 pg/mg). The result of the EtG analysis was compared with the findings of phosphatidylethanol (PEth) in femoral whole blood, as measured by high performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD; LOQ, 0.22 micromol/l). Evaluation of liver histology and anamnestic evidence of alcohol abuse of the deceased were taken in consideration for the interpretation. Measurable levels of EtG were present in 49 of the 70 autopsy cases whereas PEth was present in 36. Thirty-nine cases had EtG levels above the cutoff limit (> or = 30 pg/mg) compared with 29 for PEth (> or = 0.7 micromol/l). Fifteen cases had EtG as exclusive indicator for alcohol abuse compared with four cases for PEth. These findings suggest that measurements of EtG in hair may provide improved diagnostic information on alcohol abuse, due to a long retrospective time-window for detection and stability of EtG in hair in the decaying cadaver. However, an EtG level below the cutoff does not completely exclude previous alcohol abuse.     

Detection of ethyl glucuronide in blood spotted on different surfaces

This study aims to show that sensitive detection of ethyl glucuronide in dried blood spotted onto various surfaces after a period of 24h is feasible. At present, there is insufficient information how tightly ethyl glucuronide (EtG) binds to various materials and how easily it can be eluted. 4ml aliquots of blood samples obtained from seven volunteers after consumption of alcoholic beverages were applied to six different surfaces. After drying and a 24h-storage at 20±2°C the samples were re-dissolved in water, and EtG was subsequently analyzed by a LC-MS Paul-type ion trap. A comparison was made between dried and corresponding fluid samples. EtG was detectable in all subjects' samples following consumption of alcohol. EtG was also detectable after a storage time of four weeks at 4°C in whole blood that had been preserved with EDTA. EtG was detectable in all samples dried on different surfaces and its concentration remained relatively constant irrespective of the particular condition of the material. Detection of EtG in blood spots from the scene may indicate recent alcohol consumption in cases where collection of blood remained undone or could not be performed.     

Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers

In previous investigations hair analysis for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) proved to be suitable for the detection of excessive alcohol consumption. The aim of this study was to compare EtG and FAEE concentrations in hair of alcoholics, social drinkers and teetotallers. Hair samples from 10 alcoholics in withdrawal treatment, 11 fatalities with documented excessive alcohol consumption, four moderate social drinkers who consumed up to 20 g ethanol per day, and three strict teetotallers were analysed. After external degreasing with n-heptane, extraction with a dimethyl sulfoxide/n-heptane mixture and headspace solid-phase microextraction of the extracts, four fatty acid ethyl esters (FAEEs) (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) were analysed by gas chromatography-mass spectrometry (GC-MS) with deuterated internal standards. EtG was determined by GC-MS/NCI after ultrasonication of the samples with H2O, cleanup by SPE with aminopropyl columns and PFP derivatisation. The following concentrations were measured for the four groups: teetotallers EtG < 0.002 ng/mg, FAEE 0.05-0.37 ng/mg, moderate social drinkers EtG < 0.002 ng/mg, FAEE 0.26-0.50 ng/mg, alcoholic patients EtG 0.030-0.415 ng/mg, FAEE 0.65-20.50 ng/mg and the fatalities with alcohol history EtG 0.072-3.380 ng/mg, FAEE 1.30-30.60 ng/mg. The results confirm that by using a cut-off value of the sum of FAEE > 1 ng/mg and/or a positive EtG result in hair, excessive alcohol consumption can be identified using hair analysis. However, no significant correlation between the EtG and FAEE concentrations in the positive cases could be shown. Segmental analysis of some of the specimens did not reveal the same distribution for EtG compared to FAEE in hair, and no chronological accordance compared to the self-reported alcohol consumption could be observed for both parameters. These different results of both methods are discussed in terms of differences between EtG and FAEE in mechanism of formation and incorporation into hair and elimination from hair.     

Segmental determination of ethyl glucuronide in hair: a pilot study

Ethyl glucuronide (EtG) is a minor metabolite of ethanol. Its detection in hair is more and more studied in both clinical and forensic context for the purpose of alcohol abuse monitoring. In this pilot study, hair specimens from 15 patients included in a treatment program after alcohol abuse cessation, were segmented and analyzed for EtG. The results were then compared to their self-reported past alcohol consumption and to their blood biomarkers values (GGT, MCV, ASAT, ALAT). EtG concentrations measured in hair varied from 8 to 261 pg/mg. The pattern of EtG concentration detected in the different hair segments matched with the drinking history of patients, displaying variations (increase and decrease) in alcohol consumption and also time of cessation. Results also demonstrated the existence of a significant correlation (r(p)=0.5357; p=0.0390) between EtG concentration in hair and the amount of alcohol intake. Variations in the EtG concentrations with respect to hair segments may provide an overview of the drinking history of patients. Moreover, EtG concentration in hair may help to estimate the daily alcohol intake.     

Do drug users use less alcohol than non-drug users? A comparison of ethyl glucuronide concentrations in hair between the two groups in medico-legal cases

Two groups were selected from the remainder of hair samples that had been tested for drugs at TrichoTech for medico-legal cases: samples that tested negative (drug-negative group; N=42, age 33.4+/-7.2 years) and samples that tested positive for drugs (drug-positive group; N=57, age 32.5+/-8.8 years). A rapid, simple method to detect the ethanol metabolite, ethyl glucuronide (EtG) in hair has been developed. The hair samples were sectioned, and then submitted to overnight sonication in water. Samples then underwent SPE using anion exchange cartridges, followed by derivatisation with N,O-bis[trimethylsilyl]trifluoroacetamide (BSTFA), before confirmation by GC-MS/MS. The assay produced excellent linearity and sensitivity over the calibration range 0.02-1.0 ng/mg, assuming a 10 mg hair sample. The mean age of the two groups was not statistically different (p=0.575, Student t-test), indicating a homogeneous group. Twelve of the 57 (21.0%) hair samples of the drug-positive group tested positive for EtG, and 17 of the 42 (40.5%) hair samples of the drug-negative group tested positive for EtG. The mean concentration of EtG in the drug-positive group was 0.011 ng/mg compared to 0.107 ng/mg in the drug-negative group. When the full results of this study were subjected to statistical analysis it was shown that EtG levels in the drug-negative group were statistically higher than those found in the drug-positive group (p<0.05). This preliminary finding may be of use in the study of addiction and adds valuable data to previous studies regarding the use of EtG as a valuable marker for alcohol levels in hair.     

The influence of ethanol containing cosmetics on ethyl glucuronide concentration in hair

Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE), non-volatile, direct metabolites of ethanol have been shown to be suitable markers for the evaluation of social and chronic excessive alcohol consumption. Previous investigations have shown that the regular use of hair-care products with high alcohol content lead to an increase of FAEE concentration and consequently gave false-positive results for the determination of FAEE in hair. In this study we investigated the influence of a long-term hair treatment with EtOH containing lotion, on the EtG concentrations in hair. In this study 7 volunteer subjects (classified as either rare, social or heavy drinkers) treated the right side of their scalp every day during a one or two month period with a commercial hair tonic (Seborin), which contains 44.0% ethanol (vol%). Collection of hair specimens from both sides of the scalp was done one day before hair treatment, one week and one month after treatment (for 5 subjects also after two months of treatment). A hair segment of 3 centimeters (cm) was cut and then washed with water and acetone, and then pulverized. EtG was quantified by GC/MS after pulverization and 2h of ultrasonication in water, extraction by solid phase extraction using Oasis MAX columns and derivatization with HFBA. Measurements were done in negative chemical ionization mode using EtG-D5 as internal standard. Comparison of EtG concentration in the treated and in the non-treated hair specimens did not show any increase at the different dates of collection for the 7 subjects. In conclusion, these results show that there is no indication for an increase of EtG after use of ethanol containing hair cosmetics.     

Computer assisted modeling of ethyl sulfate pharmacokinetics

For 12 volunteers of a drinking experiment the concentration–time-courses of ethyl sulfate (EtS) and ethanol were simulated and fitted to the experimental data. The concentration–time-courses were described with the same mathematical model as previously used for ethyl glucuronide (EtG). The kinetic model based on the following assumptions and simplifications: a velocity constant k_form for the first order formation of ethyl sulfate from ethanol and an exponential elimination constant k_el. The mean values (and standard deviations) obtained for k_form and k_el were 0.00052 h^?1 (0.00014) and 0.561 h^?1 (0.131), respectively. Using the ranges of these parameters it is possible to calculate minimum and maximum serum concentrations of EtS based on stated ethanol doses and drinking times. The comparison of calculated and measured concentrations can prove the plausibility of alleged ethanol consumption and add evidence to the retrospective calculation of ethanol concentrations based on EtG concentrations.