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.     

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.     

A pharmacokinetic study of ethyl glucuronide in blood and urine: applications to forensic toxicology

This pharmacokinetic study investigated the kinetics of ethanol and its metabolite ethyl glucuronide (EtG) in blood and urine during the whole time course of absorption and elimination. There are few previous studies on the kinetics of EtG in blood, and we wanted to evaluate whether such knowledge could yield valuable information regarding the time of ethanol ingestion in forensic cases, such as, for instance, drunk driving. Ten male volunteers consumed ethanol at a fixed dose of 0.5 g/kg body weight in a fasted state. Blood samples were collected for 14 h and urine samples were collected for 45-50 h after the start of drinking. EtG reached its maximum concentration (C(max)) in blood after a median of 4 h (range 3.5-5), a median of 3 h (range 2-4.5) after C(max) for ethanol. The ethanol-to-EtG ratios in blood (ethanol in g/L, EtG in mg/L) were >1 only for the first median 3.5 h (range 2.5-3.5) after drinking. EtG elimination occurred with a median half-life of 2.2 h (range 1.7-3.1 h), and the renal clearance was 8.32 L/h (median, range 5.25-20.86). The concentrations of EtG were always much higher in urine than in blood. The total amount of EtG excreted in the urine was median 30 mg (range 21.5-39.7), representing 0.017% (median, range 0.013-0.022) of the ethanol given, on a molar basis. The information from the present study may be a valuable supplement to determine the time of ethanol ingestion. For this purpose, two subsequent increasing EtG values and a high ethanol-to-EtG ratio in blood would support information of recent drinking.     

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.     

A kinetic model describing the pharmacokinetics of ethyl glucuronide in humans

The glucuronide conjugation is a minor pathway of ethanol metabolism. The determination of ethyl glucuronide (EG) in serum and urine has gained importance in forensic and other legal decisions. To prospectively calculate the serum concentration of this non-oxidative ethanol metabolite, the computer program developed includes a parameter fitting routine. Multiple ethanol doses can be handled.The mathematical modeling was based on the following assumptions and simplifications, respectively. A single enzyme system is responsible for ethanol conjugation at one distinct site; the distribution of EG into the systemic circulation is delayed; the elimination of EG follows first-order kinetics.The concentration of EG was calculated using three kinetic parameters: a rate constant for the first-order formation of EG from serum ethanol, a transfer constant for an obstructed transfer of EG from the formation site (FS) into the central compartment (CC) and an exponential elimination constant.The program was applied to the data collected from 21 drinking experiments. The fitting algorithm optimized the three kinetic parameters, until the sum of concentration error squares of the data points was minimized. The means+/-standard deviation of the rate constant for the first-order formation of EG from serum ethanol was 0.0011+/-0.0006 h(-1), the transfer constant for an obstructed transfer of EG from the FS into the CC was 0.43+/-0.1996 h(-1) and the exponential elimination constant was 3.0+/-1.45 h(-1).Using the range of these parameters, it is now possible to calculate minimum and maximum serum concentrations of EG based on ethanol doses and drinking times. The comparison of calculated and measured concentrations can prove the plausibility of an alleged ethanol consumption. This can be crucial when the serum ethanol concentration (SEC) itself is not meaningful.     

Disappearance of ethyl glucuronide during heavy putrefaction

Adrenocorticotropic hormone (ACTH) is involved in systemic reactions to stress. The aim of the present study was a comprehensive analysis of serum and cerebrospinal fluid (CSF) levels of ACTH, and the pituitary immunohistochemistry with special regard to fatal hypothermia in routine forensic autopsy cases (n=162: 5-97 years of age; 114 males and 48 females; 4 h to 3 days postmortem, median, 19.2 h). The ACTH concentrations were independent of the postmortem time, gender, or age of the subjects. The serum ACTH level was similar to the clinical reference value for sharp instrument injury, fire fatality, and hypothermia, but was lower in other groups including hyperthermia, in particular for asphyxia and poisoning. The CSF level was usually much higher than the serum level, but was significantly lower for hypothermia and hyperthermia than in other groups (p<0.01). The rate of ACTH-immunopositivity in the anterior pituitary was low in cases of fatal hypothermia and hyperthermia, while it was high in cases of blunt injury, fire fatality, and acute ischemic heart disease. These observations showed that ACTH levels in the serum and CSF depended on the cause of death. The serum level was maintained despite a low CSF level and pituitary immunopositivity for fatal hypothermia, while the serum and CSF levels as well as pituitary immunopositivity were decreased for hyperthermia.     

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

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。     

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.     

液相色谱-串联质谱法测定血液、尿液中乙基葡萄糖醛酸苷

目的建立血液、尿液中乙基葡萄糖醛酸苷（ethyl glucuronide,EtG）的液相色谱-串联质谱（LC-MS/MS）检测方法。方法血液、尿液用乙腈沉淀蛋白,离心后取上清液用LC-MS/MS检测。结果血液、尿液中EtG的检出限均为0.05μg/mL,线性范围均为0.10～5.00μg/mL（r〉0.999）,检测方法准确度为95%～109%,日间及日内精密度〈12%。对送检案例血液中EtG进行检测,效果良好。结论本方法适用于血液、尿液中EtG的检测。     

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.     

高效液相色谱-质谱法测定人尿液中乙基葡萄糖醛酸苷

目的建立高效液相色谱-质谱法测定人尿液中乙基葡萄糖醛酸苷。方法取空白人尿液加入EtG-D5内标工作液和冷甲醇（4℃）,经涡旋、离心,取上清液于水浴氮气吹干,以沉淀杂质,采用Agilent Zorbax Bonus-RP色谱柱分离,以乙腈∶0.1%甲酸水溶液（5∶95）为流动相,采用负离子模式质谱检测,在选择离子监测模式下进行定性和定量测定。结果尿液中EtG在50～5 000ng/mL范围内线性关系良好,检出限为18ng/mL,方法回收率为90.2%～101.7%,相对标准偏差小于10%。结论所建方法简便,具有较高的回收率和精密度,适用于尿液中EtG的检测。     

Determination of ethyl glucuronide in human hair by SPE and LC-MS/MS

A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Washed and cut hair segments were extracted by ultrasonication (3h, 50 degrees C) and the extracts were cleaned-up with aminopropyl SPE columns. LC-MS/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation (ESI) using a triple quadrupole mass spectrometer (Sciex API 365) with a turboionspray source and post-column addition of acetonitrile for enhanced sensitivity. The MS/MS transitions monitored were m/z 221 -->75 for EtG and 226 -->75 for D(5)-EtG as an internal standard. The method was selective and sensitive, with a detection limit of 51 pg/mg hair at a signal-to-noise ratio of 3:1. The mean recovery was 96%, with an intra- and inter-day precision of less than 11.7% at a concentration of 200 pg/mg. The linearity was assessed in the range of 25-2000 pg/mg hair, with a correlation coefficient of 0.997. The method was successfully applied to 97 human hair samples which were taken at autopsies from persons with known alcoholism or were obtained from alcoholics who were hospitalized for ethanol withdrawal, from social drinkers and from children having not consumed any alcohol. Although, approximately two-third of the alcoholics showed EtG concentrations in hair of higher than 51 pg/mg (up to >4000 pg/mg), in one-third the EtG concentration was below the detection limit. However, only in one of five hair samples of "social drinkers", the EtG concentration was above the detection limit (51 pg/mg). No EtG has been detected in the hair of children. These investigations demonstrate that heavy alcohol consumption may be but not necessarily has to be detectable by EtG analysis in hair.     

Practical experiences in application of hair fatty acid ethyl esters and ethyl glucuronide for detection of chronic alcohol abuse in forensic cases

This article presents results from 1872 hair samples, which were analyzed for fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG). The results were evaluated in the context of self-reported drinking behavior, the use of hair cosmetics, the gender of the sample donors and hair sample length. For comparison, CDT and GGT in serum were available in 477 and 454 cases, respectively. A number of alcohol abstainers or low moderate drinkers and excessive drinkers were selected for assessment of cut-offs for FAEEs in the proximal 6cm hair segments and for EtG in the proximal 3cm hair segments. Cut-off values were assessed by ROC analysis. It was found that the cut-offs of 1.0ng/mg FAEE and 30pg/mg EtG presently used for excessive drinking lead to a low portion of false positives (4% and 3% respectively) but to a higher portion of false negatives (23% and 25% respectively). Comparison of the mean and medium concentrations in samples without any reported hair cosmetics (N=1079) and in samples with reported use of hair spray (N=79) showed an increase by the factor of about two for FAEE but no significant difference for EtG. Mean values of EtG were decreased by 80% in bleached samples (N=164) and by 63% in dyed samples (N=96). There was no significant effect of bleaching and dyeing on FAEE. Hair gel and hair wax, oil or grease showed no significant effect on both FAEE and EtG. With respect to gender and investigated hair length ambiguous results were obtained because of major differences in the compared subpopulations of male with higher alcohol consumption and mainly shorter hair, and less drinking female with longer hair. For excessive drinkers FAEEs in the 0-6cm hair segment and EtG in the 0-3cm segment decreased with increasing time of reported abstinence before sample collection. These drinkers attain the level of teetotalers only after more than 10 months of abstinence. In comparison to scalp hair, FAEEs recovered from armpit hair and leg hair were lower and from chest hair were higher. EtG in armpit hair was lower and in leg hair higher than in scalp hair. It is concluded that the combined use of FAEE and EtG essentially increases the accuracy of interpretation since both markers complement each other by a different sensitivity to sources of error.     

Fatal workplace accident involving ethyl acetate: a distribution study

The tissue distribution of ethyl acetate and ethanol in a case of acute intoxication by ethyl acetate is presented. The victim was a 39-year-old man who was found dead lying on his abdomen in the interior of a tank containing ethyl acetate. Confirmation of ethyl acetate was obtained with static headspace gas chromatography with mass spectrometry. In blood, rapid biotransformation of ethyl acetate occurs by plasma esterases resulting in acetic acid and ethanol. Quantitation of ethyl acetate and ethanol in the postmortem samples was performed using static headspace gas chromatography with flame ionization detector. N-butanol was used as internal standard. Separation of the compounds was obtained on a Supelcowaxtrade mark-10 Fused Silica capillary column. The method was linear over the specific ranges investigated and showed a within-run accuracy of 99.8 and 101.0% and a precision of 0.5 and 2.0% for ethanol and ethyl acetate, respectively. The postmortem samples were analyzed in duplicate or triplicate. Coefficients of variation were < or =4.51% for ethyl acetate and < or =0.52% for ethanol. The low ratios of the ethyl acetate concentration to the ethanol concentration found in the postmortem tissue samples confirmed the rapid in vivo biotransformation of ethyl acetate. The highest concentration of ethyl acetate was found in the testis indicating that postmortem percutane absorption may have occurred. To our knowledge, this is the first reported tissue distribution study of ethyl acetate and ethanol in a case of acute intoxication by ethyl acetate.     

Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs

While ethanol is primarily metabolized to acetaldehyde and acetic acid via alcohol dehydrogenase, a minor but increasingly important pathway in the field of forensic science involves the conjugation of glucuronic acid to form an ethyl glucuronide (EtG) metabolite. The kinetics of ethyl glucuronide formation were examined in human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferases (UGTs). The metabolite exhibited a relatively slow rate of formation in a human liver microsome mix of 75.4 pmol/(min/mg). Further investigation identified multiple UGT isoforms to be responsible for catalyzing the addition of glucuronic acid to ethanol, with UGT1A1 and 2B7 being the two most prevalent isoforms. Co-incubation with bilirubin or 3'-azido-3'-deoxythymidine (UGT1A1 and 2B7 inhibitors, respectively) inhibited the greatest amount of ethyl glucuronide formation, though other UGT inhibitors also showed some effect. Enzyme kinetics were performed in human liver microsomes and recombinant UGT enzymes. The apparent Km (Km app) and Vmax values were determined to be 0.17+/-0.08 mM and 75.98+/-5.63 pmol/(min/mg) (human liver microsomes), 0.03+/-0.01 mM and 25.22+/-3.45 pmol/(min/mg) (UGT1A1), and 0.11+/-0.04 mM and 52.03+/-9.8 pmol/(min/mg) (UGT2B7). Thus, it appears that multiple UGTs are responsible for the formation of ethyl glucuronide and that any functional differences in the enzymology underlying ethyl glucuronide formation would most likely be masked by a combination of other enzymatic pathways.     

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.     

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.     

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.