Ethyl glucuronide: unusual distribution between head hair and pubic hair

Ethyl glucuronide (EtG) is a minor metabolite of ethanol that can be detected in hair. In some specific situations, head hair can be missing, and therefore, alternative anatomical locations of hair are of interest. In this study, paired hair specimens (head hair and pubic hair) from eight social drinkers were analyzed for EtG. Each sample was decontaminated by two dichloromethane bathes (5 ml) for 2 min. After cutting into small pieces, about 50 mg of hair was incubated in 2 ml water in the presence of 10 ng of EtG-d5, used as internal standard and submitted to ultra-sonication for 2 h. The aqueous phase was extracted by SPE using Oasis MAX columns. The hair extract was separated on an ACQUITY BEH HILIC column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 221-85 and 75 and m/z 226-75 for EtG and the IS, respectively. This laboratory is using a positive cut-off at 50 pg/mg. All eight head hair specimens were negative for EtG at a limit of quantitation fixed at 10 pg/mg. Surprisingly, EtG was identified at high concentrations in pubic hair, in the range 12-1370 pg/mg. It appears, therefore, that it is not possible to document the drinking status of a subject by simply switching from head hair to pubic hair.     

Sexual abuse and anti-wrinkle cream: evidence from octocrylene

In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples. The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC-MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140pg/mg. In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one diagnostic parameter.     

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.     

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.     

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.     

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。     

A comparative study on the concentrations of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in head and pubic hair

In this study, the concentrations of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in pubic, axillary and beard hair were measured and the correlation between the concentrations of THCCOOH in head and pubic hair from same cannabis users were evaluated. The papers on body hair analysis for THCCOOH were rarely found although police officers submit body hair as a complimentary specimen to forensic laboratories in case cannabis users had no hair. Head, pubic, axillary, and beard hair were collected. All hair samples were cut into 0.5mm segments and decontaminated with methanol, digested with 1 mL of 1M NaOH at 85 °C for 30 min and extracted in 2 mL of n-hexane:ethyl acetate (9:1) two times after adding 1 mL of 0.1N sodium acetate buffer (pH = 4.5) and 200 μL of acetic acid followed by derivatization with 50 μL of PFPA and 25 μL of PFPOH for 30 min at 70 °C. The extracts were analyzed using gas chromatography tandem mass spectrometry operating in negative chemical ionization mode (GC/MS/MS-NCI). We determined the concentrations of THCCOOH in both pubic and head hair. The concentrations of THCCOOH in pubic hair were higher than those in head hair. We also evaluated the concentrations of THCCOOH in body hair (pubic, axillary and beard hair) and head hair according to the positive/negative urine test results. There was no statistically significant difference in the concentrations of THCCOOH in head and body hair according to urine results.     

Methadone concentrations in human hair of the head, axillary and pubic hair

The concentrations of methadone and its metabolites in the hair of the head, axillary and pubic hair obtained from patients receiving a daily maintenance doses, were determined. Comparison of the concentrations provides the highest values in the axillary hair, followed by pubic hair and the hair of the head.     

Ethyl glucuronide in hair - A highly effective test for the monitoring of alcohol consumption

In Germany drink driving offenders lose their license and must prove abstinence for one year in order to regain it. In this paper we assess the newly introduced ethyl glucuronide (EtG) tests in urine and hair in this alcohol abstinence monitoring. 20% (80 out of 386) of the 3cm long hair samples were tested positive for EtG in hair, compared to only 2% (92 out of 4248 samples) in urine in the same time period. Additionally 50% of the samples positive for EtG in hair had EtG values greater than 30pg/mg hair, indicating chronic alcohol consumption in the last three months. This study shows that four EtG tests in 3cm hair lengths reveal a significantly higher percentage of drink driving offenders who fail to be sober in the rehabilitation period, than do six random EtG tests in urine. Presumably, the hair test is more adequate to monitor long term alcohol abstinence than the urine test as defined by the new driving license re-granting medical and psychological assessment (MPA) in Germany.     

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.     

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.     

Tetrahydrocannabinols in hair of hashish smokers

The study investigates the presence of tetrahydrocannabinols in the head hair and the pubic and axillary hair. The hair samples were obtained from hashish smokers. The concentrations were determined by radioimmunoassay and reflect total tetrahydrocannabinols and metabolites. The values found ranged from 0.4 ng/mg hair up to 3.8 ng/mg hair. The presence of the drug in the hair samples was also demonstrated by GC/MS.     

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.     

Analysis of fatty acid ethyl esters in hair as possible markers of chronically elevated alcohol consumption by headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS)

Fatty acid ethyl esters (FAEE) are products of the nonoxidative ethanol metabolism, which are known to be detectable in blood only about 24h after the last alcohol intake. After deposition in hair they should be suitable long-term markers of chronically elevated alcohol consumption. Therefore, a method for the analysis of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate from hair was developed based on the extraction of the hair sample by a dimethylsulphoxide (DMSO)/n-hexane mixture, separation and evaporation of the n-hexane phase and application of headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS) to the extract. For use as internal standards, the corresponding D(5)-ethyl esters were prepared. The HS-SPME/GC-MS measurements were automatically performed using a multi-purpose sampler. The detection limits of the FAEE were between 0.01 and 0.04ng/mg and the reproducibility was between 3.5 and 16%. By application of the method to hair samples of 21 fatalities with known heavy alcohol abuse 0.045-2.4ng/mg ethyl myristate, 0.35-13.5ng/mg ethyl palmitate, 0.25-7.7ng/mg ethyl oleate and 0.05-3.85ng/mg ethyl stearate were measured. For social drinkers (30-60g ethanol per week), the concentrations were about one order of magnitude smaller. For 10 teetotalers negative results or traces of ethyl palmitate were found. It was shown by supplementary investigations in single cases that FAEE are also present in sebum, that there is no strong difference in their concentrations between pubic, chest and scalp hair, and that they are detectable in hair segments after a 2 months period of abstinence. From the results follows that the measurement of FAEE concentrations in hair is a useful way for a retrospective detection of alcohol abuse.     

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.     

Analytical Challenge in Postmortem Toxicology Applied to a Human Body Found into a Lake after Three Years Immersion

The body of a 30‐year‐old woman was found in Como lake at a depth of about 120 meters in her own car after 3 years of immersion. The aim of this study was to evaluate psychoactive drugs as well as alcohol biomarkers in biological matrices. The following analyses were initially performed: GC‐MS systematic toxicological analysis on biological fluids and tissues; GC‐MS analysis of drugs of abuse on pubic hair; direct ethanol metabolite determination in pubic hair by LC‐MS/MS. After 7 years, the samples, that had been stored at ?20°C, were re‐analyzed and submitted to an LC‐MS/MS targeted screening method, using multiple reaction monitoring mode. These analyses detected citalopram (150–3000 ng/mL), desmethylcitalopram (50–2300 ng/mL), clotiapine (20–65 ng/mL), and ethyl glucuronide (97 pg/mg). The methods showed an acceptable reproducibility, and the concentrations of citalopram and desmethylcitalopram calculated through the two analytical techniques did not significantly differ in biological fluids.     

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。     

Correlation of methamphetamine results and concentrations between head, axillary, and pubic hair

This study was designed to compare the qualitative results and concentrations of methamphetamine (MA) and its metabolite amphetamine (AP) in head hair and hair collected from different parts of the body (axillae and pubis). Hair from subjects (N = 14) suspected MA users was simultaneously collected. Hair preparation involved washing step, fine cutting, overnight extraction, derivatization by the trifluoroacetic anhydride, and gas chromatography/mass spectrometry (GC/MS) using selective ion monitoring. In this study, we found a good correlation of the qualitative results for MA between head hair and hair on other parts of the body, but there were some differences in concentrations of MA and AP. Namely, the concentrations of MA and AP were higher in axillary and pubic hair than in head hair.     

Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault

The quali-quantitative determination of two barbiturates, thiopental and its metabolite pentobarbital, in head and pubic hair samples of a woman who had been sexually assaulted during hospitalisation, is reported. Hair was analysed by means of solid-phase microextraction (SPME) and gas chromatography-multiple mass spectrometry (GC-MS-MS), in chemical ionisation conditions. Thiopental and pentobarbital were found in three proximal head hair segments (sample 1A: 0.30 and 0.40 ng/mg; sample 1B: 0.20 and 0.20 ng/mg; sample 3: 0.15 and 0.20 ng/mg) and pubic hair sample. Two distal head hair segments were negative for both barbiturates. Despite the lack of collection and toxicological analysis of blood or urine samples within the hospital setting, analytical findings from hair revealed the use of the anaesthetic agent thiopental to sedate the victim quickly and deeply and commit sexual assault.     

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.