Alcohol consumption during pregnancy can lead to Fetal Alcohol Spectrum Disorder, and because maternal self-reports are often unreliable, a biomarker of alcohol use during pregnancy is needed to accurately determine fetal exposure. Ethyl glucuronide (EtG) is a direct metabolite of ethanol that has been detected in the meconium of infants born to mothers who consumed alcohol during pregnancy. In the current study, a method was developed and validated for EtG detection in placental perfusate and tissue using gas chromatography-mass spectrometry. Subsequently, the ex vivo human placental perfusion model was used to investigate whether EtG crosses the human placenta. The validated GC-MS method showed sufficient sensitivity in detecting EtG in placental perfusate and tissue. EtG crossed the placenta slowly and transfer was incomplete after 3 hours of perfusion. EtG appears to cross the human placenta and, hence, to represent both maternal and fetal exposure to alcohol.

Alcohol consumption during pregnancy can lead to Fetal Alcohol Spectrum Disorder, and because maternal self-reports are often unreliable, a biomarker of alcohol use during pregnancy is needed to accurately determine fetal exposure. Ethyl glucuronide (EtG) is a direct metabolite of ethanol that has been detected in the meconium of infants born to mothers who consumed alcohol during pregnancy. In the current study, a method was developed and validated for EtG detection in placental perfusate and tissue using gas chromatography-mass spectrometry. Subsequently, the ex vivo human placental perfusion model was used to investigate whether EtG crosses the human placenta. The validated GC-MS method showed sufficient sensitivity in detecting EtG in placental perfusate and tissue. EtG crossed the placenta slowly and transfer was incomplete after 3 hours of perfusion. EtG appears to cross the human placenta and, hence, to represent both maternal and fetal exposure to alcohol.

Ethyl glucuronide (EtG) is a minor ethanol metabolite that confirms the absorption and metabolism of ethanol after oral or dermal exposure. Human data suggest that maximum blood EtG (BEtG) concentrations are reached between 3.5 and 5.5. h after ethanol administration. This study was undertaken to determine if the Sprague-Dawley (SD) rat biotransforms ethanol to EtG after a single high oral dose of ethanol. SD rats (male, n=6) were gavaged with a single ethanol dose (4g/kg), and urine was collected for 3. h in metabolic cages, followed by euthanization and collection of heart blood. Blood and urine were analyzed for ethanol and EtG by gas chromatography and enzyme immunoassay. Blood and urine ethanol concentrations were 195 ± 23 and 218 ± 19. mg/dL, whereas BEtG and urine EtG (UEtG) concentrations were 1,363 ± 98. ng equivalents/mL and 210 ± 0.29. mg equivalents/dL (X̄±standarderrorofthemean[S.E.M.]).Sixty-six male SD rats were gavaged ethanol (4. g/kg) and placed in metabolic cages to determine the extent and duration of ethanol to EtG biotransformation and urinary excretion. Blood and urine were collected up to 24. h after administration for ethanol and EtG analysis. Maximum blood ethanol, urine ethanol, and UEtG were reached within 4. h, whereas maximum BEtG was reached 6. h after administration. Maximum concentrations were blood ethanol, 213 ± 20. mg/dL; urine ethanol, 308 ± 34. mg/dL; BEtG, 2,683 ± 145. ng equivalents/mL; UEtG, 1.2 ± 0.06. mg equivalents/mL (X̄±S.E.M.). Areas under the concentration-time curve were blood ethanol, 1,578. h*mg/dL; urine ethanol, 3,096. h*mg/dL; BEtG, 18,284. h*ng equivalents/mL; and UEtG, 850. h*mg equivalents/dL. Blood ethanol and BEtG levels were reduced to below limits of detection (LODs) within 12 and 18. h after ethanol administration. Urine ethanols were below LOD at 18. h, but UEtG was still detectable at 24. h after administration. Our data prove that the SD rat biotransforms ethanol to EtG and excretes both in the urine and suggest that it is similar to that of the human.

Alcohol is the most abused drug in the United States and most frequently performed assay in forensic laboratories. Alcohol is routinely present in biological specimens from fatal residential fires and forensic toxicologists must interpret if these individuals are impaired by determination of their blood alcohol concentrations on post-incineration blood collected at autopsy. There is no known data available to confirm or refute blood alcohol concentrations and impairment in fire-related deaths. Ethyl glucuronide (EtG), a non-volatile minor ethanol metabolite, may provide a better biomarker for ethanol consumption prior to burn injury. The literature does not address the possibility that ethanol or EtG concentrations are altered in fire deaths. A Sprague Dawley rat model was employed to determine if ethanol and EtG concentrations in blood, liver, heart, and kidney were altered after burn injuries using two incineration models with varying durations and temperatures. Blood and tissues were analyzed for ethanol by gas chromatography and EtG by enzyme immunoassay. Other measurements including organ weights, lower hindquarter weights, and blood glucose concentrations were chosen for analysis to determine the mechanism by which the blood and organ ethanol and EtG concentrations are altered in burnt corpses. The rodent provided an excellent model for studying the biotransformation of ethanol to EtG and the effects of burn injury on ethanol and EtG concentrations. Our study revealed that blood ethanol concentrations were not significantly altered by burn injury but tissue ethanol concentrations were altered by burn injury. EtG concentrations were found to be altered in blood and tissue specimens in both incineration models. Our data suggest that the change in ethanol and EtG concentrations may be correlated to higher core body temperatures from burn injury and not changes in organ weight. Determining if blood ethanol concentrations are altered in burnt corpses is important for forensic toxicologists to conclude if victims were impaired at the time of death. The knowledge gained from these experiments will help forensic toxicologists by confirming the current interpretation that blood ethanol concentrations are not altered in fire deaths and provide a better understanding for the interpretation of impairment in burn deaths.

The hip-flask defence is a common claim in drunkdrinking cases. In Sweden and Norway two different models are used to determinethese cases. In Sweden one blood and two urine samples taken 60 minutes apartare used for analysis. In Norway two blood samples taken 30 minutes apart areused. Sweden focuses on the rise or fall of alcohol concentration in urine(UAC), and the ratio between UAC and blood alcohol concentrations (BAC). Norwayfocuses on the rise or fall of the alcohol metabolite ethylglucuronide (EtG) and the ratio between BAC and EtG. The aim of this study wasto test the models for multiple intakes and with different alcoholic beverages.Thirtyfive participants ingested two doses, first0.51 g/kg of beer and later either 0.25, 0.51 or 0.85 g/kg of beer, wine orvodka. Blood and urine samples were obtained before and after alcoholingestion. Alcohol was measured by GC-HS, and the alcohol metabolite byUPLC-MS/MS. The results showed that there are kineticdifferences between single and repeated intakes, that there are no significantdifferences in kinetics from different alcoholic beverages and thatthe Norwegian model appears to be the stronger one in hip-flask determination.

Obwohl EtG seit dem Jahr 2000 intensiv als Alkoholmarker in Haaren beforscht wird, bietet die Thematik weiterhin Raum für Forschung, insbesondere im Bereich der instrumentellen Analytik. Ziel der vorliegenden Arbeit ist die Beleuchtung dieser und weiterer Aspekte. Die Extraktion erfolgte überwiegend mittels der sogenannten Mikropulverisierung. Sie ermöglichte die simultane Mahlung der Haarmatrix und Extraktion des EtGs mit einem hohen Probendurchsatz. Die Selektion und anschließende Detektion erfolgte überwiegend durch HPLC-MS/MS. Die Sicherheit bei der Bestimmung des Analyten wurde durch die erfolgreiche Teilnahme an drei Ringversuchen der Society of Hair Testing (SoHT) belegt. Wiederholbedingungen wurden durch Herstellung von eigenen Haarreferenzmaterialien und die Verwendung von homogenen Fremdhaarmaterialien sichergestellt. Zur Evaluierung der Stabilität von EtG wurden zwei Haarmaterialien unter thermischen Stressbedingungen eingelagert und mit dem Gehalt von Referenzproben verglichen. Der Analyt zeigte außergewöhnliche Stabilität unter den gewählten Bedingungen. Ebenso erfolgte eine Beurteilung des Zerstörungsgrads von EtG im Haar durch oxidierende Substanzen, einhergehend mit der Entwicklung eines zerstörungsfreien Schnelltests mittels FTIR zur Detektion von oxidierten Cysteinspezies in Haaren. Das Modellsystem Barthaar wurde für zwei Experimentreihen etabliert: die Korrelation des EtG-Gehaltes im Barthaar nach Aufnahme definierter Alkoholmengen und den Nachweis von glucuronidierten Spezies im Barthaar nach Aufnahme der korrespondierenden Muttersubstanzen. Während keine eindeutige Korrelation zwischen aufgenommener Alkoholmenge und EtG-Gehalt im Barthaar hergestellt werden konnte, war es durchaus möglich, zwei glucuronidierte Metabolite von Arzneistoffen im Barthaar nach Konsum der Ausgangssubstanzen nachzuweisen. / Although EtG is subject to extended research since the year 2000, the topic still holds headroom for further experiments, especially when it comes to the field of instrumental analysis. The goal of the present thesis was the clarification of crucial analytical and further aspects. The extraction was mostly carried out using the so-called micropulverisation. It rendered the simultaneous milling of the hair matrix and extraction of EtG possible with a high sample throughput. Selection of the analyte and following detection was mainly carried out using HPLC-MS/MS. The quality of analysis was ensured by the successful participation in three interlaboratory tests carried out by the Society of Hair Testing (SoHT). Repetitive conditions were ensured by manufacturing of own hair reference materials as well as by the usage of homogeneous external hair materials. Two hair materials were treated under thermal stress conditions and the EtG values were compared to reference samples to verify the analytes stability. EtG showed extraordinary stability under the chosen conditions. Likewise, an assessment of the degree of EtG decay after oxidative treatment as well as the development of a nondestructive assay via FTIR to detect oxidized cysteine species were established. The model system beard hair was arranged for the conduction of two experimental series: the correlation of the EtG content in beard hair after defined oral consumption of ethanol and the detection of glucuronidation of the corresponding parent substances after consumption. Whilst no distinct correlation could be observed for the ethanol experiment, it was possible to provide evidence for the existence of two glucuronized metabolites of drugs after consumption of the parent compounds.

Drug facilitated assault (DFA) is an increasing problem in the UK. The crime often occurs through the surreptitious administration of a drug into a victims drink, rendering the victim unable to resist the assault. The detection of these drugs in a biological specimen from the victim is one of the most challenging facets of forensic chemistry. Drug concentrations can be very low, as often only a single dose is administered, and the pharmacodynamics of commonly employed drugs further hinders the testing process. The research presented in this work shows the development of several new assays for the detection of flunitrazepam, gamma-hydroxybutyrate (GHB) and ethyl glucuronide (EtG) in a variety of biological matrices. New methods of drug testing in blood and urine are demonstrated, as well as interesting developments in the field of hair testing. Using hair to detect drug exposure allows a much wider window of detection than the more traditional matrices of blood and urine. New methods are presented in this work using gas chromatography-tandem mass spectrometry (GCMS/MS) to detect drugs in hair. Validation data is presented along with the results of authentic DFA testing. All aspects of the drug testing procedure have been evaluated, from new extraction techniques utilising water instead of solvents, to novel clean up stages involving the unique combination of SFE and SPME. Several confirmation techniques are explored including single quadrupole, triple quadrupole and ion trap mass spectrometry. In addition to developing assays for DFA cases, the versatility of this type of analytical chemistry is explored in two population studies. The first study evaluates alcohol consumption between two groups; drugs users and non drug users in medico-legal cases. There is an anecdotal belief amongst drug clinic staff that alcohol use is lower in drugs users than it is in non drug users. This study presents the first scientific confirmation of this belief through EtG (an alcohol metabolite) testing in hair of the two groups. The second study investigates whether there is a correlation between EtG and cocaethylene (a metabolite of cocaine only produced in the presence of alcohol) in cocaine users. Results f this study suggest that there is no positive correlation between the two compounds. The research presented in this thesis aims to further the analytical science surrounding FA investigation and provide accurate, sensitive and reliable methodology for drug esting in blood, urine and hair.