Preanalytical factors influencing the results of ethanol analysis in postmortem specimens.

Excessive drinking and drunkenness are underlying factors in many fatal accidents, which make the quantitative determination of ethanol in postmortem (PM) specimens an essential part of all unnatural death investigations. The same analytical methods are used to determine ethanol in blood taken from living and deceased persons although the interpretation of the results is more complicated in medical examiner cases owing to various preanalytical factors. The biggest problem is that under anaerobic conditions ethanol can be produced naturally in decomposed bodies by microbial activity and fermentation of blood glucose. Ways are needed to differentiate antemortem ingestion of ethanol from PM synthesis. One approach involves the determination of ethanol in alternative specimens, such as bile, cerebrospinal fluid, vitreous humor and/or urine, and comparison of results with blood alcohol concentration (BAC). Another approach involves the analysis of various alcohol biomarkers, such as ethyl glucuronide, ethyl sulfate and/or phosphatidylethanol or the urinary metabolites of serotonin 5-hydroxytryptophol/5-hydroxyindoleacetic acid (5-HTOL/5-HIAA). If ethanol had been produced in the body by microbial activity, the blood samples should also contain other low-molecular volatiles, such as acetaldehyde, n-propanol and/or n-butanol. The inclusion of 1-2% w/v sodium or potassium fluoride, as an enzyme inhibitor, in all PM specimens is essential to diminish the risk of ethanol being generated after sampling, such as during shipment and storage prior to analysis. Furthermore, much might be gained if the analytical cut-off for reporting positive BAC was raised from 0.01 to 0.02 g% when PM blood is analyzed. During putrefaction low BACs are more often produced after death than high BACs. Therefore, when the cadaver is obviously decomposed, a pragmatic approach would be to subtract 0.05 g% from the mean analytical result. Any remaining BAC is expected to give a more reliable indication of whether alcohol had been consumed before death.

Highly cited forensic practitioners in the Nordic countries and their composite citation scores based on six different citation metrics.

Forensic science is a multidisciplinary field that involves the use of various scientific methods and techniques for the investigation of crimes. Forensic scientists are often required to testify in court as expert witnesses and explain the meaning of chemical, physical, and/or medical evidence to a judge and jury. This article took advantage of a citation database to identify the most highly cited forensic practitioners in four Nordic countries within the discipline of legal and forensic medicine. The starting point was 7 million scientists indexed in the SCOPUS database, each of whom had their name on at least five entries. Six different citation metrics were used to calculate a person's composite citation score. Those within the top 2% of their primary research discipline were considered highly cited.

Preanalytical Factors Influencing the Stability of Ethanol in Antemortem Blood and Urine Samples.

The quantitative analysis of ethanol in blood and other biological specimens is a commonly requested service from forensic science and toxicology laboratories worldwide. The measured blood alcohol concentration (BAC) constitutes important evidence when alcohol-related crimes are investigated, such as drunken driving or drug-related sexual assault. This review article considers the importance of various preanalytical factors that might influence changes in the ethanol concentration in blood after collection and before analysis or reanalysis after various periods of storage. When blood samples were collected by venipuncture from living subjects in evacuated tubes containing sodium fluoride (NaF) preservative, there was no evidence that the BAC increased after collection. Most studies found that the BAC decreased after collection depending on storage conditions, such as time and temperature, and the amount of NaF preservative. After the storage of blood specimens in a refrigerator (4oC) for up to 1-4 weeks, the changes in the BAC were not analytically significant. After storage for up to 12 months at 4oC, under the same conditions, the BAC decreased on average by 0.01-0.02 g%. The loss of ethanol does not appear to depend on the type of evacuated tubes used (glass or plastic), nominal volume (5 mL or 10 mL) or the amount of NaF preservative. Urine alcohol concentrations were also stable after various periods of storage, although in cases of glycosuria and urinary tract and/or Candida infections, the addition of NaF (1% w/v) was essential to prevent post-sampling synthesis of ethanol.

Prevalence and concentrations of sedative-hypnotic drugs in blood of drivers involved in road traffic crashes in the Padova region of Italy - not so easy to interpret.

BACKGROUND & OBJECTIVES: This study reports the prevalence and concentrations of sedative-hypnotic drugs as exemplified by benzodiazepines (BZD) and zolpidem (Z-hypnotic) in blood samples from drivers involved in road traffic accidents (RTA) in the Padova region of Italy. Another aim of the study was to estimate the prevalence of these drugs with concentrations in blood above the therapeutic intervals and above specific per se limits. METHODS: A total of 4066 blood samples collected from drivers involved in RTA were analysed for the presence of alcohol, drugs of abuse and medicinal drugs with sedative-hypnotic properties. Prevalence of drivers positive for BZDs and zolpidem were reported according to the reporting limit of our laboratory (1 ng/mL) in a sort of zero tolerance approach and compared with the prevalence according to analytical cut-offs used in the "European Union's research project on Driving Under the Influence of Drugs, Alcohol and Medicines" (DRUID). The impairment-based, per se limits adopted in Norway and in England and Wales and the values used to define "therapeutic ranges" in blood and in plasma/serum were also applied to the case study. RESULTS: 175 blood samples were positive for sedative-hypnotics above 1 ng/mL, with the following prevalence: diazepam 44%, nordazepam 41.8%, lorazepam 32.6%, zolpidem 28%, oxazepam 25.6%, alprazolam 16%, delorazepam 11,6%, lormetazepam 11,6%, temazepam 11.6%, clonazepam 11.6%, triazolam 6.9%, N-desalkylflurazepam 4.6%, bromazepam 2.3%. When applying DRUID analytical cut-offs, the prevalence of BZDs and zolpidem sharply decreases. Applying the impairing cut-offs used in Norway, 56% of positive samples were above the limits equivalent to a BAC of 0.2 g/L, 39% above the limits corresponding to 0.5 g/L, and 23% above the cut-off corresponding to 1.2 g/L. Only 1% of the drivers had drug concentrations above the per se concentration limits adopted in England and Wales [26]. When comparing blood levels with therapeutic ranges in plasma, bromazepam, lormetazepam and delorazepam were often found above the highest limits. The adjustment of the concentrations with the plasma-to-blood ratios causes a significant increase of cases above the therapeutic ranges in plasma. CONCLUSIONS: Sedative-hypnotic drugs are medicinal substances frequently identified in drivers involved in RTA, commonly in concentrations associated with driving impairment. Besides the concentrations of drugs in blood, several factors have to be considered to conclude that a driver was impaired. The frequent association with alcohol, cocaine and other BZDs, confirms the abuse potential of these medications.

Impact of Trauma, Massive Blood Loss and Administration of Resuscitation Fluids on a Person's Blood-Alcohol Concentration and Rate of Ethanol Metabolism.

Excessive drinking and drunkenness are tightly linked to many types of intentional and unintentional injuries involving trauma and blood loss, which often necessitate emergency medical intervention. This article considers the impact of trauma, massive blood loss, and the administration of life-saving replacement fluids on a person's blood alcohol concentration (BAC) and rate of ethanol metabolism. Both German and English language journals were reviewed and results from animal experiments, human studies, and actual victims of trauma undergoing life-saving treatment were considered. If trauma-related bleeding occurs when some ingested alcohol remains unabsorbed in the stomach, then under these circumstances continued absorption into portal venous blood is delayed, owing to altered splanchnic circulation. Hemodilution caused by administration of replacement fluids has only minimal effects on a preexisting BAC, because ethanol distributes into the total body water (TBW) compartment, which represents 50-60% of body weight. After hypovolemia there is a transfer of fluids from tissue compartments into the blood, which becomes more like plasma in composition with lower hematocrit and hemoglobin content. Unless the trauma or emergency treatment impedes hepatic blood flow, the rate of ethanol metabolism is not expected to differ from normal values, namely 0.10-0.25 g/L/h (0.01-0.025 g% per h). If ethanol is fully absorbed and distributed in all body fluids and tissues, neither massive blood loss nor administration of resuscitating fluids is expected to have any significant effect on a preexisting BAC or the rate of ethanol metabolism.

GHB pharmacology and toxicology: acute intoxication, concentrations in blood and urine in forensic cases and treatment of the withdrawal syndrome.

The illicit recreational drug of abuse, γ-hydroxybutyrate (GHB) is a potent central nervous system depressant and is often encountered during forensic investigations of living and deceased persons. The sodium salt of GHB is registered as a therapeutic agent (Xyrem®), approved in some countries for the treatment of narcolepsy-associated cataplexy and (Alcover®) is an adjuvant medication for detoxification and withdrawal in alcoholics. Trace amounts of GHB are produced endogenously (0.5-1.0 mg/L) in various tissues, including the brain, where it functions as both a precursor and a metabolite of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). Available information indicates that GHB serves as a neurotransmitter or neuromodulator in the GABAergic system, especially via binding to the GABA-B receptor subtype. Although GHB is listed as a controlled substance in many countries abuse still continues, owing to the availability of precursor drugs, γ-butyrolactone (GBL) and 1,4-butanediol (BD), which are not regulated. After ingestion both GBL and BD are rapidly converted into GHB (t½ ~1 min). The Cmax occurs after 20-40 min and GHB is then eliminated from plasma with a half-life of 30-50 min. Only about 1-5% of the dose of GHB is recoverable in urine and the window of detection is relatively short (3-10 h). This calls for expeditious sampling when evidence of drug use and/or abuse is required in forensic casework. The recreational dose of GHB is not easy to estimate and a concentration in plasma of ~100 mg/L produces euphoria and disinhibition, whereas 500 mg/L might cause death from cardiorespiratory depression. Effective antidotes to reverse the sedative and intoxicating effects of GHB do not exist. The poisoned patients require supportive care, vital signs should be monitored and the airways kept clear in case of emesis. After prolonged regular use of GHB tolerance and dependence develop and abrupt cessation of drug use leads to unpleasant withdrawal symptoms. There is no evidence-based protocol available to deal with GHB withdrawal, apart from administering benzodiazepines.

Concentrations of diazepam and nordiazepam in 1,000 blood samples from apprehended drivers--therapeutic use or abuse of anxiolytics?

Using an in-house forensic toxicology database, we selected 1000 cases of driving under the influence of drugs (DUIDs) over a 12-month period if diazepam (D) and nordiazepam (ND) were both present in the blood samples. Quantitative analysis of D and ND in blood was done by solvent extraction (butyl acetate) and capillary column gas chromatography (GC) with a nitrogen-phosphorous (N-P) detector. The limits of quantitation of this analytical method for D and ND in blood were 0.05 mg/L. The correlation between D and ND concentrations in blood was statistically significant (r = .58, P < .001), as expected for a parent drug and its primary metabolite. However, the frequency distributions were markedly skewed to the right with mean (median) and highest concentrations of 0.37 (0.20) and 6.1 mg/L for D and 0.39 (0.20) and 5.6 mg/L for ND. The mean (median) total concentration (D + ND) was 0.76 mg/L (0.50 mg/L), and the concentration ratios D/ND and ND/D were 1.29 (median 0.95) and 1.41 (median 1.06), respectively. In 90 cases (9%), the concentration of D in blood exceeded 0.83 mg/L, which corresponds to an upper therapeutic limit in plasma (∼1.5 mg/L), assuming a plasma/blood distribution ratio of 1.8:1.

Toxicological analysis of blood and urine samples from female victims of alleged sexual assault.

BACKGROUND: The toxicological analysis of blood and urine samples from victims of alleged sexual assault represents a crucial part of the forensic evidence when this crime is investigated. MATERIAL AND METHODS: We searched a national forensic toxicology database (TOXBASE) to find cases registered as sexual assault, rape, including date-rape that the police had requested the analysis of ethanol and other drugs. Between 2008 and 2010, N = 1460 such cases met this criteria. After immunological screening of urine or blood samples, all positive results were verified by more specific analytical methods, such as gas chromatography-mass spectrometry (GC-MS) for illicit drugs. A large number of prescription drugs and their metabolites were determined by capillary GC with nitrogen-phosphorous (N-P) detector. GC with flame ionization detector (FID) was used to analyze ethanol and gamma-hydroxybutyrate (GHB) in blood at limits of quantitation (LOQ) of 0.1 g/L and 8 mg/L, respectively. RESULTS: The average age (± standard deviation) of all victims was 24 ± 10.3 years and 72% were between 15 and 29 years. Ethanol and other drugs were not detected in 31% of cases (N = 459). Blood-ethanol was positive in N = 658 cases at mean, median and highest concentrations of 1.23 g/L, 1.22 g/L and 4.3 g/L, respectively. Ethanol plus drugs were present in N = 188 cases (13%) and one or more other drugs alone in N = 210 cases (14%). Cannabis (marijuana) and amphetamines were the major illicit drugs, whereas diazepam, alprazolam, zopiclone as well as newer antidepressants were the major prescription drugs identified. CONCLUSIONS: The mean age of victims of sexual assault in Sweden, the proportion of drug positive to drug negative cases, the predominance of ethanol positive cases as well as the types of other drugs showed a remarkably good agreement in two studies spanning a period of 8 years.

Driving under the influence of cannabis: a 10-year study of age and gender differences in the concentrations of tetrahydrocannabinol in blood.

BACKGROUND: Delta(9)-Tetrahydrocannabinol (THC) is the major psychoactive constituent of cannabis and its various preparations. Increasing use of cannabis for recreational purposes has created a problem for road-traffic safety. This paper compares age, gender and the concentrations of THC in blood of individuals apprehended for driving under the influence of drugs (DUID) in Sweden, where a zero-tolerance law operates. MEASUREMENTS: Specimens of blood or urine were subjected to a broad screening analysis by enzyme immunoassay methods. THC positives were verified by analysis of blood by gas chromatography-mass spectrometry (GC-MS) with a deuterium-labelled internal standard (d(3)-THC). All toxicology results were entered into a database (TOXBASE) along with the age and gender of apprehended drivers. FINDINGS: Over a 10-year period (1995-2004), between 18% and 30% of all DUID suspects had measurable amounts of THC in their blood (> 0.3 ng/ml) either alone or together with other drugs. The mean age [+/- standard deviation (SD)] of cannabis users was 33 +/- 9.4 years (range 15-66 years), with a strong predominance of men (94%, P < 0.001). The frequency distribution of THC concentrations (n = 8794) was skewed markedly to the right with mean, median and highest values of 2.1 ng/ml, 1.0 ng/ml and 67 ng/ml, respectively. The THC concentration was less than 1.0 ng/ml in 43% of cases and below 2.0 ng/ml in 61% of cases. The age of offenders was not correlated with the concentration of THC in blood (r = -0.027, P > 0.05). THC concentrations in blood were higher when this was the only psychoactive substance present (n = 1276); mean 3.6 ng/ml, median 2.0 ng/ml compared with multi-drug users; mean 1.8 ng/ml, median 1.0 ng/ml (P < 0.001). In cases with THC as the only drug present the concentration was less than 1.0 ng/ml in 26% and below 2.0 ng/ml in 41% of cases. The high prevalence of men, the average age and the concentrations of THC in blood were similar in users of illicit drugs (non-traffic cases). CONCLUSIONS: The concentration of THC in blood at the time of driving is probably a great deal higher than at the time of sampling (30-90 minutes later). The notion of enacting science-based concentration limits of THC in blood (e.g. 3-5 ng/ml), as discussed in some quarters, would result in many individuals evading prosecution. Zero-tolerance or limit of quantitation laws are a much more pragmatic way to enforce DUID legislation.

Urine as a biological specimen for forensic analysis of alcohol and variability in the urine-to-blood relationship.

This article concerns the use of urine as a biological specimen for determination of alcohol in clinical and forensic toxicology and discusses factors that might influence variability in the urine/blood concentration ratio of alcohol. A large number of human drinking experiments were conducted to determine the time course of urine-alcohol concentrations (UAC) in relation to blood-alcohol concentrations (BAC). The UAC and BAC curves were shifted in time and the BAC curve always began to decrease before the UAC started to decline. During the early absorption phase the UAC/BAC ratio was less than unity, whereas in the late absorption/distribution period the ratio was between 1.0-1.2. On reaching the post-absorptive phase, the UAC always exceeded BAC and UAC/BAC ratios averaged 1.3-1.4, increasing appreciably as BAC decreased towards zero. Alcohol-induced diuresis was most pronounced during the rising portion of the BAC curve and near to the peak value. After about 2 hours post-drinking, the production rate of urine diminished to the pre-drinking rate of about 0.5-1 mL/min. Drinking water during the post-absorptive phase of the alcohol curve produced dilute urine, as reflected in lower creatinine content and osmolality, although the concentration of ethanol remained unchanged. After subjects drank a moderate dose of ethanol (0.54-0.85 g/kg) about 2% of the dose was recoverable in the urine after 7 hours. Ethyl glucuronide, a minor metabolite of ethanol, was measured in urine samples from drunk drivers. The UAC/BAC ratio of ethanol in drunk drivers did not depend on the creatinine content of the urine and therefore the relative dilution of the specimens. When alcohol-free urine was spiked with glucose and infected with the yeast species Candida albicans, ethanol was produced by fermentation after approximately 24 hours storage at room temperature. This post-sampling synthesis of ethanol was prevented by sodium fluoride (1% weight by volume) in the urine tubes or by keeping the specimens in the cold (4 degrees C). The UAC and BAC were highly correlated (r > 0.95) in drunk drivers and in autopsy cases, although the residual standard deviations were appreciable. This speaks against attempting to estimate BAC indirectly from UAC in any individual case. The UAC/BAC ratio and the change in UAC between two successive voids can help to resolve whether a large amount of alcohol had recently been consumed. This information is useful to support or challenge allegations of drinking alcohol after driving, which has become known as the hip-flask defence.

Enhanced rate of ethanol elimination from blood after intravenous administration of amino acids compared with equicaloric glucose.

AIMS: To investigate the effect of an amino acid mixture given intravenously (i.v.) on the rate of ethanol elimination from blood compared with equicaloric glucose and Ringer's acetate as control treatments. METHODS: In a randomized cross-over study, six healthy men (mean age 23 years) fasted overnight before receiving either Ringer's acetate, glucose or the amino acid mixture (Vamin 18 g N/l) by constant rate i.v. infusion over 4.5 h. Ethanol (0.4 g/kg) was given by an i.v. infusion lasting 60 min during the time each of the treatments was administered. At various times post-infusion, blood samples were taken for determination of ethanol by headspace gas chromatography. Blood glucose and heart rate were monitored at regular intervals. Concentration-time profiles of ethanol were plotted for each subject and the rate of ethanol disappearance from blood as well as other pharmacokinetic parameters were compared by repeated measures analysis of variance. RESULTS: The rate of ethanol elimination from blood was increased significantly (P < 0.001) after treatment with amino acids (mean +/- SD, 0.174 +/- 0.011 g/l/h) compared with equicaloric glucose (0.121 +/- 0.016 g/l/h) or Ringer's acetate (0.110 +/- 0.013 g/l/h). Heart rate was also slightly higher during infusion of the amino acid mixture (P < 0.05). CONCLUSIONS: When the rate of ethanol elimination from blood is relatively slow, such as after an overnight fast, it can be increased by approximately 60% after treatment with i.v. amino acids. The efficacy of amino acid treatment was not related to the supply of calories because glucose was no more effective than Ringer's acetate. We suggest that amino acids might increase hepatic oxygen consumption, resulting in a more effective conversion of NADH to NAD+ in mitochondria. An important feature of the experimental design was ensuring hepatic availability of amino acids during much of the time that ethanol was being metabolized.