Postmortem reference concentrations of 68 elements in blood and urine.

INTRODUCTION: Fatal intoxications, both accidental and intentional, are a global issue. In the Western world, intoxications with pharmaceuticals dominate, but in other parts of the world, other substances are more common. In a forensic setting, elemental intoxications are of great importance when investigating both accidental, suicidal, and homicidal deaths. The current study presents normal postmortem reference concentrations of 68 elements in femoral blood and urine. In addition, possible sources of error such as contamination from sample tubes, preservative potassium fluoride (KF) solution, and storage time are evaluated. METHODS: Paired femoral blood and urine samples from 120 cases of death by suicidal hanging in Sweden were collected. Additionally, multiple batches of sample tubes and multiple batches of KF solution were also analyzed. Concentrations of elements were determined by double focusing sector field ICP-MS. RESULTS: Key descriptive statistics for 68 elements are provided in blood and urine. Contamination from sample tubes was minor compared to the overall mean elemental concentrations in both blood and urine. KF solution contained a large assortment of elements, but the overall contribution is relatively minor for most elements given the small amounts of solution added to samples. There were significant differences for 22 elements in blood and 17 elements in urine between samples with short and long storage time. CONCLUSION: The present study provides an important tool when evaluating postmortem elemental concentrations. It fills a needed gap between large antemortem population studies and postmortem case reports or small case series of elemental intoxications.

Post-Mortem Metabolomics: A Novel Approach in Clinical Biomarker Discovery and a Potential Tool in Death Investigations.

Metabolomics can be defined as the scientific field aiming at characterizing all low-weight molecules (so-called metabolites) in a biological system. At the time of death, the level and type of metabolites present will most likely reflect the events leading up to death.In this proof of concept study, we investigated the potential of post-mortem metabolomics by identifying post-mortem biomarkers, correlated these identified biomarkers with those reported in clinical metabolomics studies, and finally validated the models predictability of unknown autopsy cases. In this post-mortem metabolomics setting, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry data from 404 post-mortem samples, including pneumonia cases and control cases, were processed using XCMS (R). Potential biomarkers were evaluated using principal component analysis and orthogonal partial least squares-discriminant analysis. Biomarkers were putatively annotated using an in-house database and the online databases METLIN and HMDB. The results showed that clear group separation was observed between pneumonia cases and control cases. The metabolites responsible for group separation belonged to a broad set of biological classes, such as amino acids, carnitines, lipids, nicotinamides, nucleotides, and steroids. Many of these metabolites have been reported as important in clinical manifestation of pneumonia. For the unknown autopsy cases, the sensitivity and specificity were 86 and 84%, respectively. This study successfully investigated the robustness and usability of post-mortem metabolomics in death investigations. The identified post-mortem biomarkers correlated well with biomarkers reported and identified through clinical research.

Validation of an LC-MS/MS method for the determination of zopiclone, N-desmethylzopiclone and 2-amino-5-chloropyridine in whole blood and its application to estimate the original zopiclone concentration in stored specimens.

2-Amino-5-chloropyridine (ACP) is a degradation product of zopiclone (ZOP) and its two main metabolites N-desmethylzopiclone (NDZOP) and zopiclone N-oxide (ZOPNO). ACP may be formed when specimens are stored. ZOP instability in blood makes interpretation of concentrations difficult especially in cases of prolonged sample storage. This study investigated how ACP could be used to estimate the original concentration of ZOP in authentic samples. For that purpose, an analytical liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitation of ACP, ZOP, and NDZOP in blood was developed and validated. Due to poor extraction recovery, ZOPNO was not included in the analytical method. The method was then applied to investigate ACP formation, ZOP and NDZOP degradation in stored ZOP post-dosed authentic whole blood and two mathematical models were used to calculate the original concentration of ZOP. During storage, ACP was formed in amounts equimolar to the ZOP and NDZOP degradation. Results from samples in which ACP had been formed were used to test two models to estimate the original ZOP concentration. The correlation tests of the models showed strong correlations to the original ZOP concentration (r = 0.960 and r = 0.955) with p < 0.01 and explained more than 90 % of the ZOP concentration. This study showed that the equimolar degradation of ZOP and NDZOP to ACP could be used to estimate the original concentration of ZOP.

Fatal Intoxications with Zopiclone-A Cause for Concern?

INTRODUCTION: Zopiclone, a controlled substance prescribed for insomnia, has become a common toxicological finding in forensic autopsy cases. This study investigated the role and extent of zopiclone use in fatal intoxications in Sweden. METHODS: All forensic autopsy cases positive for zopiclone in femoral blood during 2012-2020 were selected. Among these cases, fatalities caused by intoxication according to the cause of death certificates issued by the forensic pathologist were identified. Intoxications where zopiclone contributed to the cause of death were included in the study. The Swedish Prescribed Drug Register was utilized to examine whether the included cases were prescribed zopiclone or not. RESULTS: In total 7320 fatal intoxications underwent a forensic autopsy during the study period, 573 of them were caused by zopiclone. Among the zopiclone fatalities, 87% (n = 494) had a prescription for zopiclone, and 8% (n = 43) were monointoxications. Most fatalities, 62% (n = 354) were suicides, and zopiclone was involved in about 17% (n = 354) of all intoxication suicides in Sweden. Women were significantly (p < 0.01) overrepresented in suicides with zopiclone, comprising 56% (n = 291) of fatalities. The median age was 55 years among zopiclone intoxications compared with 44 years amongst all fatal intoxications. CONCLUSION: This study demonstrates that the toxicity of zopiclone can be lethal both in combination with other substances and on its own. Most individuals dying in fatal zopiclone intoxications were prescribed zopiclone, which potentially indicates that a more restrictive prescribing rate could prevent future intoxication deaths, especially when caring for patients with an increased suicide risk.

Postmortem metabolomics as a high-throughput cause-of-death screening tool for human death investigations.

Autopsy rates are declining globally, impacting cause-of-death (CoD) diagnoses and quality control. Postmortem metabolomics was evaluated for CoD screening using 4,282 human cases, encompassing CoD groups: acidosis, drug intoxication, hanging, ischemic heart disease (IHD), and pneumonia. Cases were split 3:1 into training and test sets. High-resolution mass spectrometry data from femoral blood were analyzed via orthogonal-partial least squares discriminant analysis (OPLS-DA) to discriminate CoD groups. OPLS-DA achieved an R2 = 0.52 and Q2 = 0.30, with true-positive prediction rates of 68% and 65% for training and test sets, respectively, across all groups. Specificity-optimized thresholds predicted 56% of test cases with a unique CoD, average 45% sensitivity, and average 96% specificity. Prediction accuracies varied: 98.7% for acidosis, 80.5% for drug intoxication, 81.6% for hanging, 73.1% for IHD, and 93.6% for pneumonia. This study demonstrates the potential of large-scale postmortem metabolomics for CoD screening, offering high specificity and enhancing throughput and decision-making in human death investigations.

Recommended dosages of analgesic and sedative drugs in intensive care result in a low incidence of potentially toxic blood concentrations.

Background: Standard dosages of analgesic and sedative drugs are given to intensive care patients. The resulting range of blood concentrations and corresponding clinical responses need to be better examined. The purpose of this study was to describe daily dosages, measured blood concentrations, and clinical responses in critically ill patients. The purpose was also to contribute to establishing whole blood concentration reference values of the drugs investigated. Methods: A descriptive study of prospectively collected data from 302 admissions to a general intensive care unit (ICU) at a university hospital. Ten drugs (clonidine, fentanyl, morphine, dexmedetomidine, ketamine, ketobemidone, midazolam, paracetamol, propofol, and thiopental) were investigated, and daily dosages recorded. Blood samples were collected twice daily, and drug concentrations were measured. Clinical responses were registered using Richmond agitation-sedation scale (RASS) and Numeric rating scale (NRS). Results: Drug dosages were within recommended dose ranges. Blood concentrations for all 10 drugs showed a wide variation within the cohort, but only 3% were above therapeutic interval where clonidine (57 of 122) and midazolam (38 of 122) dominated. RASS and NRS were not correlated to drug concentrations. Conclusion: Using recommended dose intervals for analgesic and sedative drugs in the ICU setting combined with regular monitoring of clinical responses such as RASS and NRS leads to 97% of concentrations being below the upper limit in the therapeutic interval. This study contributes to whole blood drug concentration reference values regarding these 10 drugs.

ABCB1 gene polymorphisms are associated with suicide in forensic autopsies.

BACKGROUND: Polymorphisms in ABCB1 have the ability to affect both the function and the expression of the transporter protein P-glycoprotein and may lead to an altered response for many drugs including some antidepressants and antipsychotics. OBJECTIVE: The aim of this study was to examine the impact of the ABCB1 polymorphisms 1199G>A, 1236C>T, 2677G>T/A, and 3435C>T in deaths by suicide. PATIENTS AND METHODS: A total of 998 consecutive Swedish forensic autopsies performed in 2008 in individuals 18 years of age or older, where femoral blood was available and a toxicological screening had been performed, were investigated. Genotypes were assessed with pyrosequencing and information on the cause and manner of each death was obtained from the forensic pathology and toxicology databases. RESULTS: There was a significantly higher frequency of the T allele at positions 1236, 2677, and 3435 among the suicide cases compared with the nonsuicide cases. CONCLUSION: Our result from forensic cases suggests that ABCB1 polymorphisms are associated with an increased risk for completed suicides. The biological mechanisms involved and the clinical implications for these findings are largely unknown and need to be examined further.

Frequency of postmortem ethanol formation in blood, urine and vitreous humor - Improving diagnostic accuracy with the use of ethylsulphate and putrefactive alcohols.

PURPOSE: This study aimed to compare the frequency of postmortem ethanol formation in blood, urine and vitreous humor according to negative ethylsulphate (EtS) in blood or positive putrefactive alcohols (PA's) in either medium. Furthermore, it aimed to evaluate the interpretational value of calculated ethanol ratios in relation to EtS and PA results. METHODS: Blood ethanol positive forensic cases were included; one dataset consisting of 2504 cases with EtS analysed in blood and another dataset with 8001 cases where PA's were analysed. RESULTS: PA's were found in 24.4% of cases. EtS was negative in 15.3%, 9.4% and 7.4% of cases that were positive for ethanol in blood, urine and vitreous humor, respectively. In EtS negative cases, the concentrations of ethanol in blood, urine and vitreous humor were lower than 0.20 g/kg in 51.3%, 67.4% and 77.8%, respectively. It was 1.0 g/kg or higher in blood in 4.2% of cases. More EtS negative and PA positive cases were seen in central compared to peripheral blood. Ethanol ratios between urine or vitreous humor and blood were significantly lower in both EtS negative and PA positive cases, but large variations were observed. CONCLUSION: EtS and PA analysis improve the diagnostic accuracy of ethanol in postmortem cases. Postmortem ethanol formation in vitreous humor and urine were both more frequent than expected and we recommend the analysis of ethanol primarily in peripheral blood if available.

Postmortem Metabolomics of Insulin Intoxications and the Potential Application to Find Hypoglycemia-Related Deaths.

Postmortem metabolomics can assist death investigations by characterizing metabolic fingerprints differentiating causes of death. Hypoglycemia-related deaths, including insulin intoxications, are difficult to identify and, thus, presumably underdiagnosed. This investigation aims to differentiate insulin intoxication deaths by metabolomics, and identify a metabolic fingerprint to screen for unknown hypoglycemia-related deaths. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry data were obtained from 19 insulin intoxications (hypo), 19 diabetic comas (hyper), and 38 hangings (control). Screening for potentially unknown hypoglycemia-related deaths was performed using 776 random postmortem cases. Data were processed using XCMS and SIMCA. Multivariate modeling revealed group separations between hypo, hyper, and control groups. A metabolic fingerprint for the hypo group was identified, and analyses revealed significant decreases in 12 acylcarnitines, including nine hydroxylated-acylcarnitines. Screening of random postmortem cases identified 46 cases (5.9%) as potentially hypoglycemia-related, including six with unknown causes of death. Autopsy report review revealed plausible hypoglycemia-cause for five unknown cases. Additionally, two diabetic cases were found, with a metformin intoxication and a suspicious but unverified insulin intoxication, respectively. Further studies are required to expand on the potential of postmortem metabolomics as a tool in hypoglycemia-related death investigations, and the future application of screening for potential insulin intoxications.

Cytochrome P450-dependent disposition of the enantiomers of citalopram and its metabolites: in vivo studies in Sprague-Dawley and Dark Agouti rats.

The female Sprague-Dawley (SD) and Dark Agouti (DA) rats are considered the animal counterparts of the human extensive and poor metabolizer cytochrome P450 (CYP) 2D6 phenotypes, respectively. The aim of this work was to study possible rat strain differences in the steady-state pharmacokinetics of the (+)-(S)- and (-)-(R)-enantiomers of citalopram and its demethylated metabolites. A chronic drug treatment regimen (15 mg/kg daily) was implemented for 13 days in separate groups of SD (n = 9) and DA (n = 9) rats by using osmotic pumps. The concentrations of citalopram and two major metabolites in serum and two brain regions were analyzed by an enantioselective high-performance liquid chromatography assay. Higher serum and brain levels of citalopram and demethylcitalopram, but lower levels of didemethylcitalopram, were observed in DA rats when compared with SD rats. The enantiomeric (S/R) concentrations ratios of citalopram were lower in the DA rats when compared with the SD rats (0.53 ± 0.05 vs. 0.80 ± 0.03, P < 0.001), indicating a possibly decreased capacity in the metabolism of the (-)-(R)-enantiomer in the DA rats. This study shows that CYP2D deficiency results in steady-state pharmacokinetic differences of the enantiomers of citalopram and its metabolites.

Forensic toxicology findings in deaths involving gamma-hydroxybutyrate.

Concentrations of the illicit drug gamma-hydroxybutyrate (GHB) were determined in femoral venous blood and urine obtained at autopsy in a series of GHB-related deaths (N = 49). The analysis of GHB was done by gas chromatography after conversion to gamma-butyrolactone and quantitation of the latter with a flame ionization detector. The cutoff concentration of GHB in femoral blood or urine for reporting positive results was 30 mg/L. The deceased were mainly young men (86%) aged 26.5 +/- 7.2 years (mean +/- SD), and the women (14%) were about 5 years younger at 21.4 +/- 5.0 years. The mean, median, and highest concentrations of GHB in femoral blood (N = 37) were 294, 190, and 2,200 mg/L, respectively. The mean urine-to-blood ratio of GHB was 8.8, and the median was 5.2 (N = 28). In 12 cases, the concentrations of GHB in blood were negative (<30 mg/L) when the urine contained 350 mg/L on average (range 31-1,100 mg/L). Considerable poly-drug use was evident in these GHB-related deaths: ethanol (18 cases), amphetamine (12 cases), and various prescription medications (benzodizepines, opiates, and antidepressants) in other cases. Interpreting the concentrations of GHB in postmortem blood is complicated because of concomitant use of other psychoactive substances, variable degree of tolerance to centrally acting drugs, and the lack of reliable information about survival time after use of the drug.

The unknown known: non-cardiogenic pulmonary edema in amlodipine poisoning, a cohort study.

Context: Amlodipine is the most common calcium channel blocker (CCB) on the Swedish market, and poison center (PC) consultations for amlodipine overdoses are increasing. The clinical picture is dominated by vasodilation with relative preservation of cardiac function. CCBs selectively dilate vessels on the afferent side of the capillary network which, in states of preserved or increased blood flow may lead to edema formation, including non-cardiogenic pulmonary edema (NCPE). This complication has been considered rare in CCB poisoning. In this cohort study of nineteen amlodipine poisonings with high amlodipine blood levels, the incidence and clinical significance of NCPE in severe amlodipine poisoning are explored.Methods: During 2017-2018 the Swedish PC prospectively encouraged the gathering of blood samples in amlodipine poisonings with symptoms requiring treatment with inotropes or vasopressors. Samples were sent by mail to the Forensic Toxicology Division at the Swedish National Board of Forensic Medicine for screening and quantification of relevant toxicants. Patients with blood amlodipine levels >0.25 µg/mL were included in a cohort whose case details were gathered from medical records and PC-case notes with a special focus on signs of NCPE.Results: Nineteen patients met the blood amlodipine inclusion criteria. Four (21%) died and one patient was treated with VA-ECMO. Nine patients developed NCPE defined as a need for positive pressure ventilation (PPV) while having an echocardiographically normal left ventricular function.Conclusion: In this prospective cohort study of consecutive and analytically confirmed significant amlodipine poisonings NCPE was a common finding occurring in 47% of the whole cohort and in 64% of patients who did not go on to develop complete hemodynamic collapse.

Evaluating the hip-flask defence in subjects with alcohol on board: An experimental study.

Driving under the influence of alcohol is a major problem for traffic-safety and a popular defence argument is alleged consumption after driving, commonly referred to as the hip-flask defence. Forensic toxicologists are often called as expert witnesses in drinking and driving cases where the suspect has claimed the hip-flask defence, to assess the credibility of the explanation. Several approaches to help the expert have been introduced but the scientific data used to support or challenge this is solely based on data from controlled single doses of ethanol administered during a short time and in abstinent subjects. In reality, we believe that even in drinking after driving cases, the subject many times has alcohol on board at time of the hip-flask drink. This questions the applicability of the data used as basis to investigate the hip-flask defence. To fill this knowledge gap, we aimed to investigate how blood and urine ethanol kinetics vary after an initial drinking session of beer and then a subsequent hip-flask drink of three different doses of whiskey. Fifteen subjects participated in the study and each provided 10 urine samples and 17 blood samples over 7h. The initial drink was 0.51g ethanol/kg and the second was either 0.25, 0.51, or 0.85g/kg. Our data suggested that the difference between the ethanol concentrations in two consecutive urine samples is a more sensitive parameter than the ratio between urine and blood alcohol to detect a recent intake when ethanol from previous intakes are already present in the body. Twelve subjects presented results that fully supported a recent intake using the criteria developed from a single intake of ethanol. Three subjects showed unexpected results that did not fully support a recent intake. We conclude that data from one blood sample and two urine samples provide good evidence for investigating the hip-flask defence even if alcohol was on board at the time of the hip-flask drink.

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.

High re-arrest rates among drug-impaired drivers despite zero-tolerance legislation.

BACKGROUND: A zero-tolerance law for driving under the influence of drugs (DUID) in Sweden led to a 10-fold increase in the number of cases submitted by the police for toxicological analysis. The statutory blood-alcohol concentration (BAC) limit for driving is 0.2 mg/g ( approximately 0.02 g%). METHODS: An in-house database (TOXBASE) was used to investigate re-arrests for impaired driving over 4 years (2001-2004), which comprised 36,799 cases. The age, gender, re-arrest rate of the offenders and the concentrations of ethanol and amphetamine in blood samples were evaluated. RESULTS: We found that 44% of individuals (N=16,277) re-offended 3.2 times on average (range 1-23 arrests). Between 85 and 89% of first-time offenders were men and there was also a male dominance among the recidivists (88-93%). The mean age of drunken drivers was approximately 40 years compared with approximately 35 years for driving under the influence of amphetamine, which was the drug identified in 50-60% of DUID cases, either alone or together with other licit or illicit drugs. The median BAC was 1.5mg/g ( approximately 0.15 g%), which suggests a dominance of heavy drinkers. The median BAC was even higher in recidivists (1.6-1.7 mg/g). The median concentration of amphetamine in blood was 1.0 mg/L in recidivists compared with 0.5mg/L in the first-time offenders. About 14% of drunken drivers re-offended 1-10 times compared with 68% of DUID suspects, who were re-arrested 1-23 times. People with only a scheduled prescription drug in blood were re-arrested much less frequently ( approximately 17%) compared with those taking illicit drugs (68%). CONCLUSIONS: The appreciable increase in number of arrests for DUID after a zero-tolerance law might reflect a heightened enthusiasm by the police authorities armed with knowledge that a prosecution is easier to obtain. Zero-tolerance laws do not deter people from impaired driving judging by the high re-arrest rates. During the sentencing of hardcore offenders, the courts should give more consideration to the underlying substance abuse problem.

Driving under the influence of opiates: concentration relationships between morphine, codeine, 6-acetyl morphine, and ethyl morphine in blood.

Morphine and codeine are frequently identified in blood samples from impaired drivers. But whether these opiates reflect the use of prescription analgesics or abuse of the illicit drug heroin (diacetyl morphine) is not always obvious. Opiates, either alone or together with other drugs, were determined in 2,573 blood specimens from impaired drivers by sensitive and specific methods of analysis. The specific metabolite of heroin 6-acetyl morphine (6-AM) was quantifiable in only 52 cases (2%) at mean, median, and highest concentrations of 0.015, 0.010, and 0.10 mg/L, respectively. The mean, median, and highest concentrations of morphine were 0.046, 0.03, and 1.13 mg/L, respectively (N = 2,029). The corresponding concentrations of codeine (N = 1,391) were 0.047, 0.01, and 2.40 mg/L. Ethyl morphine was identified in 63 cases at a mean concentration of 0.055 mg/L (median 0.03 mg/L). When 6-AM was present in urine (N = 324), the mean morphine/codeine ratio in blood was 7.5 (median 6.7), and this important ratio was less than unity in only two cases. This study finds compelling evidence that approximately 90% of apprehended drivers in Sweden with morphine and codeine in their blood had used heroin.

Enantioselective pharmacokinetics of tramadol and its three main metabolites; impact of CYP2D6, CYP2B6, and CYP3A4 genotype.

Tramadol is a complex drug, being metabolized by polymorphic enzymes and administered as a racemate with the (+)- and (-)-enantiomers of the parent compound and metabolites showing different pharmacological effects. The study aimed to simultaneously determine the enantiomer concentrations of tramadol, O-desmethyltramadol, N-desmethyltramadol, and N,O-didesmethyltramadol following a single dose, and elucidate if enantioselective pharmacokinetics is associated with the time following drug intake and if interindividual differences may be genetically explained. Nineteen healthy volunteers were orally administered either 50 or 100 mg tramadol, whereupon blood samples were drawn at 17 occasions. Enantiomer concentrations in whole blood were measured by LC-MS/MS and the CYP2D6,CYP2B6 and CYP3A4 genotype were determined, using the xTAG CYP2D6 Kit, pyrosequencing and real-time PCR, respectively. A positive correlation between the (+)/(-)-enantiomer ratio and time following drug administration was shown for all four enantiomer pairs. The largest increase in enantiomer ratio was observed for N-desmethyltramadol in CYP2D6 extensive and intermediate metabolizers, rising from about two to almost seven during 24 hours following drug intake. CYP2D6 poor metabolizers showed metabolic profiles markedly different from the ones of intermediate and extensive metabolizers, with large area under the concentration curves (AUCs) of the N-desmethyltramadol enantiomers and low corresponding values of the O-desmethyltramadol and N,O-didesmethyltramadol enantiomers, especially of the (+)-enantiomers. Homozygosity of CYP2B6 *5 and *6 indicated a reduced enzyme function, although further studies are required to confirm it. In conclusion, the increase in enantiomer ratios over time might possibly be used to distinguish a recent tramadol intake from a past one. It also implies that, even though (+)-O-desmethyltramadol is regarded the enantiomer most potent in causing adverse effects, one should not investigate the (+)/(-)-enantiomer ratio of O-desmethyltramadol in relation to side effects without consideration for the time that has passed since drug intake.

Interpreting results of ethanol analysis in postmortem specimens: a review of the literature.

We searched the scientific literature for articles dealing with postmortem aspects of ethanol and problems associated with making a correct interpretation of the results. A person's blood-alcohol concentration (BAC) and state of inebriation at the time of death is not always easy to establish owing to various postmortem artifacts. The possibility of alcohol being produced in the body after death, e.g. via microbial contamination and fermentation is a recurring issue in routine casework. If ethanol remains unabsorbed in the stomach at the time of death, this raises the possibility of continued local diffusion into surrounding tissues and central blood after death. Skull trauma often renders a person unconscious for several hours before death, during which time the BAC continues to decrease owing to metabolism in the liver. Under these circumstances blood from an intracerebral or subdural clot is a useful specimen for determination of ethanol. Bodies recovered from water are particular problematic to deal with owing to possible dilution of body fluids, decomposition, and enhanced risk of microbial synthesis of ethanol. The relationship between blood and urine-ethanol concentrations has been extensively investigated in autopsy specimens and the urine/blood concentration ratio might give a clue about the stage of alcohol absorption and distribution at the time of death. Owing to extensive abdominal trauma in aviation disasters (e.g. rupture of the viscera), interpretation of BAC in autopsy specimens from the pilot and crew is highly contentious and great care is needed to reach valid conclusions. Vitreous humor is strongly recommended as a body fluid for determination of ethanol in postmortem toxicology to help establish whether the deceased had consumed ethanol before death. Less common autopsy specimens submitted for analysis include bile, bone marrow, brain, testicle, muscle tissue, liver, synovial and cerebrospinal fluids. Some investigators recommend measuring the water content of autopsy blood and if necessary correcting the concentration of ethanol to a mean value of 80% w/w, which corresponds to fresh whole blood. Alcoholics often die at home with zero or low BAC and nothing more remarkable at autopsy than a fatty liver. Increasing evidence suggests that such deaths might be caused by a pronounced ketoacidosis. Recent research has focused on developing various biochemical tests or markers of postmortem synthesis of ethanol. These include the urinary metabolites of serotonin and non-oxidative metabolites of ethanol, such as ethyl glucuronide, phosphatidylethanol and fatty acid ethyl esters. This literature review will hopefully be a good starting point for those who are contemplating a fresh investigation into some aspect of postmortem alcohol analysis and toxicology.

Gamma-hydroxybutyrate concentrations in the blood of impaired drivers, users of illicit drugs, and medical examiner cases.

Gamma-hydroxybutyrate (GHB) was determined in blood samples from impaired drivers, people arrested for petty drug offenses (non-traffic cases), and GHB-related deaths. The method of analysis involved conversion of GHB into gamma-butyrolactone and determination of the latter by gas chromatography with a flame ionization detector, and with gamma-valerolactone as the internal standard. The mean and median concentrations of GHB in blood from impaired drivers (N=473) were 90 and 84 mg/L, respectively, and offenders were predominantly men (96%) with an average age of 26 year (range 15-50 year). In 185 cases, GHB was the only drug present in blood at mean and median concentrations of 92 and 86 mg/L, respectively. People arrested for petty drug offenses (N=1061) had slightly higher GHB concentrations in their blood: median 118 mg/L for men and 111 mg/L for women. In GHB-related deaths (N=33), the mean and median concentrations were considerably higher: 307 mg/L and 190 mg/L, respectively, and the highest was 2200 mg/L. The typical signs of drug influence noted by the arresting police officers included sedation, agitation, slurred speech, irrational behaviour, jerky movements, and spitting. The short elimination half-life of GHB means that the concentrations in blood decrease rapidly and are probably a lot lower than at the time of driving, which was 30-90 min earlier.

Predominance of illicit drugs and poly-drug use among drug-impaired drivers in Sweden.

OBJECTIVE: After Sweden's zero-tolerance law came into force (1 July 1999), the number of cases of driving under the influence of drugs (DUID) submitted by the police for toxicological analysis increased more than 10-fold. This prompted an in-depth investigation into the kinds of drugs used by DUID offenders, whether licit or illicit, and the frequency of their occurrence. METHODS: All blood samples from DUID suspects sent by the police for toxicological analysis over a 4-year period (2001-2004) were investigated (N = 22,777 cases). Specimens of blood or urine were subjected to a broad screening analysis by immunoassay methods aimed at detecting amphetamines, cannabis, opiates, cocaine metabolite, and the major benzodiazepines. All positive results from the screening stage were verified by use of more specific analytical methods (e.g., GC-MS, LC-MS, GC-FID, and GC-NPD). RESULTS: Between 80 and 85% of all the blood samples contained at least one banned substance and many contained two or more therapeutic and/or illicit drugs. About 15% of cases were negative for drugs, although these frequently (30-50%) contained ethanol above the legal limit for driving in Sweden, which is 0.20 mg/g (0.02 g%). Amphetamine was the most prominent illicit drug seen in 55-60% of cases either alone or together with other drugs of abuse. Stimulants like cocaine and/or its metabolite were infrequently encountered ( approximately 1.2% of cases). The next most prevalent illicit drug was cannabis, with positive results for tetrahydrocannabinol (THC) in blood either alone ( approximately 4%) or together with other psychoactive substances ( approximately 20%). Morphine, codeine, and/or 6-acetyl morphine were identified in approximately 2% of all DUID suspects, being indicative of heroin abuse. The major prescription drugs identified in blood were benzodiazepines (10%) as exemplified by diazepam, alprazolam, nitrazepam, and flunitrazepam. Drugs for treating insomnia, zolpidem and zopiclone, were also identified in blood samples from DUID suspects over the study period. Other therapeutic agents were encountered in only 1-2% of all cases. CONCLUSIONS: The dramatic increase in DUID after the zero-tolerance law came into force probably reflects enhanced police activity and more enthusiasm to apprehend and charge individuals for this offence. Illicit drugs, particularly amphetamine and cannabis, and poly-drug use were predominant compared with use of scheduled prescription drugs. The typical DUID offender in Sweden abuses central stimulants, particularly amphetamine, and has probably done so over many years. Options for treating offenders for their underlying substance abuse problem should be considered instead of the more conventional penalties for drug-impaired driving.