Insights into the human metabolism of hexahydrocannabinol by non-targeted liquid chromatography-high-resolution tandem mass spectrometry.

Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography-high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident.

Consumption of the Sugar Substitute Stevia Leads to Cross-Reactivity of CEDIA® Buprenorphine II Immunoassay.

Buprenorphine is a semisynthetic opioid that is often used in opiate maintenance therapy. For this purpose, regular toxicological analyses of urine samples are mandatory. For fast analytical results, analyses are commonly performed by immunoassay, for example, Thermo Scientific™ CEDIA® Buprenorphine or Buprenorphine II assay. One drawback of immunoassay-based methods is the possible cross-reaction with other substances. Several drugs have already been checked for cross-reactivity to CEDIA® Buprenorphine II immunoassay. In contrast, cross-reactivities have not been checked for any food additives. In the present study, a cross-reaction of CEDIA® Buprenorphine II assay to steviol glucuronide was investigated. Steviol glucuronide is a phase II metabolite of the sugar substitute stevia. For our study, 32 urine samples of patients in rehabilitation centers were collected. These samples were tested positive with the CEDIA® Buprenorphine II immunoassay. These findings were suspicious, because it was highly unlikely that the patients in those institutions had access to buprenorphine. The absence or presence of buprenorphine in urine samples was evaluated by a validated gas chromatography-mass spectrometry method. In order to determine the concentration of steviol glucuronide in urine samples, a liquid chromatography-tandem mass spectrometry method has been developed and fully validated according to the respective guidelines of the German Society of Toxicological and Forensic Chemistry. The cross-reactivity of steviol glucuronide in the CEDIA® Buprenorphine II immunoassay was observed at concentrations above 15,000 µg/L. These findings demonstrate that food additives should also be considered as compounds that may reduce the selectivity of immunoassays and emphasize the importance of confirming implausible results by selective analytical methods.

A Novel Enzyme Immunoassay for the Detection of Buprenorphine, Norbuprenorphine and Their Glucuronides in Urine.

Buprenorphine is a commonly used opioid in pain therapy as well as in opiate maintenance therapy. Immunoassays are quick and cost-effective methods for the necessary toxicological urine analysis of maintenance therapy patients. In this study a novel enzymatic immunoassay, the Thermo Fisher Scientific CEDIA Buprenorphine II assay (Bup2) was evaluated for the detection of buprenorphine, norbuprenorphine and their conjugated metabolites in human urine samples. The Bup2 assay has a cut-off of 10 ng/mL with ±25% controls, whereas the existing CEDIA Buprenorphine assay (Bup1) has a cut-off of 5 ng/mL and ±40% controls. Both assays were analyzed on a Thermo Scientific Indiko Plus benchtop analyzer. Seven-day precision studies of Bup2 assay demonstrated excellent precision of 7.2-10.6%. No crossover between control samples and the cut-off level were observed. Urine samples of 120 patients undergoing opiate maintenance therapy were collected. Immunoassay results of Bup1 and Bup2 were confirmed by gas chromatography mass spectrometry (GC/MS) for buprenorphine and norbuprenorphine as well as for their glucuronides. Comparison showed a specificity of 0.99 between the Bup2 assay and GC/MS, whereas the Bup1 assay had a specificity 0.70 due to 21 false positive samples. The reason is a known cross-reactivity of the Bup1 assay to opiate compounds. The Bup2 assay revealed one false positive result close to the cut-off value; no specific candidate possibly causing a cross-reaction was detected by GC/MS and liquid chromatography tandem mass-spectrometry (LC/MS/MS) methods. The data presented demonstrate an excellent correlation of the Bup2 assay to GC/MS, showing improved specificity and sensitivity when compared to the Bup1 assay. Thus, the Bup2 assay is highly suitable for urine testing, even for opiate maintenance patients receiving high doses of morphine.

Association of polymorphisms in pharmacogenetic candidate genes (OPRD1, GAL, ABCB1, OPRM1) with opioid dependence in European population: a case-control study.

It is becoming increasingly evident that genetic variants contribute to the development of opioid addiction. An elucidation of these genetic factors is crucial for a better understanding of this chronic disease and may help to develop novel therapeutic strategies. In recent years, several candidate genes were implicated in opioid dependence. However, most study findings have not been replicated and additional studies are required before reported associations can be considered robust. Thus, the major objective of this study was to replicate earlier findings and to identify new genetic polymorphisms contributing to the individual susceptibility to opioid addiction, respectively. Therefore, a candidate gene association study was conducted including 142 well-phenotyped long-term opioid addicts undergoing opioid maintenance therapy and 142 well-matched healthy controls. In both study groups, 24 single nucleotide polymorphisms predominantly located in pharmacogenetic candidate genes have been genotyped using an accurate mass spectrometry based method. The most significant associations with opioid addiction (remaining significant after adjustment for multiple testing) were observed for the rs948854 SNP in the galanin gene (GAL, p = 0.001) and the rs2236861 SNP in the delta opioid receptor gene (OPRD1, p = 0.001). Moreover, an association of the ATP binding cassette transporter 1 (ABCB1) variant rs1045642 and the Mu Opioid receptor (OPRM1) variant rs9479757 with opioid addiction was observed. The present study provides further support for a contribution of GAL and OPRD1 variants to the development of opioid addiction. Furthermore, our results indicate a potential contribution of OPRM1 and ABCB1 SNPs to the development of this chronic relapsing disease. Therefore it seems important that these genes are addressed in further addiction related studies.

Cannabinoids lead to enhanced virulence of the smallpox vaccine (vaccinia) virus.

Indian hemp is used since thousands of years as herbal drug. We found that a single dose of cannabis resin was equally active as Δ9-tetrahydrocannabinol (THC) enhancing severity and duration of symptoms in vaccinia virus infected mice. Cowpox virus did not cause symptomatic disease, but some reduction of specific antibody production was observed in drug treated animals. In vitro cannabis was superior to THC alone at inhibiting mitogen stimulated proliferation of human and mouse spleen cells and peripheral blood mononuclear cells. Also resin sub-fractions other than THC, cannabidiol and cannabinol, recovered also from cigarette smoke, were found inhibitory, suggesting additional involvement of constituents other than psychoactive THC. The immunoregulatory effects must be differentiated from apoptotic effects on spleen cells and lymphocytic mouse cell lines, which were observed with resin and THC but not with cannabidiol or cannabinol. A significant contribution of cytotoxic effects seems unlikely as drug treated lymphocytes were still capable of producing cytokines after T-cell receptor-specific stimulation. Considering a recent case of unusually severe cowpox virus infection in a young drug taker these data confirm a risk of "soft drugs" for acquiring poxvirus infection or enhancing side effects of the smallpox vaccine and perhaps also other live vaccines.

[Impact of slow-release oral morphine on drug abusing habits in Austria]. / Der Einfluss von retardiertem Morphin auf die Drogensituation in Osterreich.

A well-established possibility to treat opiate addiction is the participation in opiate maintenance treatment programmes. For this purpose the opioids methadone and buprenorphine have been evaluated and are used nowadays in many countries. However, since 1998 also the use of slow-release oral morphine (SROM) has been legally permitted in Austria. Our data show that these morphine preparations are frequently abused and are dominating the black market in the meantime. Especially the intravenous consumption of SROM goes along with highly dangerous side effects that exceed the risks of needle sharing alone. Special galenics are supposed to ensure a 24 h effect of the otherwise quickly metabolised morphine. If dissolved and injected, insoluble contents such as talcum cause microembolisms, leading to severe damages of the inner organs. Furthermore, SROM, i.e. a drug prescribed by physicians, has been proved to be the main responsible substance in most drug related deaths since its permission and has nearly replaced heroin. Forensic physicians play a major role in the profound examination of these cases, including extensive toxicological analyses and interpretation of results. For instance, a differentiation between a recent morphine and heroin consumption is certainly possible, provided appropriate methods are used. A reliable estimation of the current situation of drug abusing habits is a premise for adequate therapeutic offers and preventive measures. Thus, well-founded and comparable data have to be collected. To facilitate data report a standardized report form has been developed that includes an obligatory statement regarding morphine or heroin consumption. This should help to enlighten the ongoing discussion on the role of SRM in drug abuse cases. Our results indicate that the prescription of SROM in opiate maintenance therapy has to be handled very strictly and should be reserved for special patients only. A slackening of the Austrian law concerning SROM is therefore objected.

Diazepam leads to enhanced severity of orthopoxvirus infection and immune suppression.

Benzodiazepines are drugs widely used as tranquilizers and in various other indications. We treated Balb/c mice with diazepam and infected them with cowpox (CPXV) and vaccinia virus (VACV). Disease index, weight loss and the antibody response were determined. Additionally the influence of different benzodiazepines on the mitogen response of human peripheral blood lymphocytes and spleen cells was tested. Diazepam led to earlier disease onset, prolonged duration of symptoms, higher weight loss and overall disease index in VACV infected mice. CPXV infected mice developed poxviral skin lesions only after drug administration and a significant decrease in the specific antibody response was also observed. Diazepam and alprazolam also inhibited the proliferative response of human lymphocytes/spleen cells in vitro but did not show noteworthy apoptotic effects. It is surprising that even a single dose of diazepam has a profound influence on the immune system, sufficient to facilitate symptomatic infectious disease. These data provide first evidence that commonly used drugs like Valium may augment severity of rare poxvirus infections such as CPXV or monkeypox. As VACV is still used as life vaccine against smallpox there is also a risk of enhanced side effects or possible interference with the success of vaccination.

A fatal intoxication case involving topiramate.

Topiramate belongs to a new group of anticonvulsive drugs primarily applied in treatment of epilepsy and in preventive therapy of migraines. Topiramate is structurally unrelated to other antiepileptic drugs and acts by multiple neurostabilizing mechanisms. However, the pharmacology of topiramate appears to be complex and some of its pharmacodynamic actions still remain to be elucidated. This case report documents a fatal intoxication involving topiramate. A 41-year old woman with a known history of psychiatric disorder was found unresponsive by her husband. Resuscitation efforts did not succeed and the woman was pronounced dead at the intensive care unit four hours later. At the scene, drug packages of topiramate, citalopram and flunitrazepam were found. Autopsy including histological examination revealed morphological signs of an acute intoxication and shock. A comprehensive toxicological analysis with GC-MS was performed on the deceased's autopsy samples (femoral blood, bile, kidney, gastric content). The results revealed the presence of topiramate at a concentration of 49mg/L in the femoral blood sample, thus clearly exceeding the therapeutic range. Additionally, citalopram (0.85mg/L) and flunitrazepam in traces (<2µg/L) were detected in peripheral blood. Based on the autopsy findings and toxicological results, the cause of death was primarily attributed to an intoxication with topiramate in combination with citalopram.

Comprehensive identification of active compounds in tablets by flow-injection data-dependent tandem mass spectrometry combined with library search.

A convenient mass spectrometric approach for the identification of toxicologically relevant compounds in tablets and tablet residues is presented. For comprehensive forensic-toxicological analysis electrospray ionization mass spectrometry was accomplished in positive as well as in negative ion mode on a quadrupole-quadrupole-time-of-flight instrument. Dissolved samples were introduced into the mass spectrometer by flow-injection. Mass spectra as well as tandem mass spectra were acquired. A data-dependent acquisition strategy was used to switch between the mass spectrometric modes. Identification was accomplished via search within a tandem mass spectral library. The applied database contained 8252 spectra collected from 836 compounds in positive ion mode as well as 1023 spectra collected from 103 compounds in negative ion mode. A total of 22 casework samples collected during autopsies from mouth, oesophagus or gastric contents, seized by the police, or found with patients at hospital were screened. Twelve samples contained compounds only detectable in positive ion mode (sildenafil, dihydrocodeine, diphenhydramine, oxprenolol, N-methyl-3,4-methylenedioxyamphetamine, morphine, amphetamine, caffeine, pemoline, orphenadrine, m-chlorphenylpiperazine and tramadol), six samples contained species exclusively detectable in negative ion mode (salicylic acid, acetylsalicylic acid, ibuprofen, ketorolac, valproic acid and phenobarbital), and three samples contained diclofenac detectable in both ionization polarities. One sample did not contain any compound amenable to mass spectrometric analysis. For verification all samples were additionally analyzed by GC/MS. Both methods revealed identical results for all but one sample. The beta-adrenergic blocker oxprenolol was exclusively detected by the flow-injection method.

Challenging small human hepatocytes with opiates: further characterization of a novel prototype bioartificial liver.

Bioartificial liver (BAL) systems can take over liver functions in patients undergoing liver failure until transplantation. Recently, a novel prototype rotary BAL has been developed using small human hepatocytes (SH). This study investigated the metabolism of opiates morphine and methadone in the BAL and their influence on the basic cell culture parameters, viability, and growth of SH. Opiates may be present in patients due to pain therapy, anticancer treatment, or drug abuse. Cells were cultivated in the BAL for a total of 12 days and exposed twice to 100 microg/L of morphine or methadone. Morphine and methadone concentrations were analyzed using gas chromatography with a mass spectrometry detector. Further, the production of albumin, lactate dehydrogenase release, lactate release, urea production, and glucose consumption were measured. Cell viability and growth were determined by confocal microscopy. Cytochrome P 3A4 and uridindiphosphat (UDP) glucuronosyl transferase 2B7 in SH were analyzed by western blot. The mean cell density during treatment was 5.5 +/- 0.7 x 10(6) cells/mL (n = 6) and was not altered significantly by the opiates. Cell viability stayed above 90%. Morphine was not reduced by SH and was a stress factor as determined by decreased metabolic activity. On the other hand, SH metabolized methadone showing first-order kinetics: the first-order rate constant k = 0,019, half-life t(1/2) = 36 h. Methadone metabolism led to decreased urea and albumin production. The expression of cytochrome P 3A4, mainly responsible for methadone metabolism, was proved in SH. The prototype BAL is basically suited to support liver functions, provided patients receive therapy with methadone.

On the inter-instrument and inter-laboratory transferability of a tandem mass spectral reference library: 1. Results of an Austrian multicenter study.

The inter-instrument and inter-laboratory transferability of a tandem mass spectral reference library originally built on a quadrupole-quadrupole-time-of-flight instrument was examined. The library consisted of 3759 MS/MS spectra collected from 402 reference compounds applying several different collision-energy values for fragmentation. In the course of the multicenter study, 22 test compounds were sent to three different laboratories, where 418 tandem mass spectra were acquired using four different instruments from two manufacturers. The study covered the following types of tandem mass spectrometers: quadrupole-quadrupole-time-of-flight, quadrupole-quadrupole-linear ion trap, quadrupole-quadrupole-quadrupole, and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. In each participating laboratory, optimized instrumental parameters were gathered solely from routinely applied workflows. No standardization procedure was applied to increase the inter-instrument comparability of MS/MS spectra. The acquired tandem mass spectra were matched against the established reference library using a sophisticated matching algorithm, which is presented in detail in a companion paper. Correct answers, meaning that the correct compound was retrieved as top hit, were obtained in 98.1% of cases. For the remaining 1.9% of spectra, the correct compound was matched at second rank. The observed high percentage of correct assignments clearly suggests that the developed mass spectral library search approach is to a large extent platform independent.

On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library: 2. Optimization and characterization of the search algorithm.

A sophisticated matching algorithm developed for highly efficient identity search within tandem mass spectral libraries is presented. For the optimization of the search procedure a collection of 410 tandem mass spectra corresponding to 22 compounds was used. The spectra were acquired in three different laboratories on four different instruments. The following types of tandem mass spectrometric instruments were used: quadrupole-quadrupole-time-of-flight (QqTOF), quadrupole-quadrupole-linear ion trap (QqLIT), quadrupole-quadrupole-quadrupole (QqQ), and linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LIT-FTICR). The obtained spectra were matched to an established MS/MS-spectral library that contained 3759 MS/MS-spectra corresponding to 402 different reference compounds. All 22 test compounds were part of the library. A dynamic intensity cut-off, the search for neutral losses, and optimization of the formula used to calculate the match probability were shown to significantly enhance the performance of the presented library search approach. With the aid of these features the average number of correct assignments was increased to 98%. For statistical evaluation of the match reliability the set of fragment ion spectra was extended with 300 spectra corresponding to 100 compounds not included in the reference library. Performance was checked with the aid of receiver operating characteristic (ROC) curves. Using the magnitude of the match probability as well as the precursor ion mass as benchmarks to rate the obtained top hit, overall correct classification of a compound being included or not included in the mass spectrometric library, was obtained in more than 95% of cases clearly indicating a high predictive accuracy of the established matching procedure.

Unraveling the metabolic transformation of tetrazepam to diazepam with mass spectrometric methods.

The metabolic transformation pathways of the 1,4-benzodiazepine tetrazepam (C(16)H(17)ClN(2)O, average mass: 288.772) were studied with capillary LC-QqTOF-MS and -MS/MS by analyzing human plasma and urine samples collected from healthy volunteers. Each volunteer took 50 mg of tetrazepam, given in the form of one tablet of Myolastan (Sanofi-Synthelabo, Vienna, Austria). Accurate molecular mass measurements in full-scan mode (scan range: 50-700) were used to survey the collected samples for putative metabolic transformation products. Full-scan fragment ion mass spectra were collected in subsequent LC/MS/MS experiments. Each spectrum was matched to a spectral library containing 3759 MS/MS-spectra of 402 compounds, including eighteen different benzodiazepines, to prove the structural relatedness of a tentative metabolite to tetrazepam. This "similarity search" approach provided a rapid and powerful tool to exclude non-drug-related species, even without any knowledge of the fragmentation chemistry. Interpretation of tandem mass spectrometric data was only required in order to elucidate the site of transformation. Using this strategy, 11 major classes of tetrazepam metabolites were identified. Possible metabolic routes from tetrazepam to diazepam (C(16)H(13)ClN(2)O, average mass: 284.740) via repeated hydroxylation and dehydration of the cylohexenyl moiety were discovered. No evidence for extensive hydroxylation of tetrazepam at position 3 of the diazepine ring was found. In contrast to what is commonly believed, this distinct transformation reaction may be of only minor importance. Furthermore, the occurrence of demethylation, hydration, and glucuronidation reactions was proven.

Pattern and severity of injury in avalanche victims.

In avalanche accidents, the significance of major trauma as a cause of morbidity and mortality is controversial. The aim of this retrospective study is to determine the severity and pattern of injury in avalanche victims admitted to the University Hospital of Innsbruck between 1996 and 2005. A total of 49 significant injuries were found in 105 avalanche victims; the most frequent were of the extremities (n = 20), the chest (n = 18), and the spine (n = 7). In contrast, cerebral (n = 2), abdominal visceral (n = 1), and pelvic trauma (n = 1) were rare. The severity of injury was minor or moderate in most patients, with only 9 (8.6%) being severely or critically injured. Of 105 (34.3%) avalanche victims, 36 died. Autopsy was performed in 30 of 36 nonsurvivors. The cause of death in the remaining 6 victims was concluded from clinical, radiological, and electrophysiological findings. Trauma was responsible for deaths of only 2 avalanche victims (5.6%); both had cervical spine fractures with dislocation leading to death. One death was due to hypothermia, whereas the remaining 33 fatalities (91.7%) were due to asphyxia. The incidence of life-threatening or lethal trauma was well below 10%. Asphyxia is by far the most important reason for death. Deaths from trauma were solely due to isolated cervical injuries, demonstrating that the cervical spine may be a region at particular risk in avalanche victims.

Another 'soberade' on the market: does Outox keep its promise?

OBJECTIVE: Several products are being widely promoted for reduction of the concentration of alcohol in the human body. One of these preparations, the fructose soft drink Outox, claims to noticeably increase the alcohol elimination rate (beta 60). Theories to explain this 'fructose effect' are based on the assumption that NAD+, the coenzyme for alcohol dehydrogenase, is regenerated faster in the presence of fructose. METHOD: A randomized double-blind, placebo-controlled cross-over study was performed with 30 volunteers in two drinking sessions each. Under strictly identical conditions, the same amount of alcohol was consumed, followed by the consumption of either 250 ml Outox or 250 ml placebo. Periodical measurements of blood (BAC), breath (BrAC) and urine alcohol concentration (UAC) were performed. RESULTS: Analyses revealed a significant difference (P<0.0001) between the mean alcohol levels of the Outox and the placebo drinking sessions. The overall mean BAC difference was 0.077 g/l (BAC 0.748 g/l without vs 0.671 g/l with Outox), equivalent to 10.3%. The mean BrAC difference was 0.045 mg/l (BrAC 0.314 mg/l without vs 0.269 mg/l with Outox), equivalent to 14.3%. Differences were lower for women than for men. A significant difference between the alcohol elimination rates (beta 60) was not found. CONCLUSIONS: The results show that the soft drink Outox may decrease the alcohol concentration by about 10%. However, BAC and BrAC differences are rather a consequence of slower gastric absorption of alcohol, because Outox does not increase the alcohol elimination rate. Our study demonstrates that the claim of Outox or other fructose drinks to work as a 'soberade' cannot be proven from a scientific point of view. It should be the task of physicians to warn potential consumers, especially in connection with drinking and driving.

Small human hepatocytes in rotary culture for treatment of alcohol addicts? A pilot study.

BACKGROUND: Current approaches to support alcohol addict and/or benzodiazepine-treated patients with liver failure include culturing human cells to take over basic metabolic functions for a certain time. METHODS: Small human hepatocytes (SH) were grown in a rotary cell culture system, and their potential to metabolize alcohol and the benzodiazepines oxazepam and diazepam was evaluated. Control experiments were performed with SV40-immortalized HEP cells and cell respective drug-free media. RESULTS: Our results show that SH in rotary culture are able to metabolize ethanol in reasonable amounts compared with evaporation controls (p<0.01). Moreover, SH are also able to metabolize oxazepam and diazepam which proves their ability to perform conjugation and the presence of functional cytochrome P450 enzymes. Basic metabolic activities such as glucose consumption, albumin and urea production are not significantly influenced by the drugs used, which is a precondition for clinical use of these cells. Significantly increased lactate dehydrogenase release indicates enhanced cell death in cultures of SH incubated with either ethanol (p<0.05) or diazepam (p<0.005), but stable viability at or above 90% suggests that cell proliferation is able to keep up with drug-induced cell death. CONCLUSION: Our preliminary study provides evidence that SH are basically suited to support alcohol-abusing and/or benzodiazepine-treated patients undergoing liver failure.

A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA.

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs)--generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets--enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.

Generating population data for the EMPOP database - an overview of the mtDNA sequencing and data evaluation processes considering 273 Austrian control region sequences as example.

The European DNA profiling group (EDNAP) mtDNA population database (EMPOP) is an international collaborative project between DNA laboratories performing mtDNA analysis and the DNA laboratory of the Institute of Legal Medicine (GMI) in Innsbruck, Austria. The goal is to set up a directly accessible mtDNA population database, which can be used in routine forensic casework for frequency investigations. Here we describe a safe laboratory scheme involving electronical data handling and computer-aided data transfer, which help to minimize errors originating from potential sample mix-up, data misinterpretation and incorrect transcription. The procedure is demonstrated by example of an mtDNA control region population study on 273 unrelated individuals from Austria. Our population sample was compared with five other European populations via an analysis of molecular variance (AMOVA). The inclusion of regions outside HVS-I and HVS-II increased the amount of information on the haplogroup diagnostic sites in the control region. Most of the haplotypes in Austrians fell into haplogroups H, J, K, T, and U. The random match probability in Austrians was 1:125; the average number of nucleotide differences between individuals in the Austrian database was 9.32.

Medicolegal aspects of tetrazepam metabolism.

The benzodiazepine tetrazepam is primarily muscle relaxant with comparably lower central sedating effects and is therefore commonly prescribed for muscle spasms of different origins. To evaluate tetrazepam metabolism, a study was conducted with ten healthy volunteers. Blood and urine samples were regularly collected after the intake of 50 mg tetrazepam. Toxicological analyses revealed that tetrazepam is also metabolized to diazepam and further to nordazepam, which has not yet been reported. Tetrazepam and diazepam could be detected in urine samples at least 72 h after intake, the diazepam concentration being 33% (+/-14% SD), on average, of the tetrazepam concentration. On the basis of three case histories, the importance of the detection of these newly described metabolites is shown as necessary to prevent false accusations and potential negative legal consequences for examined persons.

Elimination rates of breath alcohol.

Legal driving limits are set coequally with 0.5 g/L blood alcohol concentration (BAC) or 0.25 mg/L breath alcohol concentration (BrAC) in Austria as well as in other European countries. As mostly some time elapses between BrAC measurement and driving offence, a back calculation of alcohol concentrations is often required. The calculation of hourly BrAC elimination rates can thereby help to avoid unnecessary variances. A study with 59 participants was performed under social conditions. BrAC was determined with the legally accredited Alcotest 7110 MK III A every 30 min, and concomitantly venous blood samples were drawn. Five hundred and four BrAC/BAC value pairs were evaluated. The overall mean peak BrAC was calculated with 0.456 mg/L (+/-0.119 mg/L standard deviation). The mean hourly BrAC elimination rate was overall determined with 0.082 mg/L per h (0.050-0.114, 95% range). Mean rate of females (0.087 mg/L h(-1)) and the according 95% limits were statistically significantly higher than of males (mean rate 0.078 mg/L h(-1), p<0.04). Our results confirm the possibility to implement hourly BrAC elimination rates, provided that adequate statistical ranges and basic forensic scientific rules that have been set up for alcohol back calculations are observed.