Automatic screening in postmortem toxicology.

The systematic analysis of postmortem samples is one of the most challenging tasks in forensic toxicology. For determining cause of death, analysis of different tissues can be indispensable. Automation of these analyses would increase reproducibility and therefore lead to more reliable and comparable results. Recent developments in analytical toxicology and the availability of automation devices for various analytical stages, such as sampling, preliminary testing, sample extraction, chromatographic separation, identification, and data processing are examined and discussed. At present only parts of the analytical procedure have been automated-however, the goal should be the integration of these parts into a single, continuous process. Currently, only one "fully-automated" procedure for the comprehensive screening of blood and urine (excluding sample pretreatment, which remains separate) has been published. But it can be expected that automation of analytical procedures in forensic toxicology will indeed progress, even with regard to the very complex screening of postmortem samples.

Prediction of gas chromatographic retention indices of a diverse set of toxicologically relevant compounds.

For a set of 846 organic compounds, relevant in forensic analytical chemistry, with highly diverse chemical structures, the gas chromatographic Kovats retention indices have been quantitatively modeled by using a large set of molecular descriptors generated by software Dragon. Best and very similar performances for prediction have been obtained by a partial least squares regression (PLS) model using all considered 529 descriptors, and a multiple linear regression (MLR) model using only 15 descriptors obtained by a stepwise feature selection. The standard deviations of the prediction errors (SEP), were estimated in four experiments with differently distributed training and prediction sets. For the best models SEP is about 80 retention index units, corresponding to 2.1-7.2% of the covered retention index interval of 1110-3870. The molecular properties known to be relevant for GC retention data, such as molecular size, branching and polar functional groups are well covered by the selected 15 descriptors. The developed models support the identification of substances in forensic analytical work by GC-MS in cases the retention data for candidate structures are not available.

Systematic toxicological analysis: computer-assisted identification of poisons in biological materials.

A new software was developed to improve the chances for identification of a "general unknown" in complex biological materials. To achieve this goal, the total ion current chromatogram was simplified by filtering the acquired mass spectra via an automated subtraction procedure, which removed mass spectra originating from the sample matrix, as well as interfering substances from the extraction procedure. It could be shown that this tool emphasizes mass spectra of exceptional compounds, and therefore provides the forensic toxicologist with further evidence-even in cases where mass spectral data of the unknown compound are not available in "standard" spectral libraries.

[Fatal poisonings with antidepressive drugs and neuroleptics. Analysis of a correlation with prescriptions in Vienna 1991 to 1997]. / Letale Intoxikationen mit Antidepressiva und Neuroleptika. Analyse im Zusammenhang mit den Verordnungen in Wien von 1991 bis 1997.

As a result of the increasing use of selective serotonin reuptake inhibitors (SSRI), the number of antidepressants (AD) prescribed in Vienna doubled from 1991 to 1997. In the same period, autopsies and chemical analyses performed at the Institute of Forensic Medicine, University of Vienna, revealed a total of 164 fatal intoxications by means of AD. In this study, the number of fatal intoxications per million defined daily doses prescribed was determined and referred to as the fatal toxicity index (f-index). For both single- and multiple-substance intoxications, it proved to be significantly (p < 0.001) lower with SSRI than with tricyclic antidepressants (TCA). Single-substance intoxications (n = 30) were seen exclusively in TCA. Concerning neuroleptics (NL), the increase in prescriptions observed in the study period (plus 30%) was less pronounced, and they were found to be involved in 85 fatal intoxications. Also in NL, those of the tricyclic type (TCNL) showed a significantly (p < 0.001) higher f-index than other groups. Out of a total of 17 single-substance intoxications, 14 were caused by TCNL and none by butyrophenones or haloperidol. The present study demonstrates that the prescription of TCA or TCNL involves a relatively high risk of fatal intoxication.

Estrogen increases endothelial carbon monoxide, heme oxygenase 2, and carbon monoxide-derived cGMP by a receptor-mediated system.

Carbon monoxide, a gaseous activator of soluble guanylyl cyclase formed by a subtype of the enzyme heme oxygenase designated heme oxygenase-2 in vascular endothelium, has been found to dilate blood vessels independently from nitric oxide. Because of the parallels between nitric oxide and carbon monoxide, we speculated that estrogen might affect carbon monoxide production in vascular endothelium. Endothelial cells of human origin (umbilical vein and uterine artery) were incubated for 4 or 24 h with 10(-12)-10(-6) M 17beta-estradiol. 17beta-Estradiol, at a concentration such as that attained during the ovulatory phase of the menstrual cycle (10(-10) M), administrated for 4 h led to a 2-fold increase in intracellular carbon monoxide production and heme oxygenase-2 protein levels (P < 0.05). A reporter assay, measuring the formation of cGMP as the direct product of carbon monoxide-induced activation of soluble guanylyl cyclase in endothelial cells, also revealed a 56% increase in cellular cGMP after treatment with 10(-10) M E2 17beta-estradiol (P < 0.05). By contrast, higher 17beta-estradiol concentrations had no significant respective effects due to nitric oxide synthase inhibition of carbon monoxide release. This 17beta-estradiol effect appeared to be ER dependent, as preincubation with tamoxifen (10(-6) M) blocked the stimulatory effect of 17beta-estradiol in each instance. Our preliminary data indicate a potential role for carbon monoxide as a biological messenger molecule in estrogen-mediated regulation of vascular tone.

Different fatal toxicity of neuroleptics identified by autopsy.

Autopsies and toxicological analyses at the Institute of Forensic Medicine revealed 85 fatal intoxications with neuroleptics in Vienna from 1991 to 1997. A total of 17 cases were linked to a single neuroleptic (NL) alone, while 68 deaths were attributed to a combination of NLs with other drugs. The most frequently detected agent was prothipendyl (n=41). During the study period the number of defined daily doses of high-potency NLs prescribed increased significantly (P< or =0.001) due to increased prescribing of new atypical antipsychotics. The quantity of intermediate- and low-potency NLs dispensed remained stable. The most frequently prescribed NL was haloperidol. The relative toxicities of different NLs were calculated by dividing the number of deaths caused by this NL into the number of defined daily doses prescribed in the observation period (f-value). Single-substance intoxications and multiple-substance intoxications were distinguished. The highest f-values were associated with low-potency NLs, especially with prothipendyl, chlorprothixene and levomepromazine. Low f-values were found for the group of high-potency NLs, including flupentixol, fluphenazine, haloperidol and pimozide, as well as olanzapine. Compared to the f-values for all NLs prescribed, f-values for low-potency NLs were shown to be significantly higher concerning single-substance intoxications (P< or = 0.05) and multiple-substance intoxications (P < or = 0.001), while f-values for high-potency NLs were significantly lower (P< or = 0.05 and P< or = 0.001). We are not aware of the psychiatric diagnoses in our post-mortem sample. However, the present results indicate that careless use of low-potent NLs should be avoided in patients with a potential risk of accidental or suicidal overdose.

General unknown screening in postmortem tissue and blood samples: a semi-automatic solid-phase extraction using polystyrene resins followed by liquid-liquid extraction.

The identification of general unknown poisons in complex biological materials like postmortem blood and tissue is a great challenge for the forensic toxicologist. Therefore, a screening procedure utilizing a semi-automatic work-up with an ASPEC system was developed. A broad range of different compounds can be isolated by using non-selective and generally applicable organic polymeric sorbents such as OASIS HLB or Isolute 101. Because colloidal solutions were applied to these sorbents, the denaturation of proteins, which can result in an irreversible loss of significant compounds by adsorption and occlusion, could be avoided. Because of the process of micellar chromatography followed by liquid-liquid extraction of the crude extract, very clean fractions were obtained from such complex matrices as postmortem blood, liver, and brain samples. High recoveries (72-100%) and good day-to-day relative standard deviations (1-17%) could be achieved with both polymeric sorbents. The procedure paves the way for the identification of general unknown poisons in target organs and is therefore a useful tool in the field of forensic toxicology.

Drug-related emergencies and drug-related deaths in Vienna, 1995-1997.

The demographics of drug-related emergencies and drug-related deaths that occurred in Vienna between January 1st, 1995 and December 31st, 1997 were compared to investigate whether they represent two different subgroups of the drug-taking community. Analysis indicated that drug-related emergencies were significantly younger and that the proportion of females was higher than amongst the fatalities. In addition, emergencies were more likely than fatalities to occur in private residences than public places. No significant association between the number of contacts with the Vienna Ambulance Service and the fatal outcome of drug use could be substantiated. The results of this study indicate that drug-related emergencies and drug-related deaths represent two different subpopulations of the drug-taking community. Therefore, different strategies of prevention are considered.

Direct current auditory evoked potentials during wakefulness, anesthesia, and emergence from anesthesia.

Direct current auditory evoked potentials (DC-AEPs) are a sensitive indicator of depth of anesthesia in animals. However, they have never been investigated in humans. To assess the potential usefulness of DC-AEPs as an indicator of anesthesia in humans, we performed an explorative study in which DC-AEPs were recorded during propofol and methohexital anesthesia in humans. DC-AEPs were recorded via 22 scalp electrodes in 19 volunteers randomly assigned to receive either propofol or methohexital. DC-AEPs were evoked by binaurally presented 2-s, 60-dB, 800-Hz tones; measurements were taken during awake baseline, anesthesia, and emergence. Statistical analysis included analysis of variance and discriminant analysis of data acquired during these three conditions. About 500 ms after stimulus presentation, DC-AEPs could be observed. These potentials were present only during baseline and emergence-not during anesthesia. Statistically significant differences were found between baseline and anesthesia and between anesthesia and emergence. In conclusion, similar effects, as reported in animal studies of anesthetics on the DC-AEPs, could be observed in anesthetized humans. These results demonstrate that DC-AEPs are potentially useful in the assessment of cortical function during anesthesia and might qualify the method for monitoring anesthesia in humans.

Quality of heroin and heroin-related deaths from 1987 to 1995 in Vienna, Austria.

AIMS: To investigate the quality of heroin seized in Vienna between 1987 and 1995 and to examine whether there was a relationship between the quality of heroin and the rate of drug-related deaths. DESIGN: Reports of heroin seizure analysis and post-mortem reports of heroin-related deaths in Vienna from 1987 to 1995 were analysed. RESULTS: There were 386 seizures of heroin comprising a total weight of 25 640.12 g heroin. All the seizures were in the base form. All seizures also contained a diluent, mainly lactose. Additionally, in 95 seizures caffeine, in four seizures paracetamol and in three seizures metaqualon were detected. Of a total of 764 drug-related deaths 506 cases were classified as heroin-related deaths. In 336 cases other central nervous system-depressant drugs and/or alcohol could be detected in the blood in addition to morphine (polydrug heroin-related deaths). There was evidence of a trend towards greater polydrug involvement during the study period. The age of victims of polydrug heroin-related deaths remained unchanged over time while the age of victims of pure heroin-related deaths decreased significantly. The morphine concentration in the medulla oblongata in heroin-related deaths decreased significantly. Finally, there was no statistically significant relationship between the rate of heroin-related deaths per year and the diacetylmorphine concentration of heroin seizures in that year. CONCLUSION: The results did not substantiate the widely held belief that increases in heroin-related deaths could be explained by an increase in the quality of heroin.

Suicide by antidepressant intoxication identified at autopsy in Vienna from 1991-1997: the favourable consequences of the increasing use of SSRIs.

In the area of Vienna, any person dying under questionable circumstances is examined at the Institute of Forensic Medicine, where the cause of death is determined by means of autopsy and chemical analysis. Our study on fatal intoxications was performed in the period between 1991 and 1997, when selective serotonin reuptake inhibitors (SSRIs) were establishing themselves on the market, reaching the top of prescription statistics. Tricyclic antidepressants (TCAs) were involved in 30 single- and 127 multiple-substance intoxications, with amitriptyline and doxepin being the most frequently used drugs. SSRIs were involved in five multiple-substance intoxications. The f-value, which refers to the number of deaths per million defined daily doses prescribed, was found to be significantly (P

Suicide of a body packer.

The smuggling of illicit drugs by means of body packing has become a common problem at European airports. Europe is considered to be the fastest growing market for cocaine worldwide, and the air route is the most frequently used method of trafficking cocaine. Smuggling illicit drugs by use of body packing is considered to be a high toxicological hazard because of the risk of leakage or a package bursting. We report about the first case of suicide of a body packer by re-ingesting the content of excreted cocaine packages. The consequence of this case is that the death scene investigation and autopsy assessments in case of a body packer's death should always consider the possibility of re-ingested packages. Detention personnel should be instructed by forensic and criminalistic experts to take preventive measures.

Identification of the general unknown. Application of mass selective detectors in forensic toxicology.

One of the basic aims of forensic toxicology is the identification of previously unknown drugs and poisons. This is frequently achieved using the combination of gas chromatography and benchtop quadrupole or ion trap mass spectrometers. The influence of matrix loading on the mass spectral quality was tested, and it was found that a realistic amount of matrix changed the pattern of the spectra obtained by the ion trap mass spectrometer. Disturbed mass spectra led to unsuitable suggestions from the library search and thus rendered the identification of the "general unknown" more difficult. On the other hand, higher selectivity and lower detection limits favored the ion trap technology for target analysis because of the capability of MS-MS.

Historical development of expertise in forensic chemical analysis. General survey, illustrated by case studies from the Viennese Institute.

The history of forensic chemical analysis shows a development from the mere quotation of a concentration value to an expert opinion which helps to explain manifold circumstances of the case under consideration. The development of forensic expertises on chemical problems connected to forensic medicine reveals two essential stages: 1. A period, when sensual perceptions were the only means of investigation and 2. a period, when developments in chromatographic separation and spectrometric identification have permitted objectified results.

Drugs and driving in Vienna, Austria.

Drugs that affect the central nervous system are generally assumed to have the potential to impair driving ability. In a retrospective survey, police files and the results of toxicological urine analysis from drivers suspected of driving under the influence of drugs in Vienna from 1993 to 1996 were investigated. Decisive for police intervention was "unsafe driving" (swerving, hesitating, going too slowly, etc.), driving at high speed within the city limits, driving through red lights or stop signs, and driving at night without lights. In one-fifth of the cases drivers caused a traffic accident. Casting suspicion on driving under influence of drugs was mainly caused by impaired coordination of movements, bloodshot eyes, slurred speech, drowsiness, conspicuous behavior, and changed pupils. In the majority of the study population more than two symptoms were recorded by police. In 94% of the cases police suspicions could be confirmed by toxicological urine analysis.

Homicide by strangling or dumping with postmortem injuries after heroin poisoning?

During construction work, the corpse of a 33-year-old man, H.L., who had been missing for 2 years and 4 months, was found in the cellar of a house. Primary findings indicated an attack directed at the throat (hematomas of the soft tissue and broken larynx). The owner of the cellar claimed, however, that H.L. had died of overdose of heroin and that he had removed the body to avoid trouble with the authorities. Morphine poisoning was confirmed by chemical analysis. The case is interesting because of (a) the good condition of the corpse after over 2 years, (b) macroscopic and microscopic evidence of hematomas of the cervical soft tissue, (c) successful chemical analysis providing evidence despite a long time since death, and (d) considerations regarding the vitality of injury findings.

Suicidal chloroquine poisoning: clinical course, autopsy findings, and chemical analysis.

Suicidal overdose of chloroquine is rare. We present a 14-year-old girl who was admitted to our Emergency Department after intentional ingestion of 7.5 g of chloroquine base followed by cardiac and respiratory arrest. Despite early mechanical ventilation, cardiac support, and treatment with high doses of diazepam, the patient died. Laboratory investigation indicated disseminated intravascular coagulation confirmed by petechial hemorrhages in the leptomeninges at autopsy, a finding that has not previously been described. Postmortem tissue analysis demonstrated early drug distribution to the medulla oblongata and cerebellum.

Pharmacokinetic studies of the fragrance compound 1,8-cineol in humans during inhalation.

The present study was undertaken to investigate the pharmacokinetics of 1,8-cineol in human subjects during prolonged inhalation. The results showed that 1,8-cineol is well absorbed from breathing air, with a peak plasma concentration after approximately 18 min. The elimination of this fragrance compound from the blood is biphasic, with a mean distribution half-life of 6.7 min and an elimination half-life of 104.6 min.

[Dangerous intoxication from extreme serum concentrations of methaqualone metabolites. Detection and quantification of biosynthesis with gas chromatography-mass spectrometry]. / Lebensbedrohliche Intoxikation durch extreme Serum-konzentration eines Methaqualonmetaboliten. Nachweis und Quantifizierung mittels Biosynthese und Gaschromatographie-Massenspektrometrie.

CLINICAL COURSE: We present a potentially fatal case of acute methaqualone (M) poisoning with very low serum concentrations of M but extremely high levels of its metabolite, 2-methyl-3-(2-hydroxymethyl-phenyl)-4 (3H)-chinazoline (Met-1). A 23-year-old man was admitted to the intensive care unit 2 days after ingestion of 4-5 g M in an suicidal attempt. On admission he was somnolent and poorly responsive to painful stimuli. Physical examination revealed a heart rate of 95 bpm, a blood pressure of 125/65 mmHg, and a normal body temperature. His chest was clear to auscultation, respirations were shallow, and the skin was cyanotic. The electrocardiogram was unremarkable. The chest radiograph showed a normal heart size without pulmonary infiltrates or venous congestion. The pupils were dilated but reactive to light. The neurologic examination was further remarkable for increased limb reflexes, myoclonia, and positive pyramidal signs. During the next 2 days the patient became comatose and developed respiratory insufficiency due to non-cardiogenic pulmonary oedema, which was confirmed by chest radiograph and haemodynamic investigations by means of right heart catheterisation. He required mechanical ventilation for 6 days. Finally, he recovered completely and was discharged in good condition. DIAGNOSTICS: A lumbar puncture revealed neither blood nor pleocytosis in the cerebrospinal fluid. Cranial computed tomography was carried out on an emergency basis, but no abnormality was disclosed. An electroencephalogram did not exhibit any significant pathological findings. Testing for infectious diseases or porphyria gave negative results. Toxicological screening based on enzyme immunoassays (ELISA) was negative for alcohol, tricyclic antidepressants, benzodiazepines, barbiturates, and morphine, but gave a positive result for M. From the moment of admission daily blood samples were taken and analysed by combined gas chromatography and mass spectrometry. These showed very low levels of M but extremely high levels of Met-1. THERAPY: After gastric lavage, continuous enteric lavage with activated charcoal and mannitol was initiated to minimise intestinal absorption. Since M was hardly detectable in the serum, haemoperfusion was not regarded as indicated for drug elimination and treatment was restricted to general supportive measures. To rule out a central anticholinergic syndrome, an anticholinesterase drug (physostigmine) was administered but remained without therapeutic effect. CONCLUSIONS: The presented case is the first report of a life-threatening intoxication after M ingestion primarily caused by Met-1. It supports the significance of this metabolite for the toxic effects of the drug. A toxicological screening test based on ELISA proved helpful due to its cross-reactivity with metabolites. In cases similar to ours, resin haemoperfusion may be indicated to remove the metabolites despite low detectable concentrations of the parent substance in the serum.