Value of hair analysis in postmortem toxicology

It is generally accepted that chemical testing of biological fluids is the most objective means of diagnosis of drug use. The presence of a drug analyte in a biological specimen can be used to document exposure. The standard in postmortem drug testing is a general unknown screening, followed by the gas chromatographic/mass spectrometric confirmation conducted on a whole blood sample. In recent years, remarkable advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional biological specimens such as hair. The advantages of this sample over traditional media, like urine and blood, are obvious: collection is almost non-invasive, relatively easy to perform, and in forensic situations it may be achieved under close supervision of law enforcement officers to prevent adulteration or substitution. Moreover, the window of drug detection is dramatically extended to weeks, months or even years. The aim of this review is to document the current status of hair analysis in postmortem toxicology.     

The forensic science implications of site and temporal influences on postmortem blood-drug concentrations

The dependence of postmortem blood-drug concentrations on the collection site and on the postmortem interval before specimen collection has been studied. These studies consisted of both sequential sampling from the same collection site at defined time intervals and a comparison of the drug concentrations of postmortem blood simultaneously collected from various sites. A site and time dependence was observed for postmortem blood-drug concentrations. The heart blood-drug concentrations were, in general, significantly higher than those of peripheral specimens. As a result of this phenomenon, the analysis of peripheral blood specimens and solid tissues is often necessary before a definitive interpretation of postmortem toxicological analyses is possible.     

The use of vitreous humor as an alternative to whole blood for the analysis of benzodiazepines

In postmortem drug analysis, the most commonly used sample matrix is whole blood. However, postmortem changes can denature this matrix, resulting in a loss or degradation of drugs, thus biasing analytical findings. Vitreous humor is thought to be less affected by these changes and should, therefore, have the potential to provide a more reliable estimation of antemortem drug concentrations. To assess the usefulness of vitreous humor for the analysis of benzodiazepine drugs, vitreous humor and whole blood were obtained postmortem in 27 cases. Three benzodiazepine drugs were investigated-temazepam, diazepam, and desmethyldiazepam. For temazepam and diazepam, some correlation was found between the matrices (R2 = 0.789 and 0.724, respectively). However, for desmethyldiazepam, no correlation was observed (R2 = 0.068). Regression analysis on plots of vitreous humor versus blood concentrations produced gradients of less than 1.0 showing that, in general, levels in blood are higher than the corresponding levels in vitreous humor.     

Hyaluronidase as a liquefying agent for chemical analysis of vitreous fluid

Vitreous humor is a suitable specimen for postmortem clinical chemistry because the analytes remain relatively stable after death and they closely reflect blood levels immediately prior to death. The viscous nature of vitreous fluid, however, presents analytical problems including imprecision and inaccuracy. Various preanalytical treatments, such as boiling, high speed centrifugation, microfiltration and dilution have been used. These techniques are labor intensive and add to imprecision and inaccuracy. Because glycosaminoglycans contribute significantly to the viscosity of vitreous humor, we used hyaluronidase as a liquefying agent. We compared the results of analyses in 33 vitreous humor specimens after hyaluronidase treatment with the results after either no treatment or specimen dilution. Seventeen of the 33 specimens could not be analyzed without dilution. Even after dilution, several analytes still could not be measured. Hyaluronidase treatment negated the need for sample dilution and had no significant effect on the analyses.     

Effect of sodium fluoride on cholinesterase activity in postmortem blood

Thirty-two postmortem blood specimens, with and without sodium fluoride as preservative, were analyzed for cholinesterase activity by the Michel method. The fluoridated specimens, which contained from 0.7 to 31 mg/mL (average 6.3) of sodium fluoride, were found to exhibit cholinesterase activities that were 5 to 59% (average 25%) lower than the duplicate unfluoridated specimens. We concluded that, while this decrease is quite significant, a fluoridated postmortem blood specimen may be used for the measurement of cholinesterase activity when a non-fluoridated specimen is unavailable.     

An unusual drug death involving maggots

Toxicologic analysis of decomposed specimens provides greater analytical challenges than those encountered with fresh postmortem specimens. Despite the difficulties involved, in cases in which the cause of death is not determined at autopsy or when there is a strong indication of drug intoxication, all reasonable steps must be undertaken to perform as comprehensive a drug screen as possible. An unidentified white male was found in a field near a river. The body was decomposed and skeletonized, and 3- to 4-mm maggots were present on the body. Near the body was an empty bottle of secobarbital that had been prescribed to a female. There was no evidence of injury. Calf muscle and maggots were sent for toxicologic analysis. No volatile substances or drugs were detected in the calf muscle. Because intoxication due to secobarbital was strongly suggested from the scene investigation, the only other specimen available, the maggots, were tested for acid-neutral drugs. Secobarbital was identified by retention time and was confirmed by full-scan electron ionization gas chromatography/mass spectrometry. Based on the available information, the medical examiner ruled that the cause of death was secobarbital intoxication and the manner of death was suicide.     

The extent of postmortem drug redistribution in a rat model.

The aim of this study was to investigate the postmortem redistribution of several drugs in a rat model and to examine if any of the pharmacological properties was related to the extent of this phenomenon. One of the following drugs: phenobarbital (phenobarbitone), acetaminophen (paracetamol), carbamazepine, codeine, verapamil, amphetamine, mianserin, trimeprazine (alimemazine) or chloroquine was administered together with nortriptyline orally to rats 90 min prior to sacrifice. Heart blood was sampled immediately before sacrifice and after 2 h postmortem, as it has previously been shown that this is sufficient time for postmortem concentration changes to occur in heart blood. Blood was also sampled from the clamped abdominal inferior vena cava (representing peripheral blood) and tissue samples were taken from lungs, myocardium, liver, kidney, thigh muscle, forebrain, and vitreous humor together with a specimen from the minced carcass. Drugs were analyzed by high performance liquid or gas chromatography. For phenobarbital, acetaminophen and carbamazepine the postmortem to antemortem blood drug concentration ratios were close to 1.0 and tissue concentrations were low. The postmortem to antemortem heart blood drug concentration ratio for chloroquine (6.9 +/- 1.5) was higher than for nortriptyline (3.5 +/- 0.3), and the remaining drugs (codeine, verapamil, amphetamine, mianserin, and trimeprazine) showed ratios of the same magnitude as nortriptyline. The postmortem to antemortem blood drug concentration ratios for both heart blood and blood from the vena cava and also the lung to antemortem blood drug concentration ratio were closely related to the apparent volume of distribution for the drugs studied (p < 0.001). Accordingly, an apparent volume of distribution of more than 3-4 L/kg is a good predictor that a drug is liable to undergo postmortem redistribution with significant increments in blood levels. The postmortem drug concentration in blood from vena cava was closely related to the antemortem blood level, confirming that among the postmortem samples, the peripheral blood sample was the most representative for the antemortem blood concentration.     

Forensic Imaging‐Guided Recovery of Nuclear DNA from the Spinal Cord*,†

Abstract: Our objective is to document the recovery of DNA from the spinal cord or surrounding dura mater in 11 cases of severely burned human remains. Radiographs established that portions of charred tissue contained spine segments. Multidetector computed tomography (MDCT) revealed that each spine specimen contained an intact spinal cord remnant. A full DNA profile was obtained from seven specimens using spinal cord dura mater in six specimens and spinal cord medulla in one specimen. A partial profile was obtained from four specimens (spinal cord dura mater, 2; spinal cord medulla, 2). Bone and muscle surrounding the spinal cord appear to insulate nucleic acid containing tissue from critical thermal degradation. The spinal cord, which is easily identified by MDCT examination of remains and easily recovered at the postmortem examination, can be a source of DNA with extraction yields comparable with other tissue sources. Specimens of dura mater are preferable as processing time is faster than bone.     

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.     

The U.S. Mandatory Guidelines for Federal Workplace Drug Testing Programs: current status and future considerations

The U.S. Department of Health and Human Services (HHS) drug testing standards were published in 1988 and revised in 1994, 1998, and 2004. In 2004, significant revisions defining, standardizing, and requiring specimen validity testing on Federal employee donor urine specimens were included. In a separate notice, HHS proposed to establish scientific and technical guidelines for the Federal Workplace Drug Testing Program to: (1) permit laboratory testing of hair, oral fluid, and sweat patch specimens in addition to urine specimens for marijuana, cocaine, phencyclidine, opiates (with focus on heroin), and amphetamines [including methylenedioxymethamphetamine (MDMA), methylenedioxyethamphetamine (MDEA), methylenedioxyamphetamine (MDA)]; (2) permit use of on-site point of collection test (POCT) devices to test urine and oral fluid at collection sites; (3) permit use of instrumented initial test (screening only) facilities [IITF] to quickly identify negative specimens; and (4) add training requirement for collectors, on-site testers, and MROs. This proposal was published in the Federal Register on 13 April 2004, with a 90-day public comment period. The Substance Abuse and Mental Health Services Administration, HHS, reviewed those comments and is preparing the Final Notice that will define the requirements for such testing, including: specimen collection procedures, custody and control procedures that ensure donor specimen identity and integrity, testing facility, initial and confirmatory test cutoff concentrations, analytical testing methods, result review and reporting, evaluation of alternative medical explanations for presence of drug or metabolite in the donor's specimen, and laboratory certification issues. Voluntary pilot performance testing (PT) programs for each specimen type are on-going since April 2000 to determine how to prepare PT materials for specimens other than urine to evaluate laboratories' ability to routinely achieve accuracy and precision required. Certification programs will be developed using the current urine drug testing National Laboratory Certification Program model. The addition of accurate and reliable workplace drug testing using hair, oral fluid, and sweat patch specimens will complement urine drug testing, and aid in combating industries devoted to suborning drug testing through adulteration, substitution, and dilution. For example, hair testing may detect chronic drug use for up to 90 days and be useful in pre-employment situations; oral fluid testing may detect drug use in past hours and be useful in post-accident situations; sweat patch testing may be useful in follow-up drug testing and treatment programs; POCTs and IITFs may be most useful for quickly identifying specimens that are negative for drugs and indicate that the specimen is valid.     

Determination of deslanoside in antemortem and postmortem specimens. Unusual case report

One case of the erroneous administration of deslanoside and high level of drug in antemortem plasma and postmortem specimens has been reported owing to the unusual surrounding circumstances. Deslanoside in antemortem plasma was determined by FPIA and the analysis was done by HPLC in the postmortem tissue samples. The analytical results and methods used in the examinations are discussed in the following paper.     

Dominance of pre-analytical over analytical variation for measurement of methadone and its main metabolite in postmortem femoral blood

On the basis of simultaneously sampled postmortem blood specimens from the left and right femoral veins the pre-analytical variation of methadone measurements was evaluated and compared to the analytical variation. The material consisted of a series of 27 duplicate samples from routine autopsy cases comprising mainly drug addicts. A chiral LC-MS/MS method was used for measurement of the R- and S-enantiomers of methadone and its main metabolite 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium (EDDP). The analytical CV% was determined to be in the range 3-4% for methadone enantiomers and 4-6% for EDDP enantiomers. The total measurement uncertainty (CV(T)) was estimated from the pre-analytical variation (CV(PA)), analytical variation proper (CV(A)), and variation related to calibration (traceability) (CV(Cal)) according to the relationship CV(T) = [CV(2)(PA) + CV(2)(A) + CV(2)(cal)](0.5). Uncertainty related to calibration concerned a component related to the purity of drug reference compound and a contribution from the production of calibrator solutions (CV(Cal)<1%). Pre-analytical sampling variation was estimated from the duplicate measurements of blood samples after subtraction of the analytical component. The pre-analytical variation amounted to a CV% of 19-21% for R- and S-methadone and 30-38% for R- and S-EDDP, i.e. considerably larger than the other components. Due to the squared addition principle, the resulting total uncertainty (CV(T)) became largely identical to the CV(PA), i.e. 19-21% for R- and S-methadone and 31-38% for R- and S-EDDP enantiomers. Accordingly, CV(T) exceeded CV(A) by a factor 5 or more. Dominance of the pre-analytical component of variation may also be likely for other compounds measured in postmortem blood samples. Thus, the width of the 95%-uncertainty interval (+/-2CV(T)) for a postmortem measurement is largely determined by the pre-analytical component of variation. This should be kept in mind when judging on the uncertainty of postmortem measurement results.     

Detection of cocaine in blood stains]

Cocaine is rapidly degraded in blood samples, and its degradation was found to be highly dynamic in nature. The analysis of blood spots dried on filter paper may provide a method to minimize the break-down of cocaine and to largely preserve the analytical profile of the parent drug and its hydrolysis products at the time of sampling. The short term stability of cocaine in 100 microL blood spots prepared from unpreserved and preserved (sodium fluoride, 0.25%) blood samples was compared to the stability of the particular whole blood specimens stored in tubes at ambient temperature and at -20 degrees C. Due to dehydration, both the chemical and the enzymatic hydrolysis of cocaine and its products could be stopped in dried blood spots. More than 75% of the initial cocaine concentration could be detected in the blood spots, and the analytical profile was ensured for 17 days. Provided its practical suitability, the spot technology should offer a simple approach to detect actual impairment of motorists taken in police custody in the view of section 24a of the German traffic act as well as in cocaine associated criminal cases.     

法医毒物动力学

本文作者针对法医毒理学的任务、发展趋势和面临的挑战，提出将法医毒物动力学列为法医毒理学新的分支学科，并阐述了法医毒物动力学的概念、研究目的和任务、研究对象、研究内容、研究方法、研究方向及亟待解决的问题。内容涉及毒物动力学、死后分布、动态分布、死后再分布、死后弥散、毒物分解动力学、毒物死后产生情况等。法医毒物动力学研究将解决中毒法医学鉴定中的许多问题，如尸体或活体中毒当时机体内毒物浓度的推断、死后腐败产生毒物与生前服毒的区别、生前服毒与死后染毒的鉴别、毒物进入机体时间、途径和方式的确定。     

Postmortem DNA and RNA synthesis. Preliminary studies in human cadavers

Postmortem DNA and RNA synthesis was detected in tissue specimens harvested from two cadavers at different intervals between 2.5 and 32 h postmortem. Each tissue specimen was incubated for 1 h in a 3H-thymidine or 3H-cytidine solution. DNA- as well as RNA-synthesizing cells were found in skin tissue and bone marrow throughout the interval investigated. Cytidine incorporation decreased progressively during the course of the postmortem interval. DNA and RNA synthesis was also observed in cells of the testis, which were predominantly spermatogonia cells in the case of DNA. Low-grade RNA synthesis was detected in bowel epithelial cells up to 2.5 h postmortem; DNA synthesis was not present during the interval investigated. No supravital phenomena were observable in the splenic tissues examined.     

Alcohol loss arising from microbial contamination of drivers' blood specimens

This paper describes the circumstances in which some drivers' blood specimens containing added sodium fluoride (1% w/v concentration) deteriorated as a result of microbial contamination, accompanied by a decrease of alcohol concentration. Strains of the bacteria Serratia marcescens and a Pseudomonas sp. were isolated from the specimens and proven capable of growing at ambient temperature in blood containing sodium fluoride at 1% w/v concentration. They were shown to be active in alcohol degradation in preservatised blood, the activity being dependent on sodium fluoride concentration and storage temperature. Blood diluters were assumed to be a source of microbial cross contamination from one blood specimen to the next. It is recommended that postmortem blood specimens be analysed in separate batches from drivers' specimens when automated blood diluters are used, that the content of fluoride ions be increased to an equivalent of 2% w/v sodium fluoride, and that storage of specimens at temperatures above 4 degrees C be minimised.     

Methanol Detected in a Subdural Hematoma as an Embalming Artifact

Analysis of subdural hematomata has been used to suggest antemortem drug concentrations, with the assumption that materials within the hematoma are less subject to metabolism or degradation during any survival period and postmortem interval. We report the case of an 87‐year‐old woman whose death had not been reported to the coroner's office until postembalming. Autopsy revealed a traumatic brain injury with subdural hematoma causing a mass effect. Testing of the clot indicated a methanol concentration of 51.8 mg%. No additional analyses were detected. These findings suggest that methanol can be present in a postmortem hematoma sample, yet not represent a poisoning. Our findings also suggest that while the interior of hematomata do not necessarily represent completely “protected space” from postmortem diffusion of some blood constituents, such diffusion is not facile, and analysis may still provide useful indications of antemortem drugs present, if not actual concentrations.     

Determination of a newly encountered designer drug "p-methoxyethylamphetamine" and its metabolites in human urine and blood

A newly synthesized designer drug, para-methoxyethylamphetamine (PMEA) was unexpectedly detected in the postmortem specimens of fatality involving drug intoxication in 2005, Japan. For unequivocal identification, the isomeric discrimination of PMEA and its positional-isomers was performed by GC/MS with the trifluoroacetylation. In order to prove the intake of PMEA, the characteristic metabolites of PMEA were also identified by GC/MS analysis of the urine specimen with trifluoroacetylation. As a result, para-methoxyamphetamine, para-hydroxyethylamphetamine (POHEA) and para-hydroxyamphetamine were identified as the major metabolites of PMEA. For the quantitative analyses of PMEA and its three metabolites in body fluids, an automated column-switching LC/MS procedure was developed, and applied to the postmortem blood and urine specimens. In this fatal case, blood concentration of PMEA was estimated to be 12.2 microg/mL and this level seemed extremely high in comparison with lethal blood-levels of its analogues, representing acute-intoxication of the victim. Based on the quantitative results, PMEA was found to be extensively metabolized to POHEA via O-demethylation, partly followed by its conjugation.