[Determination of blood/serum ratios of different forensically relevant analytes in authentic samples]. / Bestimmung der Blut/Serum-verhältnisse verschiedener forensisch relevanter Analyten in authentischen Proben.

For forensic toxicological investigations only whole blood, but no serum is often available. Pharmacokinetic data are helpful for interpreting the results, but most of these studies indicate serum or plasma concentrations. In order to obtain reliable conversion factors which also take intersubject variability into account, the blood/serum ratios (B/S) of oxycodone, morphine, fentanyl, hydromorphone, zopiclone, MDMA, dexamphetamine, alprazolam, risperidone and 9-hydroxyrisperidone were determined by LC-MS/MS using authentic samples. Blood and corresponding serum samples were obtained from driving studies performed with controlled or known dosages of the above drugs. The analytes were analysed in blood and serum and the following mean B/S ratios (relative standard deviations) were determined: oxycodone 1.48 (8.19 %); morphine 1.03 (3.59 %); fentanyl 0.87 (13.9 %); hydromorphone 1.04 (8.11 %); zopiclone 0.89 (16.1 %); MDMA 1.19 (8.04 %); dexamphetamine 0.89 (10.9 %); alprazolam 0.81 (5.84 %); risperidone 0.65 (7.52 %); 9-hydroxyrisperidone 0.73 (12.3 %). These mean values are largely in line with those reported in the literature. The B/S ratios did not appear to depend on partition coefficients, whereas there was strong evidence that B/S ratios decreased with increasing plasma protein binding.

LC-MS/MS analysis of phosphatidylethanol in dried blood spots versus conventional blood specimens.

Phosphatidylethanol (PEth), which is formed extrahepatically by the action of phospholipase D on phosphatidylcholine in the presence of ethanol, has been suggested as a promising marker of alcohol misuse. Analysis of dried blood spots (DBS) is particularly advantageous for the determination of delicate analytes such as PEth. Therefore, measurement of PEth species (18:1/18:1, 16:0/18:1) in DBS versus whole blood was performed to ascertain whether respective results are directly comparable. Samples were obtained from subjects (n = 40) undergoing alcohol detoxification treatment. Analysis involved liquid-liquid extraction from both, DBS and whole blood (100 µL, respectively), with phosphatidylpropanol as the internal standard. Extracts were subjected to LC gradient separation using multiple reaction monitoring of deprotonated molecules. Results from measurements of corresponding DBS and whole blood specimens were compared by estimating the respective mean values and by a Bland and Altman analysis. Concentrations of PEth 18:1/18:1 ranged from 46.1 to 3,360 ng/mL in whole blood (mean, 461.7 ng/mL) and from 35.8 to 3,360 ng/mL in DBS (mean, 457.6 ng/mL); for PEth 16:0/18:1, concentrations were from 900 to 213,000 ng/mL (mean, 23,375 ng/mL) and 922-213,000 ng/mL (mean, 23,470 ng/mL) in blood and DBS, respectively. Estimated mean differences were -4.3 ng/mL for PEth 18:1/18:1 and 95.8 ng/mL for PEth 16:0/18:1. The Bland-Altman plot of both PEth species showed that the variation around the mean difference was similar all through the range of measured values and that all differences except one were within the limits of agreement. It could be shown that the determination of PEth species in DBS is as reliable as in whole blood samples. This assay may facilitate monitoring of alcohol misuse.

Analysis of 3,4-methylenedioxymetamphetamine: whole blood versus dried blood spots.

Analysis of dried blood spots is an increasingly accepted method in therapeutic drug monitoring, whereas its application by analogy to forensic samples has not been studied in detail. Therefore, we investigated whether determination of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolite 3,4-methylendioxyamphetamine (MDA) from dried blood spots (DBS) is as reliable as that from whole blood specimens. Analysis was performed by liquid chromatography-tandem mass spectrometry following liquid-liquid extraction of blood and corresponding DBS samples from 20 volunteers participating in a controlled driving experiment under the influence of MDMA. The assay was checked for carryover, ion suppression/enhancement, linearity of response, lower limits of detection (LLOD) and quantitation, extraction efficiency and the within-run and between-run assay imprecision for both whole blood and DBS. The LLODs were 2.0 and 1.6 ng/mL for MDMA in whole blood and DBS, respectively, using a volume of 100 µL. LLODs of MDA were determined to be 0.25 ng/mL in whole blood specimens and 0.12 ng/mL in DBS. Extraction efficiency and imprecision did not differ significantly between the two methods for both MDMA and MDA. The mean concentration ratio of corresponding whole blood and DBS samples, t-test, and the Bland-Altman difference plot were used to test hypothesis of equality. Statistical analyses revealed that methods did not significantly differ for MDMA or MDA. Thus, DBS analysis has potential as a precise and inexpensive alternative to whole blood analysis of MDMA.

A comparison between experimental and authentic blood/serum ratios of 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine.

This paper compares the blood-to-serum distribution (B/S ratio) of 3,4-methylenedioxymethamphetamine (MDMA) and its major metabolite 3,4-methylenedioxyamphetamine (MDA). B/S ratios were determined by liquid chromatography-tandem mass spectrometry analysis following liquid-liquid extraction as a function of the hematocrit value (experimental specimens) and in blood and corresponding serum samples (n = 63) from 16 healthy volunteers participating in a controlled driving experiment (authentic specimens). A regression analysis to calculate the B/S ratio was performed followed by an analysis of covariances (ANCOVA). A linear relationship between the hematocrit value and the B/S ratio of both MDMA and MDA could be established from the experimental data. For MDMA, the regressions provided mean B/S ratios of 1.22 and 1.26 for the experimental setting and the authentic samples, respectively. For MDA, the analysis determined slopes of 1.15 and 1.27 for the experimental setting and field study, respectively. ANCOVA revealed that the method of determination (experimental vs. authentic specimens) did not influence the resulting slopes. A conversion factor of 0.80 may give an adequate estimate to derive the serum concentration for MDMA if only the concentration in whole blood is known, whereas such a definitive factor could not be established for MDA because of its very low levels in authentic samples.

Determination of morphine and 6-acetylmorphine in blood with use of dried blood spots.

The use of dried blood spots (DBS) which has successfully been introduced in neonatal metabolic screening is an appropriate method to reduce virus infection risk to a minimum, facilitating regular mailing and handling of samples in the laboratory. Injection diacetylmorphine use is notably associated with a prevalence of infection and a risk of transmission of blood-borne viruses. The aim of the present study was to establish a method to determine morphine and 6-acetylmorphine (6-AM) as accurately and sensitively from DBS as from whole blood. Analysis by liquid chromatography/tandem mass spectrometry was checked for carryover, ion suppression/enhancement, linearity of response, lower limits of detection and quantification, and the within-run and between-run assay imprecision for both whole blood and DBS after liquid/liquid extraction. DBS drying time and elution were optimized, and extraction efficiency from DBS was compared with that of blood and of a solution of the pure compounds. Short-term stability of morphine and 6-AM was determined at -20 degrees C, 4 degrees C, and 40 degrees C up to 7 days from both whole blood and DBS. Furthermore, it was tested whether analysis of DBS may be as reliable as that of whole blood investigating 50 authentic samples. The lower limit of detection was 0.4 ng of morphine per spot and 0.8 ng of 6-AM per spot using a DBS blood volume of 100 microL and was 0.3 and 0.7 ng/100 microL whole blood for morphine and 6-AM, respectively. Recovery rates of the analytes from DBS did not differ from those from whole blood and were > or =55% for 6-AM and > or =25% for morphine, and the within- and between-run coefficients of variation were always < or =9%. 6-AM degraded rapidly at both 4 degrees C and 40 degrees C in whole blood; however, it seemed to be much more stable in DBS. Significant correlations between real whole-blood samples and DBS were obtained. The DBS assay has potential as a precise and inexpensive option for the determination of morphine and 6-AM in small blood samples. Further, the DBS matrix proved to excellently stabilize 6-AM.

[Fitness to drive in spite (because) of THC]. / Fahrtüchtigkeit trotz (wegen) THC.

Attention-deficit/hyperactivity disorder (ADHD) is characterized by a lack of concentration and/or an altered activation level. People with ADHD are found to violate traffic regulations, to commit criminal offences and to be involved in traffic accidents more often than the statistical norm. Furthermore, they show more deviant behaviour and have an increased co-morbidity regarding substance abuse and dependence. Hence, this disorder is of some forensic importance. The purpose of this case study is to demonstrate that in some cases people with ADHD may show unusual effects after the consumption of THC. A 28-year-old male, who showed abnormal behaviour and seemed to be significantly maladjusted and inattentive while sober, appeared to be completely normal with a very high plasma level of THC. Performance tests conducted with the test batteries ART2020 and TAP provided average and partly above-average results in functions related to driving. Thus, it has to be taken into account that in persons with ADHD THC may have atypical and even performance-enhancing effects.

[Analysis of mitochondrial SNPs in addition to conventional STR-typing in a case of aggravated theft]. / Analyse mitochondrialer SNPs als Ergänzung zur konventionellen STR-Typisierung am Beispiel eines Falles von besonders schwerem Diebstahl.

In the field of forensic DNA typing, the analysis of Short Tandem Repeats (STRs) can fail in cases of degraded DNA. The typing of coding region Single Nucleotide Polymorphisms (SNPs) of the mitochondrial genome provides an approach to acquire additional information. In the examined case of aggravated theft, both suspects could be excluded of having left the analyzed hair on the crime scene by SNP typing. This conclusion was not possible subsequent to STR typing. SNP typing of the trace on the torch light left on the crime scene increased the likelihood for suspect no. 2 to be the origin of this trace. This finding was already indicated by STR analysis. Suspect no. 1 was excluded for being the origin of this trace by SNP typing which was also indicated by STR analysis. A limiting factor for the analysis of SNPs is the maternal inheritance of mitochondrial DNA. Individualisation is not possible. In conclusion, it can be said that in the case of traces which cause problems with conventional STR typing the supplementary analysis of coding region SNPs from the mitochondrial genome is very reasonable and greatly contributes to the refinement of analysis methods in the field of forensic genetics.

Effect of psychotropic medication on the in vitro metabolism of buprenorphine in human cDNA-expressed cytochrome P450 enzymes.

OBJECTIVE: The aim of the present study was to estimate the drug interaction potential of psychtropic medication on buprenorphine (BUP) N-dealkylation using cDNA-expressed cytochrome P450 (CYP) enzymes. METHODS: BUP was incubated with psychotropic drugs and cDNA-expressed CYP 3A4 and CYP 2C8 enzymes. Seven substances were screened for their inhibition potency. To check for a mechanism-based component in inhibition, all substances were tested with and without preincubation, respectively. Norbuprenorphine (NBUP) concentrations were determined by liquid chromatography/tandem mass spectrometry, following liquid/liquid extraction. RESULTS: Midazolam and zolpidem demonstrated greatest inhibition in screening experiments. As expected, IC(50) values without preincubation were higher than those after 30-min preincubation, with zolpidem 113.1 microM and midazolam 20.25 microM. Following a 30-min preincubation period in the absence of the probe substrate BUP, the apparent IC(50) values for zolpidem and midazolam were 20.17 microM and 3.5 microM. CONCLUSION: Both midazolam and zolpidem showed a distinct inhibitory potency towards NBUP formation by CYP 3A4, implicating a decreased conversion of BUP. When preincubated, the inhibitory potency was increased, which strongly suggests a metabolically activated component in inhibition.

Tissue distribution of mirtazapine and desmethylmirtazapine in a case of mirtazapine poisoning.

An ingestion of an unknown quantity of mirtazapine in a suicide attempt leading to death is described. Sertraline and amitriptyline have been co-ingested. Because mirtazapine is reported to be relatively safe in overdose, body fluids and tissues were investigated for both mirtazapine and desmethylmirtazapine by high-pressure liquid chromatography/tandem mass spectrometry following liquid-liquid extraction. The limit of detection was sufficiently low to also apply the assay in pharmacokinetic studies. The levels of amitriptyline and nortriptyline were very low (38 and 19 ng/mL femoral venous blood) and the amount of sertraline in blood taken from the femoral vein (880 ng/mL) was considerably lower than those seen in overdosage. Accumulation of mirtazapine and N-desmethylmirtazapine was evident in fluids and tissues involved in enterohepatic circulation and excretion. The concentration determined in a brain sample suggests a contribution of the metabolite to the drug's pharmacodynamic activity. Based on literature data, significant adverse or synergistic effects among the drugs detected as well as adverse reactions such as a serotonin reaction appeared less probable. Mirtazapine exhibits alpha(1)-antagonistic properties on the cardiac-vascular system and may cause hyponatraemia. In the face of the cardiac findings at autopsy and the lack of an apparent cause of death, these effects of mirtazapine may have initiated a process leading to death.

The human brain and its neural stem cells postmortem: from dead brains to live therapy.

Contrary to the traditional dogma of being a relatively invariable and quiescent organ lacking the capability to regenerate, there is now widespread evidence that the human brain harbors multipotent neural stem cells, possibly throughout senescence. These cells can divide and give rise to neuroectodermal progeny in vivo and are now regarded as powerful prospective candidates for repairing or enhancing the functional capability of neural tissue in trauma or diseases associated with degeneration or malperfusion. Hopes primarily rest upon techniques to either recruit endogenous stem cells or to utilize exogenous donor-derived material for transplantation. In the search for suitable human cell sources, embryonic, fetal, and adult stem cells appear highly controversial, as they are accompanied by various still-unresolved moral and legal challenges. Fascinatingly, however, recent reports indicate the successful isolation and expansion of viable neural stem cells from the rodent and human brain within a considerable postmortem interval, suggesting that postmortem neural stem cells could potentially become an acceptable alternative cellular resource. This article will provide a brief overview about neural stem cells, their prominent features, and prospects for a cellular therapy, and will furthermore illuminate the cells in particular with respect to their newly discovered postmortem provenience, their advantage as a potential cell source, and several unfolding forensic considerations. Also, important ethical, social, and legal implications arising from this hitherto unpracticed cellular harvest of brain tissue from the deceased are outlined.

Representation of cerebral bridging veins in infants by postmortem computed tomography.

The postmortem diagnosis of shaken baby syndrome, a severe form of child abuse, may be difficult, especially when no other visible signs of significant trauma are obvious. An important finding in shaken baby syndrome is subdural haemorrhage, typically originating from ruptured cerebral bridging veins. Since these are difficult to detect at autopsy, we have developed a special postmortem computed tomographic (PMCT) method to demonstrate the intracranial vein system in infants. This method is minimally invasive and can be carried out conveniently and quickly on clinical computed tomography (CT) systems. Firstly, a precontrast CT is made of the infant's head, to document the original state. Secondly, contrast fluid is injected manually via fontanel puncture into the superior sagittal sinus, followed by a repeat CT scan. This allows the depiction of even very small vessels of the deep and superficial cerebral veins, especially the bridging veins, without damaging them. Ruptures appear as extravasation of contrast medium, which helps to locate them at autopsy and examine them histologically, whenever necessary.

[Visualization of bridging veins by means of postmortem computed tomography]. / Darstellung der Brückenvenen mit der postmortalen Computertomographie.

At autopsy, visualization of lesions of the bridging veins, a frequent source of subdural bleeding, is difficult due to their anatomical localization. On the other hand their demonstration is of great importance for the assignment to a chronologically defined trauma. For this reason a postmortem method using computed tomography was developed to visualize the intracranial venous system by means of X-ray contrast media. In subdural bleedings, in which the skull had not been opened up, ruptured vessels could be accurately localized with this method, so that targeted dissection was possible during the subsequent autopsy.

[An already archived latent fingerprint as a DNA source for STR typing in a murder case]. / Eine bereits archivierte Fingerspur als Ausgangsmaterial für eine STR-Analyse in einem Mordfall.

Former studies have shown that even a single skin contact, resulting in a latent fingerprint, can transfer enough DNA for genetic analysis. However, up to now latent fingerprints have usually not been used for DNA typing. In the present case the smeared trace of a hand was found in the suspect's car and archived. As it could not be evaluated in a classical manner, the evidence had to be examined by molecular genetic methods. DNA was extracted and typed in five different STR loci. Based on the yielded results, the significance of the findings is discussed.

[Weather-induced changes in cannabinoid content of hair]. / Untersuchungen zu witterungsbedingten Einflüssen auf den Cannabinoidgehalt im Haar.

Authentic hair samples from Cannabis users and a drug free hair sample which was separately spiked with tetrahydrocannabinol (THC), cannabidiol (CBD) or cannabinol (CBN) were exposed outside as well as to natural sunlight at prevailing and elevated humidity in quartz glass tubes during 8 weeks. In addition, authentic and spiked hair samples were exposed to xenon arc radiation in a light exposure cabinet for 24 hours. Stability of THC, CBD and CBN in authentic samples differed from that of the spiked hair. The radiation experiment revealed that CBN could not be measured in hair which had been spiked with THC. Under all conditions chosen the concentrations of THC, CBD and CBN decreased. At high humidity the concentrations declined more rapidly. In both authentic and spiked samples THC was most unstable compared to CBD and CBN. Therefore, in hair analysis determination of CBD and CBN seems promising to detect Cannabis exposure even under unfavorable conditions.

CD14 expression by activated parenchymal microglia/macrophages and infiltrating monocytes following human traumatic brain injury.

The immune response in the central nervous system (CNS) is under tight control of regulatory mechanisms, resulting in the establishment of immune privilege. CNS injury induces an acute inflammatory reaction, composed mainly of invading leukocytes and activated microglial cells/macrophages. The generation of this robust immune response requires binding of receptors such as CD14, a pattern recognition receptor of the immune system. CD14, a surface molecule of monocytic cells, is up-regulated after monocyte stimulation and is involved in cellular activation. To examine CD14 expression in human brain lesions we investigated sections of brains obtained at autopsy from 25 cases following closed traumatic brain injury (TBI) and 5 control brains by immunohistochemistry. Detection of CD14 in controls demonstrated constitutive expression by perivascular cells, but not in parenchymal microglial cells, equivalent to known expression pattern of ED2 in rats. Following TBI, numbers of CD14(+) cells in perivascular spaces and in the brain parenchyma increased in parallel within 1-2 days, both at the lesion and in adjacent perilesional areas. The number of CD14(+) cells in perivascular spaces and in the brain parenchyma reached maximum levels within 4-8 days and remained elevated until weeks after trauma. In contrast to activated parenchymal microglia/macrophages, resting parenchymal microglial cells lacked CD14. Thus, early CD14 expression constitutes an essential part of the acute inflammatory CNS response following trauma.