Demands on scientific studies in different fields of forensic medicine and forensic sciences. Traffic medicine--impaired driver: alcohol, drugs, diseases

Since human performance is a key factor in crash causation detailed knowledge of the involvement of alcohol, drugs and diseases is of fundamental interest for road safety. To better understand the effect of these influencing factors it is important to use a combination of witness observations and a range of experimental and epidemiological studies. Epidemiological approaches to research such as road-side-surveys, case-control-studies, culpability analysis, pharmaco-epidemiological studies are usually seen as being able to provide the best answers. This article discusses the relative merits of experimental and epidemiological approaches.     

Determination of blood/serum ratios of different forensically relevant analytes in authentic samples

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.     

Time-resolved hair analysis of MDMA enantiomers by GC/MS-NCI

The aim of the study was to determine the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA) and other amphetamine-type stimulants (ATS) in segmented hair specimens of self-declared ecstasy abusers, who took part in a double-blind placebo-controlled six-way crossover study during approximately 7 weeks, during which they received a 75 and a 100 mg dose of racemic MDMA twice. Hair specimens were washed and cut into pieces of 2 cm length. After digestion and solid phase extraction, the enantiomers were derivatized with a chiral agent (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, developed at the authors laboratory and quantified by gas chromatography coupled to mass spectrometry operating in the negative chemical ionization mode. Most of the hair specimens that were tested positive for MDMA showed a predominance of the (R)-enantiomer. The R/S ratios of MDMA varied between 1.02 and 2.75 and total concentrations ranged from 0.1 to 20.1 ng/mg. The enantiomers of its metabolite 3,4-methylenedioxyamphetamine (MDA) were also quantified in most hair segments. The R/S ratios of MDA varied between 0.60 and 1.60, while the concentrations of the enantiomers ranged from 10 to 160 pg/mg hair. When segmental analysis was performed on single hair specimens, no inversion of the R versus S ratios of MDMA and MDA was observed. The predominance of (R)-MDMA in hair was in accordance with those already published for other matrices. Furthermore, both enantiomers of amphetamine (AM) were also detected in hair segments of four volunteers and the R/S ratios ranged from 1.00 to 1.47.     

Free Movement of Goods and Property Law

Although usually considered a national competence, there is an effect of internal market law on property law. When a property right is validly created in one Member State and the object on which it rests is moved to another Member State, an internal market dimension arises. Such has been the case in the ECJ's Krantz decision 25 years ago, dealing with the question on whether the rules allowing a seizure of goods owned by someone else in another Member State and leading to a potential loss of right is in conformity with EU law. More than 25 years have passed and our thinking about the internal market as well as the free movement case‐law has changed significantly. A re‐examination of this decision leads to a different conclusion: the refusal to recognise property rights validly created in another Member State violates the free movement of goods under Article 34 TFEU.     

Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography-tandem mass spectrometry

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC-MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC-MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2-200 microg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 microg/L. Limits of quantitation were estimated to be at 2 microg/L with limits of detection ranging from 0.2 to 0.5 microg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study.     

Article 345 TFEU (ex Article 295 EC), Its Meanings and Interpretations

Research that has been conducted over the last decades shows that neither the scope of application nor the exact meaning of Article 345 TFEU (ex Article 295 EC) is clear from its wording. This article seeks to clarify its meaning through analysis of the drafting of the Article as well as the use of it by the EU's institutions and by the Member States. Article 345 TFEU, formerly Article 295 EC and, before that, Article 222 EEC, is an Article that limits, but not prevents, the application of the TFEU Treaty as a whole to the way in which rules of a Member State deal with the right of ownership of undertakings. The conclusion can be drawn that Article 345 TFEU only concerns the private or public ownership of undertakings, with which the Community shall not concern itself and which can thus be regulated by the Member States themselves. Most importantly, the Article does not concern the content of the right of ownership, nor the objects of a right of ownership. It does therefore not form an obstacle to the development of a European property law.     

Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography–tandem mass spectrometry

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC–MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept^®, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC–MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2–200 μg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 μg/L. Limits of quantitation were estimated to be at 2 μg/L with limits of detection ranging from 0.2 to 0.5 μg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept^® samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study.     

Validation of an ELISA-based screening assay for the detection of amphetamine, MDMA and MDA in blood and oral fluid

The use of amphetamine and 'ecstasy' (MDMA) has increased exponentially in many European countries since the late nineties, leading to a rapid growth in the number of clinical and forensic analyses. Therefore, a rapid screening procedure for these substances in biological specimens has become an important part of routine toxicological analysis in forensic laboratories. The objective of this study was to evaluate the Cozart amphetamine enzyme-linked immunosorbent assay (ELISA) for the screening of plasma samples and oral fluid samples (collected with the Intercept device). Authentic plasma samples from drivers (n=360) were screened, using an 1:5-fold dilution. True positive, true negative, false positive and false negative results were determined relative to the in-house routine GC-MS analysis. Samples consisted of 144 amphetamine-only positives, 141MDMA/MDA-only positives, and 74 negatives when using the limit of quantitation as the cut-off level for confirmation (10 ng/mL). Using these results, receiver operating characteristic (ROC) curves were generated and optimal cut-off values for the screening assay were calculated. Analysis showed that the ELISA is able to predict the presence of either amphetamine or *MDMA/MDA (*MDMA as its metabolite MDA) in plasma samples with 98.3% sensitivity and 100% specificity at a cut-off value of 66.5 ng/mL d-amphetamine equivalents. A similar analysis was conducted on 216 oral fluid specimens collected from a controlled double blind study. Subjects received placebo or a high (100 mg) or low (75 mg) dose of MDMA. Oral fluid samples were collected at 1.5 and 5.5h after administration. Combined results of the analysis of the high and low dose oral fluid samples indicated a screening cut-off of 51 ng/mL d-amphetamine equivalents with both a sensitivity and specificity of 98.6% (using a LC-MS/MS confirmation cut-off of 10 ng/mL). In conclusion, these data indicate that the Cozart AMP EIA plates constitute a fast and accurate screening technique for the identification of amphetamine and MDMA/MDA positive plasma samples and oral fluid specimens (collected with Intercept. It should be emphasized that method validation should be performed for each type of biological matrix.     

A potentially catastrophic outcome from a false-positive urinary drug screen immunoassay

Background: Urinary drug screens are routinely administered for patients returning from leave from forensic psychiatric wards, and are required to be negative for patients to continue to use their allocated leave. Case: A 35-year-old woman took leave from her low-secure psychiatric unit. On return, her urine screened positively for 3,4-Methylenedioxymethamphetamine (MDMA). She strongly denied she had taken it; however, all her leave was cancelled. Her clinical state greatly deteriorated, and she attempted to hang herself the following day. After stabilisation of the patient and continued denial of taking MDMA, the urine was sent for toxicology analysis. In the weeks that followed, she started self-harming and deteriorated to the point of needing electroconvulsive therapy. The toxicological report came back as a false-positive due to recently started Mebeverine. Conclusion: Clinicians must be mindful that urinary drug screens are not diagnostic, but also that not being believed can be devastating, especially for long-stay forensic inpatients.