Duarte Nuno Vieira

Hair analysis for Δ9-THC, Δ9-THC-COOH, CBN and CBD, by GC/MS-EI: Comparison with GC/MS-NCI for Δ9-THC-COOH

A sensitive analytical method was developed for quantitative analysis of delta(9)-tetrahydrocannabinol (delta(9)-THC), 11-nor-delta(9)-tetrahydrocannabinol-carboxylic acid (delta(9)-THC-COOH), cannabinol (CBN) and cannabidiol (CBD) in human hair. The identification of delta(9)-THC-COOH in hair would document Cannabis use more effectively than the detection of parent drug (delta(9)-THC) which might have come from environmental exposure. Ketamine was added to hair samples as internal standard for CBN and CBD. Ketoprofen was added to hair samples as internal standard for the other compounds. Samples were hydrolyzed with beta-glucuronidase/arylsulfatase for 2h at 40 degrees C. After cooling, samples were extracted with a liquid-liquid extraction procedure (with chloroform/isopropyl alcohol, after alkalinization, and n-hexane/ethyl acetate, after acidification), which was developed in our laboratory. The extracts were analysed before and after derivatization with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH) using a Hewlett Packard gas chromatographer/mass spectrometer detector, in electron impact mode (GC/MS-EI). Derivatized delta(9)-THC-COOH was also analysed using a Hewlett Packard gas chromatographer/mass spectrometer detector, in negative ion chemical ionization mode (GC/MS-NCI) using methane as the reagent gas. Responses were linear ranging from 0.10 to 5.00 ng/mg hair for delta(9)-THC and CBN, 0.10-10.00 ng/mg hair for CBD, 0.01-5.00 ng/mg for delta(9)-THC-COOH (r(2)>0.99). The intra-assay precisions ranged from <0.01 to 12.40%. Extraction recoveries ranged from 80.9 to 104.0% for delta(9)-THC, 85.9-100.0% for delta(9)-THC-COOH, 76.7-95.8% for CBN and 71.0-94.0% for CBD. The analytical method was applied to 87 human hair samples, obtained from individuals who testified in court of having committed drug related crimes. Quantification of delta(9)-THC-COOH using GC/MS-NCI was found to be more convenient than GC/MS-EI. The latter may give rise to false negatives due to the detection limit.

Characterization of Lesions in Hanging Deaths

Abstract:u2002 Hanging is a common method of committing suicide and a routine task in medico‐legal autopsies. The hanging mark is the most relevant external sign and its characteristics are well known, but, for unknown reasons, there are major differences in reports on internal findings. We retrospectively studied 228 consecutive cases of hanging deaths. A complete standard autopsy was performed for every case. We investigated the association between the characteristics of the hanging mark and the frequency of bone, cartilage, soft tissue, and vascular injuries with the mode of suspension. Most cases (75.3%) presented some kind of bone or cartilage fracture, but these were unrelated to any of the variables studied. Vascular lesions are clearly more infrequent: intimal injuries were found in the carotid artery (9.1%), the jugular vein (2.2%), and ruptures of the carotid adventitial layer (21.7%). These could be partially associated with the use of a hard fixed noose and body weight.

Predicting adult stature from metatarsal length in a Portuguese population

Stature can be considered one of the big four parameters to be ascertained within the biological profile in cases of forensic anthropology. However, the most reliable available methods for stature estimation require the preservation of the long bones, but since this is very often not the case, the development of alternative methods, based on distinct bones, is mandatory. Therefore, in the present work the reliability of the first two metatarsal bones in reconstructing stature is tested. The data consist of length measurements taken from the first two metatarsals removed from documented cadavers of known stature. The sample for this study consists of 220 metatarsals, namely 110 first metatarsals and 110 second metatarsals collected during the autopsies carried out in the National Institute of Legal Medicine in Portugal. The aim was to propose regression equations for the Portuguese population and test the formulae proposed by other authors to determine adult stature using metatarsal bones. We found that when estimating stature from measurement of the metatarsals, the best correlation was that obtained from the relationship with the maximum length of the 2nd metatarsal. The corresponding regression equation is as follows: S=790.041+11.689M2.

Liquid chromatography/tandem mass spectrometry for the qualitative and quantitative analysis of illicit drugs and medicines in preserved oral fluid

A qualitative and quantitative analytical method was developed for the simultaneous determination of 24 illicit drugs and medicines, in preserved oral fluid samples collected with the StatSure Saliva Sampler collection device. The samples were prepared by liquid-liquid extraction followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The chromatographic separation was performed with an Atlantis T3 (100 x 2.1 mm i.d., 3 microm) reversed-phase column using an acetonitrile/2 mM ammonium formate buffer pH 3.4 gradient and the MS/MS detection was achieved with two precursor-product ion transitions per substance. The method was fully validated, including specificity and capacity of identification, limit of detection (0.2-2.1 microg/L), limit of quantitation (0.8-6.4 microg/L), recovery (34-98%), carryover, linearity (the method was linear in the range 1-200 microg/L), intra-assay precision (coefficient of variance (CV) <20% for 20 microg/L and CV <10% for 100 microg/L) and inter-assay accuracy (mean relative error <15%) and precision (CV <20%). The method showed to be specific and sensitive. It has already been successfully used in four proficiency tests and subsequently applied to oral fluid samples collected from road traffic volunteers in the driving population of Portugal (districts of Lisbon, Coimbra and Porto), within the DRUID project.

Allelic frequency distribution of 17 STRs from Identifiler and PowerPlex-16 in Central Portugal area and the Azores archipelago

This study analyzes the allelic frequency distribution of 17 STRs contained in the AmpFlSTR Identifiler (Applied Biosystems) and PowerPlex16 System (Promega) commercial kits for two large population samples from the Azores archipelago (Portugal) (N=475) and from Central Portugal (N=2125). Likewise, it includes a comparative study among the population groups analyzed in this paper and those which history points out as originating from the first settlers of the Azores. All loci were highly polymorphic. The Central Portuguese area and the Azores archipelago population samples are in Hardy-Weinberg equilibrium for the 17 markers analyzed.

No Cash No Whiplash?: Influence of the Legal System on the Incidence of Whiplash Injury

Whiplash injury has been a continuous source of controversy due to its association with litigation. We studied the incidence of whiplash associated disorder (WAD) in two similar socioeconomic areas and carried out a retrospective study based on the hypothesis that the Spanish law 30/1995 might have an affect on the incidence and duration of cervical symptoms and the persistence of impairment. More than 10,000 patients injured in traffic accidents were studied over a period three years. Of these, only patients with an initial diagnosis of whiplash injury were included in the study. Patients with other injuries were excluded. The patients were classified into two groups: Galicia-Spain and North-Central Portugal (depending on where the accident took place and the medico-legal evaluation procedure in force). Statistical analysis was made using SPSS 13.0 and Statistix 8.0. We found a statistically significant difference between Spain and Portugal in the incidence of WAD and in the duration of symptoms. The incongruities caused by the compulsory application of Spanish law arise from the fact that evaluation on a points scale of impairment does not always reflect the functional state of the injured person.

PMICALC: An R code-based software for estimating post-mortem interval (PMI) compatible with Windows, Mac and Linux operating systems

In legal medicine the correct determination of the time of death is of utmost importance. Recent advances in estimating post-mortem interval (PMI) have made use of vitreous humour chemistry in conjunction with Linear Regression, but the results are questionable. In this paper we present PMICALC, an R code-based freeware package which estimates PMI in cadavers of recent death by measuring the concentrations of potassium ([K+]), hypoxanthine ([Hx]) and urea ([U]) in the vitreous humor using two different regression models: Additive Models (AM) and Support Vector Machine (SVM), which offer more flexibility than the previously used Linear Regression. The results from both models are better than those published to date and can give numerical expression of PMI with confidence intervals and graphic support within 20 min. The program also takes into account the cause of death.

Illicit drugs in alternative biological specimens: A case report

Postmortem tissues (e.g. liver, kidney) have been long used in forensic applications especially in those cases where blood is unavailable. The aim of this paper is to demonstrate the importance of the information provided to the forensic toxicologist at the time of carrying out the toxicological analysis, especially in cases where the samples commonly used in forensic toxicology are unavailable. This work describes the toxicological findings in a violent death resulting from a man who was hit by a train. Vitreous humor, liver and kidney were sent for toxicological analysis, once it was not possible to obtain blood and urine. The validated procedures used in the routine casework of Forensic Toxicology Laboratory of the Centre Branch of the National Institute of Legal Medicine, were applied in the analysis of liver, kidney and vitreous humor, using gas chromatography-mass spectrometry after solid-phase extraction and gas chromatography-flame ionization detector for the analysis of drugs of abuse and ethanol, respectively. Morphine, codeine, cocaine, benzoylecgonine and ecgonine methyl ester were found in the liver and in the kidney and no ethanol was found in the vitreous humor. The method validation included the study of specificity, selectivity, limits of detection, recovery and carryover. Although blood and urine are the most common and preferred matrices used for toxicological studies involving drugs of abuse, sometimes the choice of specimen is determined by the case under investigation. The forensic pathologist must be aware that relevant information must be provided so that the toxicological analysis can be conducted in accordance with case history, particularly when the only samples available for analysis are these unconventional specimens, since the interpretation of the obtained results is more difficult.