Segmental analysis for cocaine and metabolites by HPLC in hair of suspected drug overdose cases

Hair samples of eight postmortem cases were analyzed in segments of 1 to 3 cm for cocaine, benzoylecgonine and cocaethylene. Samples were prepared for analysis by digestion in 0.1 M HCl and subsequent extraction with mixed-mode solid-phase extraction columns. Measurement was made by reversed-phase, narrow-bore HPLC and fluorescence detection using two laboratory-made internal standards. The concentrations were in the region of 0.29-316 ng/mg of hair for cocaine, 0.43-141 ng/mg of hair for benzoylecgonine and 0.93-1.83 ng/mg of hair for cocaethylene. All eight investigated cases had cocaine-positive segments. In six of the cases, all segments were positive, suggesting regular cocaine use and two showed in-between negative segments indicating an interruption or a change of the abuse intensity. The results showed a second, remarkable observation, i.e. enormous concentration differences (factor >150) for both cocaine and benzoylecgonine between the different subjects. Furthermore, interindividual cocaine/benzoylecgonine ratios ranged from 0.02 to 8.43. We believe these observations could in part be attributed to both some of the still existing limitations in the analytical approach(es), especially the mandatory hair washing steps, and in our still too limited knowledge of the hair incorporation processes. Nevertheless, in some cases, segmental analysis proved to be an important tool to distinguish, together with postmortem examination, deadly chronic abuse from single acute drug overdosage.     

The statistical variability of blood alcohol concentration measurements in drink-driving cases

Like many other places in the world, Hong Kong has drink-driving legislation which prohibits a driver from having in his blood alcohol exceeding a prescribed limit while in control of a motor vehicle. The accuracy of measuring this alcohol concentration is obviously of prime concern as an erroneous result can avert the administration of justice. The common practice is to deduct all errors from the measured value and compare the deducted value with the prescribed limit, so that the benefit of all errors of the measurement is given to the driver. It is therefore important for any laboratory responsible for measuring blood alcohol concentrations to identify and quantify all errors associated with the measurement. The present study examined 900 blood alcohol determinations carried out by the Hong Kong Government Laboratory (HKGL) on cases of suspected drink driving. The determinations were performed by 5 different analysts with two different sets of instruments during 1995-1997. Statistical analysis indicated that the instruments had no bearing on the random error or variability and that even though analyst was a significant factor on variability, the deviation from the mean so caused was only 0.3% and of no practical significance. When the systematic error introduced by the tolerance limits of the certified alcohol standards (purchased from the Laboratory of Government Chemists, UK) was taken into account, the total uncertainty (random plus systematic errors) of an alcohol determination at 99.5% confidence level was found to be 4%. It is recommended that laboratories engaged in blood alcohol determination should adopt similar statistical treatment of their analytical results to find out the error and to ensure that the results are independent of analyst and instrument used.     

Alternative primers for DYS391 typing: advantages of their application to forensic genetics

The amplification of the STR DYS391, using the primers described in the Genome Data Base (GDB: G00-365-251), shows not only an additional band to the Y-specific one in males with a size range of 26 bp less than those of DYS391 locus alleles, but also a polymorphic pattern in females in the same size range as the additional band observed in males. The DYS391 pattern in families reflects a Y-specific linked locus and also a polymorphic X locus with an X-linked pattern of inheritance. A first screening in the X homologous locus allowed the identification of five different alleles. Allele frequencies were explored in different population groups for both the Y locus and the homologous locus in the X chromosome showing a similar allele distribution pattern in the X and Y homologous loci. An alternative reverse primer was designed to amplify the Y-chromosome specific STR in order to improve the specificity and applicability of this system to forensic genetics. Comparative results of the amplification with the new and the previously described primers proved that with this new primer there is a substantial increase in the specificity of the amplification. Moreover, a smaller fragment is amplified with a size out of the range of the alleles of the other Y-STRs usually used in forensic applications, therefore simplifying its inclusion in multiplex systems.     

Hair analysis by using radioimmunoassay, high-performance liquid chromatography and capillary electrophoresis to investigate chronic exposure to heroin, cocaine and/or ecstasy in applicants for driving licences

The present paper describes an integrated diagnostic strategy to check the physical fitness of subjects, formerly users of illicit drugs, to obtain a driving license, after having quit their addiction. According to the Italian law, applicants for a driving license with a history of drug abuse must give evidence to have quit this behaviour and to show no risk of relapse in the future. To prove this, at our institute, they undergo medical examination, hair analysis and a urinalysis program on eight seriate samples, collected over about 40 days. About 700 subjects per year are investigated with this strategy. The hair samples are screened for opiates (morphine), cocaine and ecstasy, the most abused illicit substances in our region, by using commercial radioimmunoassays adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negatives are confirmed by high-performance liquid chromatography. Further confirmation of results can be carried out by capillary electrophoresis (and/or GC/MS or MS/MS). In 1998, the prevalence of positives for morphine, cocaine and ecstasy was 4.8, 11.3 and 2.6%, respectively. In this year, for the first time, the percentage of hair samples positive for cocaine was greater than that for opiates. The results of this integrated diagnostic strategy are presented and discussed, with particular emphasis on the comparison between hair analysis on a single sample and seriate urinalyses (on eight samples).     

The use of supercritical fluid extraction for the determination of amphetamines in hair

A laboratory study interested in the analysis of human hair for drugs-of-abuse was conducted to determine if drugs could be detected and quantified from hair. Supercritical fluid extraction (SFE) techniques followed by GC-MS analysis were applied to extract amphetamines from hair. The group of amphetamines included methylenedioxyamphetamine (MDA), methylenedioxymetamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and internal standard mephentermine (MP). To validate information on amphetamine use in hair, powdered hair samples free from drugs were collected and soaked in a known amphetamine standard solution. Authentic fortified case hair samples taken from known drug users known to have consumed amphetamines were also analyzed for amphetamine. Results from this study show that amphetamine use can be detected in spiked and authentic fortified human hair using SFE techniques for qualitative and quantitative reproducible results.     

Beyond traditional paternity and identification cases. Selecting the most probable pedigree

The paper extends on the traditional methodology used to quantify DNA evidence in paternity or identification cases. By extending we imply that there are more than two alternatives to choose between. In a standard paternity case the two competing explanations H(1): "John Doe is the father of the child and H(2): "A random man is the father of the child, are typically considered. A paternity index of 100000 implies that the data is 100000 more likely assuming hypothesis H(1) rather than H(2). If H(2) is replaced by "A brother of John Doe is the father", the LR may change dramatically. The main topic of this paper is to determine the most probable pedigree given a certain set of data including DNA profiles. In the previous example this corresponds to determining the most likely relation between John Doe and the child. Based on DNA obtained from victims of a fire, bodies found in an ancient grave or from individuals seeking to confirm their anticipated family relations, we would like to determine the most probable pedigree. The approach we present provides the possibility to combine non-DNA evidence, say age of individuals, and DNA profiles. The program familias, obtainable as shareware from http://www.nr.no/familias, delivers the probabilities for the various family constellations. More precisely, the information (if any) prior to DNA is combined with the DNA-profiles in a Bayesian manner to deliver the posterior probabilities. We exemplify using the well published Romanov data where the accepted solution emerges among 4536 possibilities considered. Various other applications based on forensic case work are discussed. In addition we have simulated data to resemble an incest case. Since the true family relation is known in this case, we may evaluate the method.     

Unusual infant death: dog attack or postmortem mutilation after child abuse?

An unusual form of fatal child abuse is reported in which investigations by the police and the medical examiner were able to distinguish blunt force head trauma followed by postmortem dismemberment from a fatal dog attack. A discussion of the approaches used to ascertain the correct diagnosis is presented, as well as an overview of dog attacks on humans.     

An analysis of the usefulness of specific stages in the pathologic investigation of sudden infant death

Retrospective analysis of autopsy findings in 60 infants who had been found unexpectedly dead in their cribs or beds in South Australia from 1994 to 1998 was undertaken to determine the diagnostic usefulness of individual stages in the postmortem investigation. Positive findings occurred in 2 of 43 scene examinations (3%), 2 of 60 external examinations (3%), 2 of 11 radiologic examinations (18%), 8 of 60 internal examinations (13%), 7 of 60 histologic examinations (12%), and 3 of 58 microbiologic examinations (5%). No positive findings were detected on toxicologic screening. Not every case underwent each diagnostic step. This gave alternative diagnoses to sudden infant death syndrome (SIDS) in 15 cases (25%). This study demonstrates an increase in the percentage of cases of unexpected infant death due to causes other than SIDS; it also shows the diagnostic yield of individual stages in the postmortem evaluation of such cases. Negative findings were important in giving validity to the diagnosis in the 45 cases that were ultimately designated as SIDS.     

Plastic bag asphyxia in southeast Scotland

Death resulting from plastic bag asphyxia has been recognized for >40 years, but relatively little is known about either its epidemiology or its pathophysiology. Over 15 years (1984-1998), 30 deaths were attributed to plastic bag asphyxia among the 14,560 autopsies performed in the Forensic Medicine Unit in Edinburgh. These 30 deaths involved 20 male and 10 female subjects, with an age range of 13 to 81 years. Eleven had some alcohol measurable in the blood, with four having levels >80 mg/dl. Only one individual appeared to have ingested a drug overdose, but inhaled substances within the plastic bag may have contributed to death in five cases. The absence of childhood accidental deaths may reflect successful preventive measures. The 3 accidental deaths involved adults (including 2 who died of autoerotic asphyxia), and the remaining deaths were 27 suicides. Of those who committed suicide, most (59%) had chronic psychiatric illness rather than chronic debilitating or terminal physical illness. In contrast with reports from the United States, publicity associated with "self-deliverance" did not result in an increased number of deaths from plastic bag asphyxia (4 deaths in this series). Analysis of the circumstances of all the deaths revealed them to be difficult to predict and hence prevent.