Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.     

Methamphetamine-related fatalities in forensic autopsy during 5 years in the southern half of Osaka city and surrounding areas

To outline the recent features of methamphetamine-related fatalities from the medico-legal point of view, a retrospective investigation of forensic autopsy cases involving methamphetamine during a 5-year period (1994-1998) in the southern half of Osaka city and surrounding areas (about 1.57 million population) was undertaken. Among 646 autopsy cases, methamphetamine was detected in 15 victims (nine males, six females; 16-71 years of age; most frequently in males in their thirties). Primary scenes of fatal events were concentrated in the middle of the city. About half of them were transferred from emergency medical centers (survival time, up to 30 h). The cause and manner of death were: methamphetamine poisoning (n=4), homicide (n=4), accidental falls and aspiration from drug abuse (n=4), fire death (n=1), myocardial infarction (n=1), and cerebral hemorrhage (n=1) under drug influence. Usually injection scars and fresh puncture sites were found. Blood methamphetamine concentrations were 2.29-17.05 micromol/dl in the fatal poisoning, 0. 44-3.80 micromol/dl in deaths from other extrinsic causes (trauma), and 1.35-2.17 micromol/dl in cardio- and cerebrovascular strokes. Common complications were cardiomyopathy, cerebral perivasculitis and liver cirrhosis/interstitial hepatitis. Fatal and nonfatal methamphetamine poisonings are separately dealt with by the administrative medical examiner's office and in emergency medical centers. Tightly cooperative approaches of clinical and medico-legal experts are required for the effective social and medical management of drug abuse.     

Extraction and amplification of authentic DNA from ancient human remains

A total of 36 ancient human remains from 12 individuals (three tooth/bone samples each) and one sample each of three individuals from the newest time was typed in a blind study using the amelogenin sex test. Prior to this molecular sex determination the sex of the individual was determined morphologically. The success rate of the amelogenin amplifications was >90%. For every individual an unambiguous molecular sex typing result was obtained. Furthermore, the morphological and molecular sex determinations were in accordance with each other, giving evidence for the authenticity and ancient origin of the polymerase chain reaction (PCR) amplifications.     

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.     

Synthesis of 2,3- and 3,4-methylenedioxyphenylalkylamines and their regioisomeric differentiation by mass spectral analysis using GC-MS-MS

3,4-methylenedioxyamphetamine (MDA) derivatives are increasingly abused central nervous system stimulants with neurotoxic properties. In recent years a number of controlled substance analogs (designer drugs) with high structural variety reached the illegal market making their identification an arduous task. The underivatized compounds give very similar or even virtually identical electron impact mass spectra containing mainly intense C(n)H(2n+2)N(+) immonium ions. Using tandem mass spectrometry (MS-MS) the additional structural information contained in the collision induced dissoziation (CID) mass spectra of molecular ions using electron impact (EI) and especially chemical ionization (CI) allowed an unequivocal differentiation of 18 studied regioisomeric 1-(methylenedioxyphenyl)-2-propanamines and 1-(methylenedioxyphenyl)-2-butanamines.Further synthetic methods are presented for 1-(3,4-methylenedioxyphenyl)-N-propyl-2-butanamine, N-isopropyl-1-(3,4-methylenedioxyphenyl)-2-butanamine and four 1-(2, 3-methylenedioxyphenyl)-2-butanamines. N-alkylated 1-(3, 4-methylenedioxyphenyl)-2-butanamine compounds (e.g. MBDB) are also known to be abused psychoactive agents (entactogenes) without the sympatomimetic effects of the 3,4-methylenedioxyamphetamines.     

Report of the European Network of Forensic Science Institutes (ENSFI): formulation and testing of principles to evaluate STR multiplexes

This paper describes a collaborative exercise organised under the auspices of the European Network of Forensic Science Institutes (ENFSI). The purpose of this EU (European Union) funded group is to carry out research to enable STR loci to be compared between European laboratories, ultimately leading to the formation of a pan-European database. Accordingly, an exercise was designed to evaluate a prototype STR multiplex system manufactured by Applied Biosystems (ABD). Each laboratory was sent 12 samples to analyse along with a multiplex kit. Of specific interest was the definition of parameters to define the efficiency of the system. Stutter, split allelic peaks (differing by one base), pull-up, heterozygous balance and between locus balance were all objectively measured. Once the important parameters are defined it is possible to directly compare performances of different multiplexes and the different laboratories carrying out the tests. Since the multiplex used was a prototype system, this exercise cannot be regarded as a proficiency test.