Comparison of nonradioactive microtiter plate enzyme immunoassays for the sensitive detection of fentanyl

Fentanyl is a very strong opioid with analgesic properties that are approximately 80 times stronger than those of morphine and therefore is used in major surgery and treatment of pain in tumor patients. Cases of fentanyl abuse by intravenous injection, inhalation, oral or nasal application have been reported especially in the USA. Therapeutic levels of fentanyl are as low as 1 ng/ml of serum and therefore a screening test must have a detection limit below that concentration. Recently three non-radioactive enzyme immunoassays (EIAs) have become commercially available from COZART, STC and DIAGNOSTIX, all of them supplied by MAHSAN Diagnostika for evaluation with serum samples from forensic and clinical cases. A calibration curve is obtained with samples that contain 0, 0.1, 0.5, 1 and 5 ng fentanyl per ml of negative serum. The calibration curve of COZART is especially in the low range, steeper than those of STC and DIAGNOSTIX. The cut-off for all these EIAs, however, can be set at 0.5 ng/ml. After the administration of therapeutic doses, fentanyl concentrations were between 3 and more than 5 ng/ml as determined with the EIAs. The presence of the typical drugs of abuse, e.g. heroin, methadone, cocaine, cannabinoids and amphetamines including the derivatives of methylenedioxyamphetamine, don't generate false-positive results. No cross-reactivity was also observed at toxic levels of benzodiazepines and paracetamol and therapeutic levels of barbiturates, phenothiazines, antidepressants and analgesics. The EIAs tested so far appear to be suitable for the detection of fentanyl at therapeutic levels. False-positive results or cross-reactivity towards other compounds have not been observed.     

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.     

Homicide by ligature strangulation in Finland: offence and offender characteristics

In Finland, homicide by ligature strangulation is a rare event. The purpose of this study was to investigate recent homicide cases by ligature strangulation and to analyse offence and offender characteristics. All forensic psychiatric evaluation statements of offenders accused of ligature strangulation homicides during the 7-year period 1996--2002 (n=19) were reviewed retrospectively. Thirty-five percent of the victims were females. In four cases, the victim was the offender's intimate partner; in one case, it was the stepson; in one case, a father and in the rest of the cases (n=13), an acquaintance. None of the cases involved planning, sadism or sexual elements. In 89% of the cases, the offender was either intoxicated or on drugs at the time of the offence and in 94% of the cases, the victim was found at the scene of the killing. Of the offenders, 63% had crime history, and approximately half of the offenders had experienced parental alcohol abuse, domestic violence or previous trauma. Most of the offenders were unemployed at the time of the offence. In all, 89% were diagnosed as having personality disorder and two were schizophrenic.     

STR analysis of artificially degraded DNA-results of a collaborative European exercise

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.     

Sudden death associated with a multifocal type II hemangioendothelioma of the liver in a 3-month-old infant

Sudden unexpected death in an infant caused by or associated with neoplasm is rare. We describe a case of a sudden death in an apparently healthy 3-month-old female, in which the autopsy revealed a multiple type II infantile hepatic hemangioendothelioma (IHE). This uncommon tumor has, untreated, a relatively high mortality rate, mainly due cardiac failure resulting from massive arteriovenous shunts, but the association with sudden infant death is very rare.     

Amniotic fluid embolism as cause of death in a car accident--a case report

Amniotic fluid embolism (AFE), a relatively rare complication in pregnancy, has a high mortality rate. We describe a case of a 38-week pregnant woman with such an embolism leading to almost immediate death after a blunt abdominal trauma inflicted in a motor vehicle accident and probably associated with improper positioning of a seat belt. It has been assumed that the pathophysiology of amniotic fluid embolism is related to an anaphylactoid reaction and that mast cell degranulation indicates this mechanism. Moreover, immunohistochemical antitryptase staining of pulmonary tissue samples in our case revealed mast cell degranulation.     

Effect of hair care and hair cosmetics on the concentrations of fatty acid ethyl esters in hair as markers of chronically elevated alcohol consumption

Fatty acid ethyl esters (FAEE) can be used as alcohol markers in hair. It was investigated in this study whether this diagnostic method is disturbed by hair care and hair cosmetics. Traces of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate were detected in all of 49 frequently applied hair care products by headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The highest concentration was 0.003% in a hair wax. From experiments with separated hair samples of alcoholics as well as from the evaluation of the FAEE concentrations and the data about hair care of 75 volunteers (alcoholics, social drinkers and teetotalers) follows that usual shampooing, permanent wave, dyeing, bleaching or shading are of minor importance as compared to the drinking amount and other individual features. However, false positive results were found after daily treatment with a hair lotion containing 62.5% ethanol, with a deodorant and with a hair spray. As an explanation, it is assumed that FAEE are formed in the sebum glands also after regular topical application of products with a higher ethanol content.     

Detection and assay of cis- and trans-isomers of 4-methylaminorex in urine, plasma and tissue samples

The 4-methylaminorex (4-MAX) is an amphetamine-related psychostimulant drug that has appeared on the clandestine market with a street name of "U4Euh". This compound exists as four stereoisomers, trans-4R,5R, trans-4S,5S, cis-4R,5S and cis-4S,5R, of which the cis forms have been classified as Schedule I substances in the US. The increasing variety of designer drugs has highlighted the importance of detection, identification, and quantitative measurement of these drugs, including 4-MAX, in biological samples. In the present study, the isomers of 4-MAX were detected in urine of rats treated with the drugs by some but not all of the on-site immunoassays tested, mainly as amphetamine or methamphetamine. To facilitate identification of 4-MAX by laboratories specialized in drug analysis, the electron-ionization mass spectrum and TLC data for underivatized 4-MAX using a routine laboratory drug-screening procedure is provided. In addition, a GC/MS method is described for the quantitative determination of cis- and trans-4-MAX as tert-butyldimethylsilyl-derivatives in plasma, urine and tissue.