Frequency of positive diazepam-screening in post-mortem examinations (author's transl)]

The post-mortem blood specimens of 389 forensic autopsies were analyzed for diazepam. The age of the cases investigated was above 10 years and the survival time was less than 12 hours. Eighteen samples corresponding to 4.6% were found to be diazepam-positive. These 18 samples were distributed equally between men and women. The proportion of diazepam-positive samples was increased in the groups of suicide and poisoning (alcohol and opiates). The association between diazepam intake and poisoning was statistically highly significant. No correlation was found between diazepam intake and age. Alcohol was found to occur significantly more often in the group of the diazepam positive cases as compared to the diazepam negative group.     

Screening for drugs in forensic blood samples using EMIT urine assays

A screening method for the detection of drugs in haemolysed whole blood has been evaluated. Methanolic extracts of 300 forensic blood samples known to be positive or negative for drugs were analysed with EMIT d.a.u. assay kits for amphetamine, cannabinoids, opiates and benzodiazepines (the latter to analyse for diazepam and the main metabolite N-desmethyldiazepam). There were very few false positive results, except for the amphetamine assay in postmortem blood samples, where 9% were false positive. For amphetamine and cannabinoids a few false negatives were found, these were from samples with very low drug concentrations. No false negatives were found for opiates and diazepam. The present modification of the EMIT d.a.u. method seems to be a good method for screening of drugs in forensic blood samples, except for amphetamine in postmortem samples. The method is simple and requires only 0.5 ml blood.     

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid

The purpose of this study was to evaluate the efficiency of the Cozart RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen at -20 degrees C until analysis by GC-MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC-MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

Determination of Malathion levels and its effect on the development of Chrysomya megacephala (Fabricius) in South China

The present study determines the concentration of Malathion in rabbit tissues and Dipteral larvae feeding on those tissues. Malathion was found in all muscle and liver samples of the test rabbit, as well as larvae fed on it. Samples from the control rabbit and pupae from all colonies were negative for Malathion. Correlations were found between administered dosage, tissue concentrations and younger or prepuparial larvae. Effects of Malathion on the development rate of Chrysomya megacephala (Fabricius) were also reported. C. megacephala is the most common fly species found on corpses in South China during the early stages of decomposition. Significant differences in larval growth rate were both observed among the colonies fed on muscle and liver. The presence of Malathion in both muscle and liver appears to retard the normal growth rate of C. megacephala in larval stage. Larvae from all colonies fed on tissues from rabbits treated with Malathion were smaller and attained maximum length later than those from the control colony. Duration of the larval and pupal stages was both significantly prolonged for larvae on tissues from rabbit receiving Malathion than those from the control colony. The difference of the duration of the larval and pupal stages together from the muscle colonies would alter the postmortem interval estimation by up to 36 h. As for liver colonies, it would alter the postmortem interval estimation by up to 28 h. A significantly different duration of the larval and pupal stages from the muscle colonies would alter a postmortem estimate by up to 28 h relative to the liver colonies.     

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid

The purpose of this study was to evaluate the efficiency of the Cozart^® RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart^® RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart^® RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography–mass spectrometry (GC–MS). The samples were stored frozen at −20 °C until analysis by GC–MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC–MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

Simultaneous hair testing for opiates, cocaine, and metabolites by GC-MS: a survey of applicants for driving licenses with a history of drug use

A sensitive GC-MS method for the simultaneous determination of opiates, cocaine, and metabolites in hair at a cut-off level of 0.1 ng/mg was adopted to assess past exposure to these drugs in applicants for driving licenses with a history of drug use. The sampling protocol consisted of collection of one hair (sample A, 5-cm length) and one urine sample. When hair and urine (EMIT Syva, cut-off levels: 0.3 mg/l for opiates, 0.15 mg/l for cocaine, GC-MS confirmation of positives) were both positive or negative the protocol was concluded. In the other cases, the assessment of 'current exposure' to drugs was carried out, in order to avoid seriated random urinalysis, by collecting a second hair sample (sample B) 6 weeks later and analysing the proximal 1-cm segment. Out of the 214 'A' hair samples analyzed, 14 (6.5%) tested positive for morphine and/or 6-acetylmorphine (6AM), and 26 (12%) for cocaine and/or benzoylecgonine (BE), whereas none of the samples tested positive for both drugs. Levels between 0.1 and 1 ng/mg of the single analytes were found in eight out of the 14 morphine-6AM positives (57%) and in 18 out of the 26 cocaine-BE positives (69%). The time course of positive cases showed a progressive decrease of morphine-6AM positives and a corresponding increase of cocaine-BE positives within the study period September 1995-February 1999. No cases with positive urine and negative hair were observed. Among the 40 positive cases, seven (four and three for opiates and cocaine, respectively) were found to be 'currently exposed to drug', four by urinalysis (three and one) and three by analysis of the hair sample B (1 and 2).     

Validation of the Cozart microplate EIA for analysis of opiates in oral fluid

The purpose of this study was to determine the performance characteristics of the Cozart microplate EIA for detecting opiates in oral fluid from patients in a drug misuse treatment program. Oral fluid samples were collected using the Cozart RapiScan Collection System from 216 donors who were receiving treatment for their addiction and were monitored for drug abuse. A further 40 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory by using the Cozart microplate EIA for opiates and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. The intra-assay precision for the Cozart microplate oral fluid EIA for opiates over 40 assays was 0.43% to 9.13% CV (within assay) and 2.9% to 9.1% CV (within day). A total of 109 samples were positive for various opiates. The Cozart microplate EIA for opiates in oral fluid, using a cut-off of 30 ng/ml morphine equivalents in neat oral fluid, had a sensitivity of 99.1 +/- 2.1% and a specificity of 94.4 +/- 2.2% versus GC-MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

Pholcodine interference in the immunoassay for opiates in urine

The excretion in urine after single oral therapeutic doses of morphine derivatives was analysed with radioimmunoassay (RIA) and homogeneous enzyme immunoassay (EMIT) for opiates. In contrast to the rapid excretion of ethylmorphine and codeine, pholcodine showed positive results for opiates 2-6 weeks after intake when the urines were analysed with the RIA-method. When analysed with the EMIT-method, positive results were obtained for pholcodine for approximately 10 days. As pholcodine is a common component in cough mixtures, its prolonged excretion could represent a hazard in interpreting the results from drug analyses of urines.     

Determination of drug levels in larvae of Lucilia sericata (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

This study concerns the determination of morphine concentrations in fly larvae reared on rabbits administered different concentrations of morphine and a correlation between concentrations of the drug in larvae and tissues. Three rabbits (R1, R2 and R3) were given dosages of 12.5, 25.0 and 50.0 mg/h of morphine over a 3 h period via continuous ear artery perfusion. These dosages and time of perfusion were calculated to create tissue concentrations of morphine similar to those encountered in human death due to overdose. Morphine blood level plateau was attained after 1 h of perfusion. A fourth rabbit was used as a control. To evaluate drug concentrations, tissues were sampled using a coelioscopic technique. Approximately 400 eggs of Lucilia sericata, all of the same age category, were placed in eyes, nostrils and mouth of each rabbit carcass. Larvae and puparia were regularly collected from each rabbit for toxicological analysis. The concentrations of the drug in the tissues sampled were determined to be similar to those normally encountered in human overdoses and were correlated with the dosage of morphine that had been administered. Morphine was detected in all larvae and pupae fed on tissues from carcasses administered morphine, except for puparia from the colony fed on the R1 animal which received 12.5 mg/h dosage of morphine. All samples from the control rabbit were negative for morphine. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were 30 to 100 times lower than the concentrations found in the tissues. A correlation between the tissue concentrations and larval concentrations was found in only 3rd instar larvae (80 to 140 h following hatching). No correlations were found between administered dosages, tissue concentrations and younger larvae, prepuparial larvae or puparia.     

Immunochemical methods in detection of opiates in tissues and organs

Conditions of isolation of opiates from tissues and analysis of the resultant extracts by immunochemical methods are discussed. The main characteristics of OPIATES U kits (Pharmatech) for polarization fluorescent immunoanalysis of cadaveric material (liver) are determined. Immunochemical methods for forensic chemical analysis for opiates are experimentally compared.     

Gender-related differences in the pharmacokinetics of opiates

Previous studies have documented gender-related differences in a number of aspects of the pharmacology of opiates, including their analgesic activity, stimulative properties and generation of physical dependence. The current experiments were carried out with the aim to examine whether male–female differences exist in the blood and brain levels of opiates attained after their intraperitoneal injection to male and female Wistar rats. The tests were performed 5, 15, 45 and 120 min after the animal treatment with seized heroin. Gas chromatography–mass spectrometry (GC–MS) method was developed to quantitatively determine opiate alkaloids in blood and brain regions (known for their high concentration of μ-opiate receptors): cortex, brainstem, amygdala and basal ganglia. Maximal contents of opiates in blood of animals of both genders were found in the second measurement time (15 min), the values measured in the males being significantly higher, which suggests a faster passage of the opiates from blood to brain tissue in female animals. The highest content of opiates in the brain tissue of female animals was measured 15 min and with male animals 45 min after the treatment, which also indicates faster distribution of opiates from blood to brain in the female compared to male rats. The highest proportion of opiates was found in the basal ganglia of the animals of both genders. The obtained results offer the possibility of selecting this part of the brain tissue of both males and females as a representative sample for identifying and assessing contents of opiates.     

Prevalence of psychoactive substances in truck drivers in the Nord-Pas-de-Calais region (France)

A previous study conducted in 1995 showed that psychoactive drug use by workers was higher in safety/security workstations than in the rest of the labour force. In order to verify this finding, we conducted a new study in 2003-2004 in the Nord-Pas-de-Calais region, restricted to truck drivers. The aim of this study was to allow harmonizing the professional practice of the occupational physicians, proposing drug prevention and drug testing policies, validating the analytical methods and the guidelines in case of positive testing results. One thousand truck drivers were studied. Urines were tested for amphetamines, cannabinoids, cocaine, opiates, benzodiazepines, buprenorphine and methadone by immunoassay. Urine ethanol determinations were performed by an ADH method. Positive urines for drugs of abuse, methadone or buprenorphine were then tested by gas chromatography or liquid chromatography coupled to mass spectrometry. Out of the 1000 drivers, cannabinoids were detected in 85 cases, opiates in 41 cases, amphetamines in 3 cases and cocaine in only one case. Buprenorphine was detected in 18 cases, methadone in 5 cases and benzodiazepines in 4 cases. Urine ethanol was positive in 50 cases. We found only one case with 6-monoacetylmorphine. Other positive opiates were metabolites of antitussives. The relatively low number of benzodiazepine positive urines could be explained by the lack of sensitivity of the test we used. All these results confirm those of the previous study for cannabinoids and ethanol in safety/security workstations. Positive results for methadone and buprenorphine are eight times higher than in the general population. In conclusion, the authors think that it will be of a great interest to test urine of truck drivers for other classes of psychoactive drugs, using a liquid chromatography-mass spectrometry method.     

Impairment in drivers due to cannabis in combination with other drugs.

Blood samples from 425 suspected drugged drivers who were clinically impaired and negative for alcohol were analysed. Fifty-six percent of the samples were positive for tetrahydrocannabinol (THC). Tetrahydrocannabinol-positive blood samples were analysed for amphetamines, barbiturates, benzodiazepines, cocaine metabolites and opiates. Eighty-two percent of the samples were found to be positive for one or more drugs in addition to THC, and the concentrations of these drugs were often high. Thus, THC in combination with other drugs seems to be a much more frequent reason for impairment than THC alone among Norwegian drugged drivers.     

Width as an alternative measurement to length for post-mortem interval estimations using Calliphora augur (Diptera: Calliphoridae) larvae

The length of fly larvae collected from corpses is often used to help provide an entomological estimate of time since death. However, 'head-curling' by larvae can affect the accuracy of length measurements. To investigate a possible resolution to this problem, larvae of Calliphora augur were grown on sheep's liver at two constant temperatures (20 and 25 degrees C). Replicate samples were collected at set time intervals until pupation. Body length and width were measured for individual larvae and examined as predictors of age. It was found that body width, as measured at the junction of the fifth and sixth abdominal segments, is comparable with body length for age prediction of maggots of C. augur grown at these temperatures. Furthermore, conversion of width to length can be done with 95% accuracy from a simple linear model.     

Optimized conditions for simultaneous determination of opiates, cocaine and benzoylecgonine in hair samples by GC--MS

The present paper describes a qualitative and quantitative method for the simultaneous detection of opiates, cocaine and benzoylecgonine from human hair samples. Every step of the analytical procedure was studied to find the optimized conditions. Nine different incubation systems were examined. The influence of different pH values of samples on the isolation of analytes from the incubation media by Bond Elut cartridges and the stability of the compounds of interest in the different incubation media and conditions were investigated. The extracting power of different incubation media was studied as well. The phosphate buffer 0.1 N at pH 5 was chosen as the extraction medium in an optimized procedure for simultaneous determination of opiates, cocaine and benzoylecgonine in hair samples. The method developed was validated. Recoveries were 90% for morphine (M), 81% for 6-monoacetylmorphine (6-AM), 90% for codeine (CD), 86% for cocaine (C) and 90% for benzoylecgonine (BE). Relative standard deviation for inter-day precision was better than 12%. The limits of detection resulted as 0.05 ng/mg for M and C, as 0.08 for 6-AM and as 0.2 ng/mg for BE. Forty hair samples collected from drug abusers admitted to centers for detoxification treatment were analyzed obtaining 23 positive results for opiates and/or cocaine. Twelve hair specimens longer than 10 cm were analyzed following a sectional approach. In the six positive cases, it was interesting to find that the 6-AM/M ratio generally decreased for each sample from the proximal segment to the distal segments. Moreover, the 6-AM/M ratio was generally lower than 1 in the intermediate and distal segments.     

Practical aspects of roadside tests for administrative traffic offences in Germany

In the context of the European project ROSITA, the Institut of Legal Medicine Homburg/Saar has co-operated with the Saarland traffic police in order to assess different roadside drug tests for their functionality and reliability in traffic controls, and for their analytical force of evidence. In 254 cases within the time period from June 1999 to December 1999, police officers performed a (voluntary) roadside drug testing in saliva/sweat, or urine, to confirm or refute their initial suspicion that a driver had used drugs. Whereas in 45 cases the tests gave negative results (which were confirmed by lab urinalysis), in 209 cases the police officers ordered blood samples after a positive outcome of the tests.In 203 of the 209 positive cases, the results could be confirmed by GC/MS analysis. Regarding the prevalence of used drugs, a single consumption was found in 156 cases (113 cannabis, 38 amphetamines/methamphetamines, three opiates, two cocaine), and a consumption of two drugs was found in 44 cases (34 cannabis+amphetamines/methamphetamines, five cannabis+opiates, three cannabis + cocaine, two cocaine+amphetamines/methamphetamines). In three cases, multi-consumption was found.In six cases, the performed tests gave an incorrect prediction to the police officer at the roadside.The roadside tests gave 97.6% correct assistance to the police officers in the right direction (79.9% correct positive predictions and 17.7% correct negative predictions). As a consequence, the performed tests can be seen as a positive and needful tool for the police to get an immediate response to their initial suspicion and to take the right steps concerning a following legal action.     

Use of drugs of abuse in less than 30-year-old drivers killed in a road crash in France: a spectacular increase for cannabis, cocaine and amphetamines

A collaborative study was conducted in France in order to determine the prevalence of cannabinoids, opiates, cocaine metabolites and amphetamines in blood samples from drivers killed in road accidents in 2003 and 2004 and to compare these values with those of a previous study performed during the period 2000-2001 involving 900 drivers. Blood samples were provided from 2003 under 30-year-old drivers, killed in a traffic accident. Drugs of abuse were determined by gas chromatography-mass spectrometry using the same analytical procedures in all the 12 laboratories. The most frequently observed compounds were by far cannabinoids, that tested positive in 39.6% of the total number of samples. Delta9 tetrahydrocannabinol (THC), the most active of the principle constituents in marijuana (cannabis sativa), was detected in the blood of 28.9% drivers and was the single drug of abuse in 80.2% of the positive cases. It was associated with amphetamines in 7.4% and with opiates and cocaine in 1.9 and 4.8%, respectively. Amphetamines were present in 3.1% of the total number of samples, cocaine metabolites in 3.0% and opiates in 3.5%. When comparing these results with those of a previous study performed 3 years before, a significant increase is observed for THC (28.9% versus 16.9%), cocaine metabolites (3.0% versus 0.2%) and amphetamines (3.1% versus 1.4%). This study demonstrates the critical necessity of implementing in France as soon as possible systematical roadside testing for drugs of abuse.     

Assessment of exposure to opiates and cocaine during pregnancy in a Mediterranean city: preliminary results of the "Meconium Project"

For the first time in Europe, the "Meconium Project" aimed to estimate the prevalence of drug use by pregnant women and the effects of exposure to illicit drugs during pregnancy on the fetus and infant. Between October 2002 and February 2004, 1151 (79%) dyads among the 1439 mother-infant dyads from the Hospital del Mar, Barcelona, Spain, met eligibility criteria and agreed to participate in the study. We present preliminary results on the first 830 meconium samples and 549 mother-infant dyads, for which statistical analysis of socio-economic and demographic characteristics and newborn somatometry was completed. The meconium analysis showed an overall 7.9% positivity for drugs of abuse, with 6-monoacetylmorphine and cocaine being the analytes, most frequently found in samples positive for opiates and cocaine. Structured interview disclosed 1.3, 1.8 and 1.3% of mothers exposed to opiates, cocaine and both drugs, while only one mother declared ecstasy consumption. Meconium analysis showed that prevalence of opiates, cocaine and combined drugs exposure was 8.7, 4.4 and 2.2%, respectively, and confirmed the case of ecstasy use. Arecoline, the main areca nut alkaloid, was found in meconium specimens from four Asiatic newborns, whose mothers declared beetle nut consumption during pregnancy. Parental ethnicity was not associated with drug use, nor was the social class, although a higher tendency toward drug consumption was observed in professional and partly skilled mothers. Drug consuming mothers showed a higher number of previous pregnancies and abortions (p<0.05) when compared to non-consumer mothers (meconium negative test), probably due to a lack of family planning. Consumption of opiates and cocaine during pregnancy was associated with active tobacco smoking, a higher number of smoked cigarettes and cannabis use. Exposure status and smoking behavior correlated with significantly lower birth weight in newborns from mothers exposed only to cocaine and to opiates and cocaine simultaneously. Of the four newborns exposed to arecoline, one showed a low birth weight, low intrauterine growth, hyporeflexia and hypotonia.     

Postmortem evaluation of barbiturate poisonings

The evaluation of barbiturate intoxication as the cause of death is often difficult when the concentration in body fluids and organs is not extremely high. The problem arises because of the great capacity of barbiturates to produce tolerance after chronic use, a property that is often unknown. Therefore, the most abused barbiturates were studied to assess whether chronic intake causes morphological liver changes or not. It was found that the chronic abuse of drugs containing seco-, cyclo-, brallo-, and/or pentobarbital produces hypertrophy of the smooth endoplasmic reticulum of hepatocytes corresponding to that of phenobarbital. Neither acute barbiturate overdose (without a history of abuse) nor chronic abuse of opiates causes similar liver changes. In conclusion, barbiturate tolerance can be evaluated postmortem by light microscopic examination of the liver.