Brain concentrations of cocaine and benzoylecgonine in fatal cases

Since cocaine in blood rapidly hydrolyzes to benzoylecgonine, cocaine concentrations determined in postmortem blood may not reflect the presence or concentration of cocaine in the body at the time of death. The interpretative value of the determination of cocaine and benzoylecgonine in brain tissue was investigated. Cocaine and benzoylecgonine were quantitated by coextraction and formation of the propyl derivative of benzoylecgonine followed by selected ion monitoring gas chromatography/mass spectrometry (GC/MS) using electron ion impact ionization. Cocaine and benzoylecgonine were found to be evenly distributed throughout the brain. Cocaine and benzoylecgonine concentrations were stable in frozen brain tissue (-4 degrees C) on reanalysis after 1 to 3 months of storage, and in refrigerated tissue (10 degrees C) after 30 days of storage. Blood, brain, and liver concentrations of cocaine and benzoylecgonine in 37 cocaine overdose cases and 46 cases in which cocaine was incidental to the cause of death were reviewed. The ratios of cocaine/benzoylecgonine in the toxic cases (brain mean 14.7 and blood mean 0.64) were clearly different from those found in the incidental cases (brain mean 0.87 and blood mean 0.27). The brain/blood ratios of cocaine and benzoylecgonine concentrations generally were characteristic of the time elapsed since cocaine dosing. In cocaine overdose cases, the mean ratio was 9.6 for cocaine and 0.36 for benzoylecgonine. These are within the range found in animal studies for brain/blood ratios of cocaine and benzoylecgonine 0.5 to 2 h after cocaine administration. In incidental cases, the brain/blood ratios were mean 2.5 for cocaine and 1.4 for benzoylecgonine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative measurement of cocaine and its metabolites in blood, cerebrospinal fluid and the brain

This study investigated the transfer of cocaine and its metabolites from plasma into the cerebrospinal fluid. The concentration of cocaine and its metabolites in plasma and cerebrospinal fluid was determined by radioimmunoassay because this method only, the sum of the drug and metabolites restored. In sheeps a sublethal cocaine hydrochloride dose (2,4 mg/kg b. wt.) was administered intraarterial daily for up to 8 days. In the first hours after administration the concentration of cocaine in cerebrospinal fluid was low. It is supposed that a barrier against the transport of cocaine from blood into cerebrospinal fluid exists. After intrathecal administration a delay of transport could from CSF to blood not be seen.     

Effect of murine retroviral infection on hair and serum levels of cocaine and morphine.

LP-BM5 retrovirally infected female C57BL/6J mice were administered cocaine, morphine or both by daily intraperitoneal injection for 9 weeks. Drug concentrations were measured by radioimmunoassay in serum and in hair extracts. Hair samples obtained from all drug-treated mice were positive for the drug injected, while none of the saline-treated mice had detectable drug levels in hair or serum. The average morphine concentration obtained from non-infected mice was 11 ng/mg hair whereas the amount found in the LP-BM5-infected mice was significantly higher (20 ng/mg hair). Mice injected with both morphine and cocaine were given 50% of the regular dose of each drug and drug levels in the hair of these animals were approximately half that of mice injected with the full dose of the single drug. Non-infected mice treated with both drugs had a mean value of 7 ng morphine/mg hair and 374 ng cocaine/mg hair while retrovirus-infected mice had significantly higher concentrations, 10 ng morphine/mg hair and 1160 ng cocaine/mg hair (P less than 0.001). Serum concentrations of cocaine and morphine were significantly higher (P less than 0.01) in the retrovirus-infected animals from 40 min to 1.5 h. The increased concentrations of cocaine and morphine in serum during retrovirus infection are accompanied by a significant increase in the amount of drug incorporated into the hair matrix. This change indicates that retroviral infection may influence the disposition of these drugs in the systemic circulation.     

Validity testing of the TDx Cocaine Metabolite Assay with human specimens obtained after intravenous cocaine administration

Clinical specimens obtained from human subjects after intravenous cocaine administration were analyzed by the TDx Cocaine Metabolite Assay (TDx) and by GC/MS for benzoylecgonine. The TDx results were significantly correlated with results by GC/MS assay with no evidence of bias in the TDx assay. All cocaine metabolite positive specimens (greater than or equal to 300 ng/ml) were confirmed by GC/MS. Detection times to the last positive specimen by TDx assay and GC/MS assay of four subjects after a 20-mg intravenous dose of cocaine ranged from 29.3 to 39.1 h and 27.9 and 36.6 h, respectively. Overall, the TDx assay was found to be highly specific and accurate for the detection and measurement of benzoylecgonine in urine.     

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4°C and -18°C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4°C and -18°C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18°C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4°C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4°C and had declined by 81% following storage at -18°C. At 4°C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18°C was essentially stable for the study period whereas at 4°C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4°C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18°C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH.     

Cocaine- and methamphetamine-related deaths in San Diego County (1987): homicides and accidental overdoses

Cocaine- and methamphetamine-related homicides and fatal accidental overdoses in San Diego County were studied retrospectively for the 1987 calendar year. Cocaine was involved in 66 cases (39 homicides, 27 accidental overdoses), methamphetamine in 32 cases (23 homicides, 9 accidental overdoses), and a combination of cocaine and methamphetamine in 10 cases (4 homicides, 6 accidental overdoses). The composite for cocaine-related deaths was a 30-year-old black man in whom was also found at least 1 other drug, usually ethanol or morphine. The composite for methamphetamine-related deaths was a 32-year-old Caucasian man who used methamphetamine with at least 1 other drug (usually ethanol). For cases involving both cocaine and methamphetamine, the composite was a 36-year-old Caucasian man in whom was also found at least 1 other drug, usually ethanol, codeine, or morphine. Mean tissue concentrations of cocaine and benzoylecgonine were significantly higher in accidental overdoses than in homicides except for cocaine concentrations in liver, which did not differ significantly between the two groups. For methamphetamine-related deaths there was no significant difference between mean tissue concentrations in accidental overdoses and in homicides. Cocaine or methamphetamine or both were involved in approximately one third of homicides in San Diego County in 1987, and when fatal accidental overdoses were included, cocaine was involved in twice as many cases as methamphetamine.     

An epidemic of cocaine associated deaths in Utah

This article describes 15 cocaine associated deaths in Utah in a 3 1/2-month period in early 1984, compared to 6 cocaine associated deaths for all of 1983. Brief case histories of the 15 cases are presented along with data on age, sex, county of death, cause of death, association of the cocaine with the death, cocaine analytical methodology, and a summary of cocaine concentrations in blood and urine. In four of the cases, cocaine by injection was the cause of death, whereas the other cases had cocaine or metabolite detected incidentally. The epidemiology of a single, well-defined cocaine source is described and related to this transient, remarkable increase in cocaine associated deaths.     

Cocaine fatality: an unexplained blood concentration in a fatal overdose

A case is presented of a 26-year-old female who died as a result of cocaine intoxication. A blood cocaine concentration of 330 mg/l, about 1.5 times greater than the highest concentration previously reported, was found. Blood benzoylecgonine and ecgonine methyl ester concentrations were 50 and 18 mg/l, respectively. The unusually high blood concentrations of cocaine and the metabolites are suggestive of a massive administration, however, the history suggests a series of recreational uses. The manner of death was undetermined.     

Testing human hair for drugs of abuse. IV. Environmental cocaine contamination and washing effects

Active cocaine use results in sequestration of parent drug in hair. In addition, hair has unique physicochemical properties that permit absorption of cocaine from the environment. When hair is tested for evidence of cocaine, it is important to consider whether the positive test resulted from active drug use or environmental contamination. In a series of laboratory experiments, it was found that exposure of ‘cut’ hair to cocaine vapor (‘crack’ smoke) and to aqueous solutions of cocaine hydrochloride resulted in significant contamination of hair samples. Similar results were obtained with two subjects who were exposed to cocaine vapor in an unventilated room. The amount of contamination adsorbed by hair depended upon both time and extent of exposure. Washing the hair samples with methanol removed >70% of the cocaine contaminant after cocaine vapor exposure, but was less effective (<50%) following contamination with aqueous cocaine. Shampoo treatment cycles (overnight soaking) progressively removed increasing amounts of cocaine from the contaminated hair, but residual cocaine remained after 10 cycles. Studies were also performed to determine the usefulness of benzoylecgonine as a marker of active cocaine administration. Small amounts of benzoylecgonine (ca. 1 ng/mg) were formed in hair as a result of environmental contamination with cocaine. Also, it was found that benzoylecgonine could be adsorbed from illicit cocaine contaminated with benzoylecgonine. It was concluded that positive hair test results should be interpreted cautiously due to the possibility of environmental contamination from cocaine and related constituents.     

Morphine and 6-acetylmorphine concentrations in blood, brain, spinal cord, bone marrow and bone after lethal acute or chronic diacetylmorphine administration to mice

The aim of this study was to evaluate postmortem incorporation of opiates in bone and bone marrow after diacetylmorphine (heroin) administration to mice. Mice were given acute (lethal dose of 300 mg/kg) or chronic (10 and 20 mg/kg/24 h for 20 days) intraperitoneal administration of diacetylmorphine. The two metabolites of diacetylmorphine, 6-acetylmorphine (6-AM) and morphine, were extracted from whole blood, brain, spinal cord, bone marrow and bone (after hydrolysis) using a liquid/liquid method. Quantification was performed by gas chromatography-mass spectrometry (GC/MS). Results showed that after acute administration, opiates were present in all studied tissues. Morphine concentrations appeared to be higher than those of 6-AM in blood (52.4 microg/mL versus 27.7 microg/mL, n=12), bone marrow (87.8 ng/mg versus 8.9 ng/mg, n=6) and bone (0.85 ng/mg versus 0.43 ng/mg, n=6), but 6-AM concentrations were higher than those of morphine in brain (14.0 ng/mg versus 7.4 ng/mg, n=12) and spinal cord (27.8 ng/mg versus 20.8 ng/mg, n=12). No correlation was found for both compounds between blood concentrations and either brain, spinal cord, bone or bone marrow concentrations while a significant one was found between brain and spinal cord concentrations either for morphine (r=0.89, n=12, p<0.001) or 6-AM (r=0.93, n=12, p<0.001), the concentration being higher in spinal cord than in brain. When bones were stored for 2 months, only 6-AM remained in bone marrow but not in bone. After chronic administration, mice being sacrificed by cervical dislocation 24 h after the last injection, no opiate was detected in any studied tissues. Further studies are required, in particular in human bones, but these results seem to show that 6-AM could be detect in bone marrow several weeks after the death and could be an alternative tissue for forensic toxicologist to detect a fatal diacetylmorphine overdose, even if no correlation between blood and bone marrow was observed. On the other hand, neither bone tissue nor bone marrow will allow the confirmation of a chronic diacetylmorphine use.     

Gas chromatographic detection of cocaine and cocaethylene in hair of mice chronically injected with cocaine or cocaethylene and fed ethanol.

GC and GC/MS analysis was used to detect cocaine and cocaethylene in hair extracts of mice injected with 20 mg/kg cocaine hydrochloride or an equivalent dose of cocaethylene fumarate twice daily for 3 weeks. Some mice were fed liquid Lieber-DeCarli diets containing ethanol (26% of total calories) and injected twice daily with the same doses of cocaine or cocaethylene or combination of cocaine and morphine (5 mg/kg). The average concentrations of cocaine in different experimental groups were in the range of 0.9-2.4 ng/mg of hair and for cocaethylene, 2.4-2.8 ng/mg of hair. There were no significant differences in hair concentrations of cocaine among groups receiving cocaine treatment, nor were there significant difference in cocaethylene concentration in hair in the two groups administered cocaethylene. In hair extracts of mice treated with cocaine and ethanol, levels of cocaethylene were below the limit of detection.     

A rare case of lethal methadone intoxication of a 3-week-old infant

The authors describe a rare case of lethal methadone intoxication of a 3-week-old infant. Born of a drug-addicted mother who was undergoing methadone substitution therapy, the newborn presented withdrawal symptoms and was immediately admitted to a paediatric ward where it was treated for 3 weeks, without administration of methadone. The infant died during bottle feeding within 24 h following discharge home.A post-mortem examination was performed as well as histopathological, toxicological and genetic tests were carried out in order to establish the cause and mode of death.The autopsy revealed obstruction of the lower air passages by stomach contents along with classical features of asphyxia. Toxicological tests revealed high concentrations of methadone and its metabolite in blood and urine as well as presence of methadone in stomach contents, and the DNA polymorphism analysis of the (sex-specific) amelogenin locus gave a negative result.On the basis of the results of the post-mortem examination, the laboratory tests and the police investigation, it was concluded that the drug was ingested with the bottle milk, and the possibility of administration of methadone at the hospital or its ingestion with the breast milk was precluded. Though both the cause and mode of death were established, the public prosecutor discontinued proceedings in the case due to inability to find the perpetrator.     

Tissue disposition of cocaine in man: a report of five fatal poisonings

The disposition of cocaine in five cases of fatal poisoning are presented. The highest concentrations of cocaine were found in urine, kidney, spleen, brain, lung and skeletal muscle. Cocaine concentrations in these organs far exceeded those in blood. Cocaine was detected in all other specimens tested including: bile, heart, liver, vitreous and adipose tissue. These results are in agreement with limited, previously reported, tissue data, and indicate that when urine is not available, kidney, spleen, brain and/or lung should be the specimen of choice for cocaine detection.     

阿维菌素在急性中毒死家兔体内的再分布研究

目的考察阿维菌素在急性中毒死家兔体内的再分布。方法按最小致死量一次性灌胃250mg/kg阿维菌素,HPLC法检测家兔死后0h、24h、48h和72h中阿维菌素的含量。结果给家兔一次性灌胃250mg/kg阿维菌素的临床死亡时间为120.6±9.2min(±s,n=10);测定了阿维菌素的致死血浓度和致死组织浓度;家兔死后0h~72h心血和各主要脏器组织中阿维菌素含量存在体内再分布现象;确定肝、肾、肺为最佳组织检材。结论阿维菌素在急性中毒死家兔体内的再分布数据,对法医办理此类案件具有重要参考价值。     

The evidential value of intra-alveolar haemosiderin-macrophages in cases of sudden infant death syndrome (SIDS)

This study aimed to determine the effect of cocaine on the development and growth of immature and adult blowflies, in an attempt to better understand the impacts of such effects on postmortem interval (PMI) estimation. Twice the lethal dose of cocaine was injected into rabbits. The control animals were injected only with saline solution. Experimental and control rabbits were autopsied, and portions of their livers were exposed to newly eclosed larvae of Chrysomya putoria and Chrysomya albiceps. Larvae were weighed individually every 6 h, up 54 h of exposure. The larvae were then placed on an artificial diet to continue their development. Pupariation time, adult emergence and adult longevity were also analysed. The larvae of both species that fed on the cocaine-containing livers developed faster than those that fed on the livers of the control animals, leading to the conclusion that cocaine influences and stimulates larval growth. The difference in growth between the control and treated flies was best observed from 12 h of exposure onward. This finding has important implications for forensic investigations.     

Hair analysis of opiates in mothers and newborns for evaluating opiate exposure during pregnancy

The increasing interest in toxicological hair analysis as a marker of human exposure to xenobiotics such as illicit substances or therapeutic drugs, has been made feasible by the extension of mass spectrometry, a highly sensitive method of detection. A newborn exposed to drugs in utero can suffer from a varying degree of withdrawal syndrome, a few days after birth. If of opiate origin, the withdrawal syndrome can be treated with morphine, among other therapeutics, but it is not easy to diagnose because of atypical symptoms presented by neonates and especially when maternal drug addiction has not been revealed. To assess and measure toxicological factors linked with the appearance and the severity of this syndrome, maternal and neonatal matrices such as urine, meconium and hair were collected during a protocol approved by the ethical committee. Opiates in particular were measured with GC-MS and potential combined dependences (cannabis, cocaine, amphetamine, LSD and benzodiazepines) and/or substitutive therapeutics (methadone or buprenorphine) were also assessed in 17 mother/neonate couples. Gestational opiate exposure profiles were drawn up and linked with the observed withdrawal syndromes. A withdrawal syndrome seems to appear more frequently after foetal exposure to an association of opiates/substitutive molecules (8 out of 10 withdrawal syndromes observed in this study), although the impact of cocaine and benzodiazepines must also be taken into account. The results obtained in neonatal hair make it possible to affirm foetal drug exposure and are in accordance, for the majority, with the appearance of a neonatal withdrawal syndrome (NWS). Neonatal hair analysis could contribute to assess in utero exposure to opiates, particularly when results in urine and meconium are negative or when these matrices are not available.     

Cocaine and benzoylecgonine excretion in humans.

Maximal urinary excretion of unchanged cocaine occurred within 2 h of the intranasal absorption of 1.5 mg/kg body weight of cocaine hydrochloride, and diminished rapidly thereafter. Excretion of benzoylecgonine was maximal 4 to 8 h following administration of the drug and diminished slowly over an interval of several days. Peak cocaine and benzoylecgonine concentrations observed were 24 and 75 microgram/ml, respectively. Benzoylecgonine/cocaine ratios were too varied to allow estimation of cocaine concentrations from benzoylecgonine concentration data or vice versa. Benzoylecgonine concentrations generally exceeded the corresponding cocaine values by a wide margin, but excretion of free cocaine in the absence of benzoylecgonine was observed in one subject. Cocaine was generally detected for only approximately 8 h, and for a maximum of 12 h, whereas benzoylecgonine was generally detected by chromatographic or enzyme immunologic assays for 48 to 72 h. Benzoylecgonine was positively identified in urine by raidoimmunoassay for 96 to 144 h after dosing.     

Identification of N,N-dimethylamphetamine formed by methylation of methamphetamine in formalin-fixed liver tissue by multistage mass spectrometry

Methamphetamine is methylated in the presence of unbuffered formalin solutions within hours at room temperature. The product, N,N-dimethylamphetamine, is also found in human liver exposed to methamphetamine followed by incubation with formalin. In the present study, a direct mass spectrometric method was developed to identify N,N-dimethylamphetamine in human liver before and after treatment with formalin. Human liver samples were obtained from four deaths that were investigated by the West Virginia Office of Chief Medical Examiner. Full toxicological analysis was conducted on samples from the decedents and methamphetamine was among the positive findings in each case. The method used to expose liver tissue to formaldehyde involved treating a small piece of liver from each case with formalin solution (20% v/v) for 24 h at room temperature. The formalin treated tissues were homogenized and the resulting suspension was sonicated for 5 min, and then centrifuged. Supernatant aliquots were directly analyzed by electrospray ionization (ESI) mass spectrometry without chromatographic isolation. Positive ion multistage mass spectra recorded in MS, MS/MS and MS/MS/MS (MS3) modes were used to confirm the presence of N,N-dimethylamphetamine and methamphetamine in the mixture. Liver tissue not treated with formalin did not contain a detectable level of N,N-dimethylamphetamine. Decreases in methamphetamine concentrations in liver tissue resulting from treatment with formalin were measured using deuterium-labeled methamphetamine as internal standard. The method can be completed in less than 2 h on thawed tissue. The results suggest that the process of fixing tissues with formalin may lead to false negative findings for methamphetamine.     

Elevated morphine concentrations determined during infant death investigations: artifacts of withdrawal of care

Abstract:  Three cases are reported of elevated postmortem blood morphine concentrations (189–3036 ng/mL) that were observed during the course of death investigations involving three children ranging in age from 1 week to 2 years, all of whom underwent withdrawal of life support. In all three cases, the presence of opiates in postmortem blood was indicated by immunoassay (ELISA) and quantitative confirmatory analysis of free morphine concentrations in postmortem blood was performed by solid-phase extraction followed by gas chromatography/mass spectrometry (GC/MS) in the selected ion monitoring mode. While the practice of withdrawing life support from terminally ill patients, with the accompanying administration of narcotics/analgesics has been reported in the medical literature, it has not been adequately described in the forensic literature. The implications of this practice on the forensic toxicological interpretation of morphine findings are discussed. To our knowledge, this is the first report of postmortem morphine concentrations arising directly from administration in conjunction with withdrawal of care in pediatric patients.