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.     

Drugs in postmortem adipose tissues: evidence of antemortem deposition

INTRODUCTION: Drug concentration measured in postmortem adipose tissue may or may not reflect antemortem concentration. To examine the possibility of whether the presence of basic drugs in adipose tissue is the result of postmortem change, we examined: tissues with and without livor mortis, concentration gradients within the adipose layer, and the stability of drug concentrations during the postmortem period. CASE REPORTS: Five drug-related deaths with case histories and analytical data are presented. Adipose tissues with and without livor mortis from the thigh area of the same decedent were analyzed for cocaine. The cocaine concentration of the tissue exhibiting 4+ livor was equivalent to the concentration observed in tissue without livor. Analyses of cross sections of adipose tissues containing cocaine and methamphetamine disclosed that drug concentrations were equally distributed throughout the layer, from just beneath the dermis to directly above the muscle. When morphine and temazepam concentrations were measured in adipose tissues collected from similar sites, but at different times, from the same cadaver, they remained essentially the same over 3 days (approximately 80 h). CONCLUSIONS: Since concentrations were the same in areas with and without livor mortis, the possibility of redistribution into adipose from blood or vascular channels is eliminated. The absence of a concentration gradient within the adipose layer rules out diffusion or permeation from muscle into the adipose layer, and the failure of morphine or temazepam concentration to change over time indicates that drugs in the adipose tissue are stable during the postmortem interval. Our findings support the notion that drugs identified in postmortem adipose tissue are there because of antemortem deposition and not because of any postmortem change or event.     

Distribution of morphine and morphine glucuronides in body tissue and fluids--postmortem findings in brief survival

An intoxication following administration of morphine, tramadol and atracurium in a suicide case is reported. The route of administration and the amount of the particular drug were known from the investigation of the death scene and the findings of the postmortem examination. Tramadol was present in the gastric contents as well as in blood, liver, kidney and brain samples, whereas the drug could not be detected in muscle. All body fluids and tissues investigated contained morphine as well as its 3- and 6-glucuronides with the exception of muscle tissue. The concentrations of morphine and its glucuronide metabolites were determined by LC/MS following solid phase extraction. Interestingly, the concentration of M6G in brain, liver and kidney were close to the concentration of M3G in the particular tissue. This phenomenon might be explained by a preferential hydrolysis of M3G or by a preferential formation of M6G postmortem. Measurement of morphine and M6G in femoral blood and cerebrospinal fluid may be a useful indicator in rapid deaths.     

The stability of tetramine, morphine and meperidine in formalin solution.

The stability of tetramine, morphine and meperidine in formalin solution is an important factor for drug analysis in forensic investigation. In this paper, the tissues (liver, kidney, lung and heart) from poisoned rabbits were immersed in 50 ml 10% formalin solutions for 4 months before examination. We compared the levels of tetramine, morphine, meperidine and the main metabolite normeperidine, measured by GC/NPD or GC-MS, in frozen rabbit tissues, formalin-fixed rabbit tissues, and formalin solution. There was a decrease in the levels of tetramine, morphine, meperidine in formalin-preserved tissues compared with the levels of these drugs in the frozen tissues. It is suggested that the formalin-fixed tissues and formalin solution should be analyzed at the same time to assure the accurate results.     

Morphine extraction in necrophagous insects remains for determining ante-mortem opiate intoxication.

To study the potential use of insects remains as toxicological indicators, we measured morphine in desiccated entomological materials using a radioimmunoassay method (Coat-A-Count Serum Morphine, Dade Behring, France) following enzymatic hydrolysis.First, enzymatic cuticle extraction of morphine (pronase digestion preceded by a 2h incubation with a dithiothreitol solution) was performed on various substrates (Calliphoridae puparial cases and desiccated adults, and desiccated pupae of Dermestidae) which were then tested to identify possible interferences with the radioimmunoassay procedure.This same procedure was performed on puparial cases and desiccated adults of Lucilia sericata (Diptera, Calliphoridae) previously reared on minced meat containing various morphine concentrations. Morphine was detected only in cuticle powder from insects reared on meats containing 100 and 1000mg/kg. Higher concentrations were measured in puparial cases. Rearings on psoas from eight heroin overdose victims confirmed previous experimental results.Remains of necrophagous insects, particularly puparial cases, are often preserved for a long time, and consequently can serve as late alternative specimens for toxicological analysis when suitable tissues are not available.     

Postmortem disposition of morphine in rats

The antemortem and postmortem distribution of morphine was studied in rats for the purpose of establishing whether drug distribution is altered after death. Samples were examined for free and total morphine concentration, pH and water content at 0-96 h after death. Morphine was administered antemortem at various intervals. All groups of rats studied showed a significant (P less than 0.05) increase in postmortem cardiac blood morphine concentrations. These changes, which are detectable within 5 min after death are likely to be related to an observed, rapid decrease in cardiac blood pH from 7.34 +/- 0.02 to 6.74 +/- 0.05. Significant increases in free morphine levels were, also, observed 24 and 96 h after death in liver, heart and forebrain while urine morphine levels decreased. The liver showed the greatest increase (20-fold) in free morphine levels 96 h after death, while hindbrain levels did not significantly change. Bacterial hydrolysis of morphine glucuronides accounted only in part for the observed increase in free morphine concentration. Postmortem fluid movement and pH-dependent drug partitioning was detected. It would appear that several mechanisms are responsible for postmortem drug distribution. Understanding the mechanisms and patterns responsible may eventually lead to better choices of postmortem tissue which may better represent antemortem drug levels.     

MALDI-TOF-IMS在生物医学中的研究进展及法医学应用展望

基质辅助激光解吸/电离-飞行时间质谱成像(matrix-assisted laser desorption/ionization time-offlight imaging mass spectrometry,MALDI-TOF-IMS)技术通过对生物组织切片的扫描,分析切片中的未知组分,获取组织的分子成像图,已成为研究生物标志物、脂类分布、药物代谢等方面的有力手段。本文对该技术在蛋白质鉴定、临床应用、药物研发、脂类研究及脑损伤方面的应用进行了综述。     

Determination of drug levels in larvae of Protophormia terraenovae and Calliphora vicina (Diptera: Calliphoridae) reared on rabbit carcasses containing morphine

Two species of blow flies (Diptera: Calliphoridae) were reared on tissues from rabbits administered different dosages of morphine. These species, Protophormia terraenovae and Calliphora vicina are among the first wave of insects colonizing a dead body. Two series of 3 rabbits were given dosages of 10, 20, and 40 mg/h of morphine over a 3 h period via ear artery perfusion. A morphine blood level plateau was attained after 1 h of perfusion. Two other rabbits were used as controls. Samples of tissues collected from rabbits using a coelioscopic technique were determined to have morphine concentrations similar to those encountered in human overdoses and were correlated with dosages of morphine administered. All samples from control rabbits were negative for morphine. Larvae and puparia of both species were regularly collected from each rabbit for toxicological analysis. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were significantly lower than concentrations found in the tissues. There was a decrease in concentration in morphine observed in transition from feeding 3rd instar larva to puparium. A correlation between larval concentration and tissue concentration was found only in feeding 3rd instar larvae.     

Effects of morphine in decomposing bodies on the development of Lucilia sericata (Diptera: Calliphoridae)

This study concerns the effects of morphine in tissues on the rate of development of Lucilia sericata (Diptera: Calliphoridae) using those tissues as a food source. Lucilia sericata is a species of fly commonly found on human corpses in Europe during the early stages of decomposition and thus of forensic interest. Three rabbits were administered 12.5, 25.0 and 50.0 mg/h of morphine chlorhydrate via ear perfusion over a period of 3 h. These dosages and duration of perfusion were calculated to give tissue concentrations of morphine similar to those encountered in fatal human overdoses. A fourth rabbit was used as a control. Following administration of the drug, rabbits were sacrificed and 400 eggs of Lucilia sericata, all of the same age, were placed in the eyes, nostrils and mouth of each rabbit. Developing larvae were sampled daily to determine growth rate and weight. Puparia and emerging adult flies were also sampled. Data were analyzed using analysis of variance (ANOVA) and Student's T-test. Results of this study show that an underestimation of the postmortem interval of 24 h is possible if the presence of morphine in tissues is not considered. This study demonstrates again the necessity of considering the possible effects of drugs in tissues on insect growth rates when estimating the postmortem interval using entomological techniques.     

不同死后时间组织中吗啡免疫组化检测的初步研究

目的检测不同死后时间组织中的吗啡。方法应用抗吗啡兔血和SABC法对不同死后时间大鼠肝肾组织中吗啡进行了检测。结果初步发现死后27h仍可在肝肾组织普通石蜡切片上检出阳性染色。结论免疫组化技术可以用以检测死后组织中的毒物。     

Morphine perfused rabbits: a tool for experiments in forensic entomotoxicology

In order to establish an animal model for entomotoxicological studies, the kinetics of morphine elimination from blood after a single intravenous injection of morphine and the concentration of morphine in tissues following a continuous perfusion were studied. The aim of these experiments was to obtain controlled morphine tissue concentrations similar to those encountered in fatal human heroin overdoses. These tissues can be used as a food source for developing fly larvae in entomotoxicological studies. In the single injection experiment, seven rabbits were administered 1 or 2 mg/kg body weight of morphine chlorhydrate via the main ear artery. Blood samples of 200 microL were removed regularly via a catheter. Morphine concentration was determined using RIA techniques. Morphine was found to be first rapidly distributed and then slowly eliminated, following a two-exponential equation. Elimination of morphine from blood can be described as a two-compartment model. Constants of the equation were determined using the Kaleidagraph program. Using those constants, the main pharmacokinetics parameters were calculated. Results of these parameters showed the following: clearance from 13.3 to 16.2 L.h.1, half-life of the distribution phase from 0.6 to 0.9 min, and half-life of the elimination phase from 21 to 26 min. These results were used to calculate the rate of perfusion of morphine for rabbits to obtain desired, controlled, and constant concentrations of morphine in tissues. In the second experiment, three rabbits received a perfusion of morphine intravascularly at a rate of 2 mg/kg/h for a period of 3 h. These rabbits were sacrificed and analyses performed on several abdominal and thoracic organs. Results showed that the concentrations of morphine differed according to the organ analyzed, but were reproducible for organs between animals. These concentrations were similar to those normally encountered in cases of human death due to heroin overdoses.     

吸毒案件中的尿吗啡检测

目的　研究吸毒人员停止使用毒品海洛因后 ,尿吗啡含量自然递减的一般规律。方法　对确证的 1 82名吸毒 (海洛因 )人员在停止使用毒品后连续 9天采集尿样 ,用放射免疫分析法检测尿吗啡含量。结果　 1 82名吸毒 (海洛因 )人员在停止吸毒后第 8天 ,尿吗啡平均含量降至 30ng/ml。 结论　在办案中 ,吸毒 (海洛因 )人员的尿样检测 ,建议最好在末次使用海洛因 8天之内     

Immunohistochemical contribution to the study of morphine metabolism in Calliphoridae larvae and implications in forensic entomotoxicology

Morphine was detected by immunohistochemistry on sections of third stage larvae of Calliphora vomitoria (Diptera, Calliphoridae) reared on minced beef meat previously treated with morphine hydrochloride. The detection was performed with an avidin-biotin-peroxidase-complex method. Positive specimens showed specific staining of the haemolymph and a more intense immunoreaction in an area located at the limit between exocuticle and endocuticle. These results constitute an evidence of morphine accumulation inside the cuticle of Diptera larvae during their development. During the pupariation, the larval cuticle is transformed into the sclerotized puparium. This study consequently points out the possibilities of analyzing empty pupariae when suitable tissues or living necrophagous insects are absent.     

Drug deposition in adipose tissue and skin: evidence for an alternative source of positive sweat patch tests

In a series of licit and illicit drug-related deaths, qualitative and quantitative analyses on extracts of adipose tissue and skin were performed by GC/MS. In all cases, the adipose tissue was found to contain drugs at concentrations lower than, approximately equal to, or even greater than the concentrations of the same analytes found in the blood, which may reflect a consequence of long-term chronic exposure, or acute intoxication, or some combination of both. Approximately one cubic inch of skin with adipose tissue was removed from the mid to lower abdominal region adjacent to the midline incision during autopsy. The drugs were recovered from the specimens following incubation and alkaline, acidic, and alkaline chloroform back extraction of one to three grams of tissue. Deuterated analogs of the analytes were added to the matrix at the beginning of the incubation period. Cocaine and free morphine (from heroin) were readily identified in several cases. The presence of these illicit drugs in adipose tissue raises significant forensic questions, especially the use of 'sweat patches' to monitor recent cocaine or heroin use in chronic drug users.     

Interpretation of GC-MS opiate results in the presence of pholcodine

Although the cross reactivity of pholcodine with opiate immunoassays has been well documented there is little published information the potential for pholcodine interference with chromatographic analyses. Wilson and Smith [Ann. Clin. Biochem. 36 (1999) 592] recently described the 'misidentification' of morphine in quality control specimens that had been spiked with pholcodine. This report describes a sensitive, rapid gas chromatography-mass spectrometry (GC-MS) method for the detection and quantitation of pholcodine and morphine, together with 6-monoacetylmorphine (6-MAM), codeine and dihydrocodeine in urine. This method was used to analyse urine specimens collected from volunteers given single and multiple doses of pholcodine to establish the significance this drug on the analytical results obtained when performing drug screening according to the proposed UK and EU legally defensible workplace drug testing guidelines. The maximum urinary free morphine concentration achieved following a single 10mg oral dose of pholcodine was 1.39 mg/l at 2-4h post dose. Following multiple 10mg oral doses of pholcodine the maximum urinary free morphine concentration was determined as 0.4 mg/l at 170 h after the final dose was administered. This apparent anomaly in the morphine concentrations obtained following single and multiple pholcodine doses can be explained in part by differences in the concentration of the specimens, and may be overcome by applying a correction factor for specimen dilution using their creatinine concentration. The data from this study suggests that even following one single 10mg dose of pholcodine, free morphine concentrations greater than both the proposed UK workplace drug testing guidelines threshold of 0.3mg/l total morphine and the proposed European Union threshold of 0.2mg/l total morphine can be achieved. This highlights the need for caution when interpreting confirmatory opiate data, especially in medicolegal and clinical cases, and in cases where the use of pholcodine is suspected.     

Determination of drug levels in two species of necrophagous Coleoptera reared on substrates containing morphine

Two species of necrophagous Coleoptera: Dermestes frischi (Dermestidae) and Thanatophilus sinuatus (Silphidae), were reared on substrates containing different amounts of morphine. Colonies of D. frischi were reared on rabbit carcasses which had been given 10, 20, and 40 mg/h of morphine hydrochloride via ear artery perfusion over a 3 h period prior to death. A fourth rabbit served as a control. T. sinuatus was reared on minced beef spiked with morphine hydrochloride to give concentrations of 1,000, 2,500, 5,000, and 10,000 ng/g and one control colony. These dosages were calculated to create tissue concentrations of morphine similar to those encountered in human deaths due to morphine overdose. Larvae. pupae, and adults (except for T. sinuatus) were analyzed for morphine content. All developmental stages of D. frischi were positive for morphine and concentrations correlated with cadaveric tissue concentrations during larval stages and to a lesser extent in the adult stage. For T. sinuatus, the best correlations were found in 2nd and 3rd instar larvae. This study demonstrates the potential for use of necrophagous Coleoptera, as well as Diptera larvae, as alternate specimens for toxicological analyses.     

Fatal intoxications in Denmark following intake of morphine from opium poppies

In Denmark it is legal to grow opium poppies for the production of poppy seeds and until 1986 for decoration purposes, too. Danish poppy capsules contain 0.3-5 mg morphine per capsule and the content of morphine in opium exuded from the capsules may amount to 24%. This has resulted in misuse as both fresh and dried poppy capsules have been used for the production of "opium tea". During the period 1982-1985, seven casualties occurred among drug addicts in Denmark which were solely or partly caused by these opium poppies.     

Forensic applications of sodium rhodizonate and hydrochloric acid: a new histological technique for detection of gunshot residues

Demonstration of the presence of lead residues deriving from gunshot in skin and underlying tissues is essential for the correct forensic analysis of numerous legal cases. Optical microscopy remains the fastest, cheapest diagnostic technique, even though its sensitivity and specificity are poor because of the scarce quantity of histological tissue that can be examined and possible environmental lead pollution. To confirm the presence of lead from gunshot residues, we applied to histological sections of human skin a technique proposed by Owens and George in 1991 for macroscopic detection of lead on the clothing of shooting victims, involving a reaction with sodium rhodizonate and subsequent confirmation by color change on application of HCl. Our results demonstrate the technical possibility of using this macroscopic technique even on histological samples and support the need for further studies on a larger series of cases correlated with the type of ammunition and firing distance.     

Heroin, morphine, and hydromorphone determination in postmortem material by high performance liquid chromatography

A procedure has been developed for the simultaneous determination of heroin, morphine, and hydromorphone from postmortem tissues by reversed phase high performance liquid chromatography (HPLC) using electrochemical detection. This method permits the direct determination of unmetabolized heroin from antemortem or postmortem urine as evidence of illegal drug use. Presumptive confirmation of heroin was based on the ability to hydrolyze the HPLC heroin fraction to morphine. Heroin was also confirmed in urine by gas chromatographic/mass spectroscopic (GC/MS) analysis of the HPLC fraction. Analysis of postmortem blood, gastric contents, urine, and injection site tissues have revealed the presence of morphine and hydromorphone, while heroin has only been identified in urine.     

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.