Identification of N-piperidinyl etonitazene in alternative keratinous matrices from a decomposing cadaver.

This short report describes research on N-piperidinyl etonitazene, also known as etonitazepipne, in keratinous matrices (hair and nails) after death related to a suspected opioid overdose. Etonitazepipne belongs to the family of benzimidazole opioids, a class of new synthetic opioids that has penetrated the illicit drug market. Analysis in the case under study showed the presence of etonitazepipne in both hair and nails, confirming that the substance accumulates in the body with repeated intake.

Comparative analysis between CDT in serum and Ethyl glucuronide in hair to define the best reliable tool for the diagnosis of alcohol abuse.

The increase in alcohol consumption in society has not only led to a number of medical issues but has also become a matter of considerable legal importance. Thus, there is both scientific interest and the necessity to diagnose alcohol abuse in the application of the provisions of the law through laboratory tests that ensure maximum objectivity. The purpose of this work is to study and compare the diagnostic performance of two of the main markers of alcohol abuse, serum carbohydrate-deficient transferrin (CDT) and Ethyl glucuronide (EtG) in a group of 336 driving under the influence (DUI) of alcohol offenders. Thus, it is possible to establish the best marker of alcohol consumption in order to assess the fitness to drive of DUI subjects.EtG was detected in 55 hair samples, while CDT was detected in 5 blood samples. Of the EtG-positive subjects 96,4% had CDT values below the cut-off. While CDT refers to an alcohol consumption of approximately the previous 10 days, EtG allows to detect an excessive alcohol consumption of the last few months. Because of these two different time-windows, EtG proves to be more reliable, since it is more difficult for subjects to change their drinking practice to test negative to toxicological analysis. The determination of Ethyl glucuronide on hair matrix is a valuable tool for the diagnosis of alcohol abuse, with high sensitivity and specificity and certainly greater reliability than traditional markers such as CDT, being a direct marker of alcohol consumption.

A study into the nature and extent of polydrug use in driving recidivism behavior.

OBJECTIVE: Polydrug use has become a frequent pattern of drug consumption in Europe, and this is considered a particularly dangerous risk factor for impaired driving. In Italy, persons whose license has been revoked or suspended due to the use of psychoactive drugs can reapply for a new driving license, depending on the judgment of the relevant local medical committee (CML). To regain a revoked license, offenders must remain drug free throughout an observation period. An important problem with enforcement of impaired driving is recidivism. The aim of the present study is to analyze the influence of polydrug use on driving recidivism. METHOD: We report the findings of several years' experience at the forensic toxicology laboratory of the University of Macerata. Hair samples collected over a 7-year period by the CML from drug users were analyzed for cocaine, opiates, and cannabis using gas chromatography-mass spectrometry. RESULTS: Three hundred thirty-five of the tested subjects were recidivists. Recidivism was more frequent among monodrug users (81%) compared with polydrug users (19%), but logistic regression showed that polydrug use is certainly a risk factor for recidivism compared to monodrug use (odds ratio [OR] = 1.99). The sex and age distribution of recidivist subjects showed a strong predominance of males in both groups, but there were no sex differences. There were more recidivist polydrug users than recidivist monodrug users in the younger age groups (OR = 2.012). Cocaine use was most prevalent in the recidivist monodrug group. All drugs analyzed were demonstrated to be a risk factor for recidivism among monodrug users, whereas only the cocaine and cannabis combination was shown to be a risk factor for recidivism among polydrug users (OR = 1.65 versus cocaine; OR = 1.30 versus Δ9-tetrahydrocannabinol). Almost all polydrug users became monodrug users, and cocaine was the most frequently detected drug in the subsequent test during the monitoring phase. CONCLUSIONS: Our results show that polydrug use increases the risk of impaired driving recidivism and represents a considerable threat to road safety.

Validation of a new method for the detection of Ethyl glucuronide in larvae of Lucilia sericata as a marker of ante-mortem alcohol consumption.

Larvae and insects are an important and alternative biological matrix in the development anpost-mortem forensic toxicology. They are very useful when conventional matrices are not available, in particular when the loss of biological fluids, due to the decomposition of corpses or to a traumatic death, occurs. The purpose of this study is to develop and validate an analytical method in Ultra High-Performance Liquid Chromatography at High Resolution Mass Spectrometry (HPLC/HR-MS) for the research and quantification of Ethyl glucuronide (EtG) on larvae. The criteria taken into consideration for the validation are linearity, quantitation limits (LOD and LLOQ), accuracy, precision, carryover, interferences and ionization suppression/enhancement. The method was shown to be linear within the tested range, with a coefficient of determination higher than 0.99. LOD was 2 pg mg-1, while LLOQ was 5 pg mg-1. Accuracy, precision and ionization/suppression enhancement fulfilled the criteria indicated in the guidelines used for the validation. The establishment and validation of this method allowed the identification of Ethyl glucuronide on the larvae of Lucilia sericata (Calliphoridae) of a subject found death in an advanced state of decomposition.

Detection of Three Opioids (Morphine, Codeine and Methadone) and Their Metabolites (6-Monoacetylmorphine and 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) in Larvae of Lucilia sericata Species by UHPLC-TF-MS and Validation.

Insects on corpses could be a useful tool for the detection of exogenous substances such as drugs of abuse. The identification of exogenous substances in carrion insects is critical for proper estimation of the postmortem interval. It also provides information about the deceased person that may prove useful for forensic purposes. High-performance liquid chromatography coupled with Fourier transform mass spectrometry is a highly sensitive analytical technique that can identify substances even at very low concentrations, such as in the case of searching for exogenous substances in larvae. In this paper, a method is proposed for the identification of morphine, codeine, methadone, 6-monoacetylmorphine (6-MAM) and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the larvae of Lucilia sericata, a common carrion fly widely distributed in temperate areas of the world. The larvae, which were reared on a pig meat substrate, were killed once they reached their third stage by immersion in hot water at 80 °C and aliquoted into 400 mg samples. The samples were fortified with 5 ng of morphine, methadone and codeine. After solid-phase extraction, the samples were processed with a liquid chromatograph coupled to a Fourier transform mass spectrometer. This qualitative method has been validated and tested on larvae from a real case. The results lead to the correct identification of morphine, codeine, methadone and their metabolites. This method could prove useful in cases where toxicological analysis must be conducted on highly decomposed human remains, where biological matrices are very limited. Furthermore, it could help the forensic pathologist to better estimate the time of death, as the growth cycle of carrion insects can undergo changes if exogenous substances are taken.

Validation of an HPLC-HR-MS Method for the Determination and Quantification of Six Drugs (Morphine, Codeine, Methadone, Alprazolam, Clonazepam and Quetiapine) in Nails.

Keratinized matrices, including nails, are among the most resistant matrices that can be analyzed in cases where remains are deeply decomposed and relatively non-invasive for living people. In order to exploit these new matrices in the search for exogenous substances, it is necessary to develop analytical technologies capable of achieving high levels of sensitivity. In this technical note, an easy method is presented for the simultaneous extraction and quantification of three narcotic substances (morphine, codeine and methadone), two benzodiazepines (BDZs) (clonazepam and alprazolam) and an antipsychotic (quetiapine) from nail matrix by analysis in ultra-high-performance liquid chromatography at high-resolution mass spectrometry. The method has been validated following the Standard Practices for Method Validation in Forensic Toxicology of the Scientific Working Group for Forensic Toxicology. Nail specimens from eight authentic postmortem (PM) cases and 13 living donor samples were extracted and analyzed. Of the eight PM samples, five resulted positive for at least one of the three substances searched. Ten of the 13 living donor specimens were positive for at least one of the targeted BDZs or quetiapine.

Detection of Morphine and Opioids in Fingernails: Immunohistochemical Analysis and Confirmation with Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry.

This study aimed to investigate the detection of morphine in fingernails from forensic autopsies using immunohistochemistry (IHC), with confirmation by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). A primary antibody specific to morphine and a secondary antibody conjugated to horseradish peroxidase (HRP) was used. IHC on specimens of Subjects A and B (both drug addicts) resulted in the detection of morphine on a cell layer of the nail plate matrix. UHPLC-HRMS and GC-MS analysis showed that Subject A had a morphine concentration of 0.35 ng/mg in the fingernail and 472 ng/mL in the blood, while Subject B reached 1.23 ng/mg in the fingernail and 360 ng/ml in the blood. Most of those matrices were positive for codeine, methadone, EDDP, and 6-MAM. The use of IHC in Subject C (a former addict) showed no positivity for morphine in the fingernail, while the UHPLC-HRMS analysis confirmed its absence in the fingernail and blood. Additionally, an analysis of the scalp or pubic hair of the subjects was carried out using UHPLC-HRMS. The results suggest that IHC can be used to establish the site of accumulation of morphine in the nail matrix; for postmortem diagnosis; and that basic substances can be detected by UHPLC-HRMS. There are no previous studies on the use of IHC as a technique for forensic purposes in unconventional matrices, such as nails.

Correlation study between anatomopathological data and levels of blood morphine concentrations in heroin-related deaths.

Heroin-related mortality is a complex phenomenon involving several factors that make an individual more susceptible to opioid toxicity. Among these, pre-existing pathological conditions play an important role. The current paper reviewed 51 autopsied cases performed over the last 30 years, each subject ("frequent heroin user") having taken heroin intravenously alone before death. For each case, total blood morphine concentration determined by gas chromatography-mass spectrometry (GC-MS) was obtained from the archives. For histopathological evaluation we examined microscopic sections of the heart, lung, brain, liver and kidney, and for each organ a score table with a numerical value from 1 to 5, as an indicator of increasing severity, was drawn up. Finally, we created a scoring system based on the Injury Severity Score (ISS), generated by the sum of the squares of the three highest organ scores. We investigated the relationship between total blood morphine value (mg/l) and pathological score by inserting the two variables in a linear regression model: Score = a + b·Morphinemia (p-value <0.001). The results demonstrated a statistically significant correlation between the two variables: individuals with low total blood morphine values tended to have a higher pathological score. Our study provides useful assessment tools for forensic practice, confirming the need to combine two important disciplines in drug death investigation: forensic pathology and toxicology.

Determination of Cyanide by Microdiffusion Technique Coupled to Spectrophotometry and GC/NPD and Propofol by Fast GC/MS-TOF in a Case of Poisoning.

A man was found dead in a hotel located near Rome (Italy). The man was still holding a syringe attached to a butterfly needle inserted in his left forearm vein. The syringe contained a cloudy pinkish fluid. In the hotel room the Police found a broken propofol glass vial plus four sealed ones, an opened NaCl plastic vial and six more still sealed, and a number of packed smaller disposable syringes and needles. An opened plastic bottle containing a white crystalline powder labeled as potassium cyanide was also found. Systematic toxicological analysis (STA), carried out on blood, urine and bile, evidenced only the presence of propofol in blood and bile. So the validated L-L extraction protocol and the GC/MS-TOF method for the confirmation of propofol in the biological fluids optimized in our laboratory was applied to blood, urine and bile. The concentration of propofol resulted to be 0.432 µg/mL in blood and 0.786 µg/mL in bile. The quantitative determination of cyanide in blood was carried out by microdiffusion technique coupled to spectrophotometric detection obtaining a cyanide concentration of 5.3 µg/mL. The quantitative determination was then confirmed by GC/NPD and the concentration of cyanide resulted to be 5.5 µg/mL in blood and 1.7 µg/mL in bile. Data emerging from autopsy findings, histopathological exams and the concentrations of cyanide suggested that death might be due to poisoning caused by cyanide, however, respiratory depression caused by propofol could not be excluded.

Toxicological Analysis of Opiates from Alternative Matrices Collected from an Exhumed Body.

In this case study, the body of a 45-year-old man was exhumed after 1 year at the request of the public prosecutor to assess whether the death was caused by drug consumption. Toxicological analyses were performed on several matrices, including liver, kidney, and the alternative matrices hair and teeth. The systematic toxicological analysis (STA), which consisted of basic and acid liquid/liquid extraction and gas chromatography-mass spectrometry (GC-MS) analysis, showed the presence of opiates in each of the matrices analyzed. Subsequently, to confirm and quantify the presence of opioids, samples of each of the matrices were subjected to solid-phase extraction and specific GC-MS analysis. The case presented demonstrates the possibility of drug detection in an exhumed body that has been buried for 1 year, despite the problems of quantitative interpretation of the data, and that toxicological results could be useful along with other forensic evidence.

Determination of Drugs of Abuse in a Single Sample of Human Teeth by a Gas Chromatography-Mass Spectrometry Method.

The purpose of this study was to develop and validate a gas chromatography-mass spectrometry method to detect drugs of abuse in a single sample of tooth. Pulverized samples of dental materials were subjected to acid hydrolysis to detect opiates, cocaine and their metabolites. The residual dental materials from these analyses were subjected to basic extraction to detect cannabis products (Δ9-tetrahydrocannabinol, cannabidiol and cannabinol). The method showed a good linearity between 0.05 and 2 ng/mg for all substances. The limit of detection ranged from 0.02 to 0.03 ng/mg, and the limit of quantification was 0.05 ng/mg. The application of the method to samples of teeth obtained from drug addicts was successful. It can be applied in post-mortem cases, especially when limited amounts of sample are available.

Driving under the effect of drugs: Hair analysis in order to evaluate recidivism.

Under Italian law drug addiction and regular drug abuse are incompatible with driving ability. One important problem with the enforcement of the impaired driving law is the large number of people that re-offend. To regain their license, offenders must be drug-free for the duration of an observation period, according to the judgement of a medical commission. The exclusion of illicit drug use is determined by toxicological analysis. A few studies exist that have used a hair matrix to monitor recidivism. Hair is an attractive matrix for monitoring drug recidivism, due to the large time window for drug detection, and to the non-alterability of this matrix. We report the results of several years of experience at our forensic toxicology laboratory in the use of hair analysis for the assessment of past exposure to drugs in persons suspected of driving under the influence of drugs. 5592 subjects were analyzed for opiates, cocaine and delta-9-tetrahydrocannabinol (Δ9-THC) using a GC/MS method. 1062 (19.0%) subjects resulted positive. From this group, the individuals that resulted positive at least at the second control were considered recidivists (243, 22.9%). 79.7% of recidivist subjects were positive for cocaine and metabolites, 14.9% for morphine and metabolites, 5.4% for Δ9-THC. We also studied the time frame of the abuse, as well as gender and age distribution of recidivist subjects. Furthermore, we analyzed risk factors associated with recidivist behaviour. Our results show that cocaine consumption was the only factor that showed significance with regard to increased likelihood of being a recidivist.

Behavioural and pharmacological characterization of a novel cannabinomimetic adamantane-derived indole, APICA, and considerations on the possible misuse as a psychotropic spice abuse, in C57bl/6J mice.

The novel adamantane derivative APICA (N-(adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide) was recently identified as a cannabinomimetic indole of abuse. Despite its novel structure, APICA recalls cannabinomimetic indoles, such as representative member JWH-018. In present study, the effects of APICA (1-3mg/kg, i.p.) were tested in C57BL/6J mice, in the Tetrad task which includes the assessment of: body temperature; locomotor activity and behavioural reactivity; nociception; motor coordination; declarative memory. Furthermore, pre-treatment with the CB1 antagonist AM251 (3mg/kg, i.p.) or the CB2 antagonist AM630 (3mg/kg, i.p.) was carried out to characterize APICA activity. Our results show that APICA was able to dose-dependently decrease locomotor activity and behavioural reactivity in the open field, whereas only the highest dose was able to induce hypothermia, analgesia, motor incoordination and recognition memory impairment, with respect to vehicle (p<0.01; p<0.001). The pretreatment with the CB1 antagonist AM251 elicited an increase in body temperature, total distance travelled in the open field, latency to fall down in the Rotarod, and a decrease in tail flick latency (p<0.05; p<0.01). On the other hand, pretreatment with AM630 did not induced significant differences on APICA effects. This study supports preliminary reports on APICA cannabinomimetic properties, extending its detrimental effects on cognitive function. Moreover, these properties can be attributed to the CB1 receptor activity, indicating APICA as a selective CB1 receptor agonist.

Detection of Cannabinoids by ELISA and GC-MS Methods in a Hair Sample Previously Used to Detect Other Drugs of Abuse.

A forensic standard procedure is described that combines enzyme-linked immunoassay for screening and gas chromatography-mass spectrometry (GC-MS) for confirmation to detect drugs of abuse in a sample before used to detect opioids and cocaine. We used two equal aliquots of the same previously selected cannabinoid positive hair samples, one of which was subjected to acid hydrolysis. Afterward, both the aliquots were subjected to basic extraction and then to immunoassay screening. After derivatization, the GC-MS parameters were the same for both the aliquots for the determination of the cannabinoids (Δ(9)-tetrahydrocannabinol, cannabidiol and cannabinol). The results show that there were no statistical differences between the nonpreviously treated and the pretreated hair samples for the quantification of the three cannabis products for immunochemical procedure. No differences between the two groups were shown as for GC-MS confirmation procedures. All substances showed a good linearity between 0.05 and 2 ng/mg. The limit of detection ranged from 0.02 to 0.03 ng/mg, and the limit of quantification was 0.05 ng/mg for all substances. To our knowledge, this is the first time that screening and confirmation procedures have been applied on the same sample of hair to detect more than one drug of abuse.

Unexpected variation of the codeine/morphine ratio following fatal heroin overdose.

Postmortem samples from 14 cases of suspected heroin overdose were subjected to a preliminary systematic toxicological analysis in order to highlight the presence of unknown exogenous compounds (e.g., drugs of abuse, alcohol) that may have played a role in the mechanism of death. This analysis unveiled histories of poly-drug use in seven of the cases under investigation. Moreover, the concentrations of morphine and codeine in the brain were also investigated, and the results were compared with the data obtained from the blood specimens. The concentration of morphine in blood ranged from 33 to 688 ng/mL, while the concentration of codeine ranged from 0 to 193 ng/mL. However, in the brain, the concentration of morphine was found to be between 85 and 396 ng/g, while the levels of codeine ranged from 11 to 160 ng/g. The codeine/morphine ratio in the blood ranged from 0.043 to 0.619; however, in the brain, the same ratio was found to be between 0.129 and 0.552. In most cases, a significantly higher codeine/morphine ratio was found in the brain, suggesting the accumulation of codeine in brain tissue due its high lipophilicity as compared with morphine.

The Eluana Englaro Case: cause of death after the withdrawal of artificial nutrition and hydration in a subject in a permanent vegetative state and with quadriplegia.

A 38-year-old woman, who had been in a permanent vegetative state with quadriplegia for 17 years following a motor vehicle accident, died 87 h after the judicially authorised suspension of artificial nutrition and hydration. Medico-legal investigations, requested by the Judicial Authorities and focusing on the evaluation of clinical and necroscopic data, aimed to explain the cause of death, to exclude any lethal cause other than one deriving from the withdrawal of artificial nutrition and hydration and to verify the level of perceived discomfort and the correctness in the application of the supportive protocol during the end of life phase. The inability of quadriplegic patients to compensate critical hyperthermic and haemodynamic disturbances induced by dehydration was considered to be the cause of a rapid demise after the withdrawal of artificial sustenance.

Effects of A2A adenosine receptor blockade or stimulation on alcohol intake in alcohol-preferring rats.

RATIONALE: A(2A) adenosine receptors (A(2A)ARs) have been proposed to be involved in drug addiction; however, preclinical studies about the effects of A(2A)AR ligands on alcohol consumption have provided inconsistent results. OBJECTIVES: The present study evaluated the effect of intraperitoneal injections of the A(2A)AR antagonist ANR 94, and the A(2A)AR agonists CGS 21680 and VT 7 on voluntary drinking and operant self-administration of 10% ethanol in Marchigian Sardinian alcohol-preferring (msP) rats. RESULTS: Voluntary ethanol drinking was increased by ANR 94 in acute and subchronic experiments, while it was reduced by A(2A)AR agonists. The effect of CGS 21680 was abolished by a low dose of ANR 94, confirming its mediation by A(2A)ARs. Ethanol self-administration was reduced by CGS 21680 and VT 7, while ANR 94 slightly but significantly increased it. Blood alcohol levels were not modified by A(2A)AR agonists, indicating that their effect is not related to ethanol pharmacokinetics. The effect of VT 7 on ethanol drinking was behaviourally selective; ethanol and food intake were reduced, but water intake was increased, and total fluid intake was not different from that of controls. Moreover, VT 7 did not affect locomotor activity. CGS 21680 (0.1 mg/kg) did not modify total fluid intake, but 0.2 and 0.3 mg/kg reduced total fluid intake and locomotor activity. CONCLUSION: These results provide evidence that A(2A)AR agonists reduce ethanol consumption in msP rats, which represent an animal model of alcohol abuse related to stress, anxiety and depression. A(2A)ARs may represent a potential target for treatment of alcohol abuse.

[Antonio Filippo Ciucci: aspects of forensic toxicology]. / Antonio Filippo Ciucci: profili di tossicologia forense.

The Authors have studied the aspects of forensic toxicology contained in the book "II Filo di Arianna" (Ariadne's thread) written by Antonio Filippo Ciucci, and published in 1682. This book is considered the first treatise of legal medicine written in vulgar (ancient italian). The Ciucci's book contains many elements of originality, in particular for what concerns the poisoning diagnosis, the role of the institutional actors of sanitary body of this historical period, and their activity of export.

Blood cyanide determination in two cases of fatal intoxication: comparison between headspace gas chromatography and a spectrophotometric method.

Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC/NPD). In the former, hydrogen cyanide (HCN) was quantitatively determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC/NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC/NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5-10 and 0.05-5 mug/mL, respectively. Detection limits were 0.2 mug/mL for VIS and 0.05 mug/mL for HS-GC/NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases.