Toxicological Analysis of Illicit Drugs Seized in Naples (Italy) and First Detection of Synthetic Cannabinoids during COVID-19 Pandemic.

The COVID-19 pandemic has consistently raised the number of drug seizures, in United States as in Europe. The COVID-19 pandemic has also changed the typology of seizures from "more traditional" drugs to New Psychoactive Substances (NPSs), depending on geographical area. In Europe, the most frequent NPSs are synthetic cannabinoids (SCs) and cathinones, nonetheless synthetic opioids and phenethylamines are widely used. The aim of the study is the detection of NPS and other substances of abuse available in the black market, by quali/quantitative methods in LC-MS/MS and GC-MS. From 2018 to 2021, 268 seizures occurred in total and were analyzed by the Forensic Toxicology Laboratory (FTL) of Naples (Italy). The distribution of analyzed seizures over the years is the following: 53 in 2018, 61 in 2019, 89 in 2020 and 65 during the first semester of 2021. Cannabis was the most detected drug both in hashish and marijuana seizures, followed by cocaine > heroine > prescribed drugs > ketamine−amphetamine MDMA. No NPSs were seized until June 2021, when NPSs were found in two different seizures: Case #1 showed a bar of Cannabis resin containing a low level of Δ9THC = 0.57% associated to SC AB-FUBINACA in 4.5%; Case #2 showed a vegetal resinous substance (Δ9THC = 0.27%) with SC 5F-APINACA (4.1%) associated with methadone (1.0%). The detection of NPSs is alarming evidence that can lead to an increase in the risk of overdose or other negative and unpredictable consequences, such as violent or self-harming behavior in unaware users of cannabis derivatives considered of "natural" origin.

Helium Suicide, a Rapid and Painless Asphyxia: Toxicological Findings.

Suicide by helium inhalation has become increasingly common in the last few decades in Europe and the US because it produces a quick and painless death. Inhaled-gas suicides can easily be assessed through death scene investigation and autopsy. However, helium is a colorless and odorless inert gas that unfortunately cannot be detected using standard toxicological analysis. A successful gas analysis was performed following the suicide of a 17-year-old female. For the detection of helium, central/peripheral blood samples and gaseous samples from the esophagus, stomach, and upper and lower respiratory airways (from the trachea and the primary left and right bronchia) were collected with a gastight syringe, ensuring minimal dilution. Qualitative analyses were positive in all gaseous samples. Quantitative analyses were performed using a special gas-inlet system with a vacuum by which the sample can be transferred to a mass spectrometer, reducing the risk of contamination. Helium concentrations were 20.16% from the trachea, 12.33% from the right lung, and 1.5% from the stomach. Based on the high levels of helium, the cause and manner of death were assessed as asphyxia suicide by inhalation of helium. Therefore, toxicological analyses should always be applied in order to gain evidence of inhaled gas in gaseous samples.

Long-Term Detection in Hair of Zolpidem, Oxazepam and Flunitrazepam in a Case of Drug-Facilitated Sexual Assault.

Drug-facilitated sexual assault (DFSA) cases are pretty common in forensic toxicology. In this case report, a 56-year-old female tourist claimed to have been sexually assaulted by five men after having had a drug-spiked alcoholic drink. Urine samples were collected at 38, 44 and 45 h after the alleged rape. After 7 months, hair strands (28 cm in length) were also sampled to perform the segmental hair testing. The urine samples and decontaminated hair segments were tested for different groups of basic, acidic and neutral substances (γ-hydroxybutyrate or GHB, Z-drugs, barbiturates, benzodiazepines, hypnotics, antipsychotics and drugs of abuse). Gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods were applied for the qualitative and quantitative analyses. Toxicological analyses performed on the urine samples showed inconclusive findings. Zolpidem, flunitrazepam and oxazepam were detected in the hair segments corresponding to the time frame of the alleged assault. The endogenous levels of GHB were detected along the hair shaft. No drugs were detected in the proximal and distal hair segments or in washing solutions. This DFSA case demonstrated that the segmental toxicological analysis of hair, even when performed 7 months after the sexual assault, can provide evidence consistent with a single exposure to psychoactive drugs, at the time of the offense.

Fatal poisoning by ingestion of a self-prepared oleander leaf infusion.

An unusual case of poisoning by the ingestion of oleander leaves is reported. A 71 year old male laboratory technician committed suicide at home in this unusual manner. At the death scene a steel pan and other paraphernalia, used for the extraction of oleandrin and other cardiac glycosides from the leaves of the Nerium oleander plant were found.Toxicological investigations for oleandrin, oleandrigenin, neritaloside, and odoroside were performed by LC-MS/MS on all biological samples (peripheral blood, vitreous humor, urine, liver, gastric contents) and on the yellow infusion found at the death scene.In all samples, toxic levels of oleandrin were detected (blood 37.5 ng/mL, vitreous humor 12.6 ng/mL, urine 83.8 ng/mL, liver 205 ng/mg, gastric content 31.2 µg/mL, infusion 38.5 µg/mL). Qualitative results for oleandrigenin, neritaloside, and odoroside were obtained. Oleandrigenin was present in all tissue samples whereas neritaloside and odoroside were absent in the blood and vitreous humor but present in urine, liver, gastric content, and in the leaf brew.The purpose of this study was the identification of oleandrin and its congener oleandrigenin, detected in the vitreous humor. The blood/vitreous humor ratio was also calculated in order to assess of the likely time interval from ingestion to death. According to the toxicological results death was attributed to fatal arrhythmia due to oleander intoxication. The manner of death was classified as suicide through the ingestion of the infusion.

Fatal inhalation of volcanic gases in three tourists of a geothermal area.

The study reports the environmental, toxicological and histopathological forensic investigations applied on three victims of accidental death (father, mother and son), due to the fall in a volcanic pothole, during the touristic visit of the "Solfatara park", near Naples (Italy). At autopsy greenish skin discolouration was observed and all bodies showed the classical signs of asphyxial deaths, such as cyanosis and hemorrhagic pulmonary edema. Focal micro-hemorrhages were found in the brain at intracranial and subpial levels. The hemogasanalysis and spectrophotometric test on blood for Methemoglobin (MetHb), Carboxyhemoglobin (HbCO) and Sulfhemoglobin (SHb) showed pCO2, SHb and MetHb above the physiological levels. On biological specimens, toxicological analyses performed by GC/MS revealed high concentrations of hydrogen sulfide (H2S) and of thiosulfate (TS), its main metabolite. The monitoring of toxic gases on the death scene showed an unsafe environment, into the pothole, able to cause the sudden loss of consciousness of the victims with subsequent asphyxiation (knockdown effect). In particular, at the bottom of the hole, the maximum levels of H2S and carbon dioxide (CO2) were 2200 ppm and 98% respectively. For the family members, the cause of the death was assessed as acute poisoning by H2S and CO2. The fatalities, happened in quick succession as for a domino effect, were pretty similar to the asphyxial deaths by confined spaces, frequently observed in occupational setting. Fatalities secondary to accidental volcanic gases inhalation, such as H2S and CO2 in geothermal areas, have been already described but often without a forensic approach. To the best of our knowledge this is the first case that reports the accidental poisoning by volcanic gases involving three people, with different caracteristic of age and sex, allowing the correlation between toxicological and pathological results with the true levels of asphyxiating gas, measured on the death scene.

Alcohol and drugs use among drivers injured in road accidents in Campania (Italy): A 8-years retrospective analysis.

A recent update of the Italian Road Traffic Law (RTL 41/2016), established severe penal sanctions when drivers, driving under the influence of alcohol (DUI) or drugs (DUID), are involved in road accident that results in death or injuries. A study was carried out to assess the trends of consumption of alcohol, illicit drugs or pharmaceutical among injured drivers suspected for DUI or DUID from 2009 to 2016 in the region of Campania (Italy). Confirmation toxicological analyses were performed on 780 blood samples and 1017 urine samples collected from 1797 injured drivers. These drivers all tested positive for alcohol or drug use through immunoassay screening applied at hospital emergency units and their biological samples transferred to the Forensic Reference Laboratory (FRL) for confirmation analysis. The GC/HS-FID methodology was used to test Blood Alcohol Concentration (BAC). Qualitative and quantitative analyses for drugs were performed using the GC/MS or LC-MS/MS methodology. The BAC >0.5g/L was confirmed in 91.5% of drivers suspected for DUI cases and in 93% of DUID respectively. In DUI cases, results show an increasing incidence of road accidents involving drivers with BAC above 1.5g/L while at concentrations above 0.8g/L alcohol and drugs are both used. Among the suspected DUID cases, the intake of alcohol in association with drugs has consistently increased over time and positive results on blood samples was confirmed for multiple drugs (20%) or cannabis and cocaine alone (18%) followed by benzodiazepines (6%) and methadone (3.5%) respectively. The majority of injured drivers suspected for DUID (1017 cases) did not authorize blood sampling, therefore only urine was analyzed showing the prevalent use of cannabis, followed by multiple drug>cocaine>benzodiazepines>opiates. Among 1797 drivers, suspected at screening for DUI or DUID, 15.4% of cases (64 blood and 213 urine samples) were not confirmed by GC/HS, GC/MS or LC-MS/MS analysis. In forensic toxicological investigations, it is mandatory to satisfy the best quality standards, which is not achievable if immunochemical screening is only performed on urine. Therefore, only confirmed positive results of alcohol or drugs on blood samples can represent conclusive evidence to demonstrate the DUI or DUID related offences. An improvement of the protocols currently applied in Italy for the assessment of DUI or DUID crimes is needed and the confirmation analysis on blood should be considered mandatory.

Ethical Implications in Vaccine Pharmacotherapy for Treatment and Prevention of Drug of Abuse Dependence.

Different immunotherapeutic approaches are in the pipeline for the treatment of drug dependence. "Drug vaccines" aim to induce the immune system to produce antibodies that bind to drugs and prevent them from inducing rewarding effects in the brain. Drugs of abuse currently being tested using these new approaches are opioids, nicotine, cocaine, and methamphetamine. In human clinical trials, "cocaine and nicotine vaccines" have been shown to induce sufficient antibody levels while producing few side effects. Studies in humans, determining how these vaccines interact in combination with their target drug, are underway. However, although vaccines can become a reasonable treatment option for drugs of abuse, there are several disadvantages that must be considered. These include i) great individual variability in the formation of antibodies, ii) the lack of protection against a structurally dissimilar drug that produces the same effects as the drug of choice, and iii) the lack of an effect on the drug desire that may predispose an addict to relapse. In addition, a comprehensive overview of several crucial ethical issues has not yet been widely discussed in order to have not only a biological approach to immunotherapy of addiction. Overall, immunotherapy offers a range of possible treatment options: the pharmacological treatment of addiction, the treatment of overdoses, the prevention of toxicity to the brain or the heart, and the protection of the fetus during pregnancy. So far, the results obtained from a small-scale experiment using vaccines against cocaine and nicotine suggest that a number of important technical challenges still need to be overcome before such vaccines can be approved for clinical use.

Clinical and Forensic Diagnosis of Very Recent Heroin Intake by 6-acetylmorphine Immunoassay Test and LC-MS/MS Analysis in Urine and Blood.

AIM: The study evaluates the suitability of a specific immunoassay screening test for 6-acetylmorphine (6-AM) in the setting of suspected very recent heroin consumption for forensic and clinical purposes. MATERIAL AND METHOD: The EMIT® II Plus 6-AM immunoassay was applied in 65 cases that had already tested positive for morphine in urine or blood. Biological samples (n.65 urine and n.53 blood) were obtained from workplace drug tests (WDT n. 5), tests for driving under the influence of drugs (DUID n. 30), vehicle accidents (n. 10), overdoses (n. 12) and heroin-related deaths (n. 8) cases. The 6-AM screening assay results were confirmed with the LC-MS/MS analysis in relation to the cut-off set at 10 ng/mL for both urine and blood. RESULTS: Among the 65 urine samples (all morphine-positive), 38 samples were 6-AM-positive and 27 were 6-AM-negative with 100% agreement between the positive/negative results of the two assays. Among the 53 blood samples (34 positive and 19 negative for the morphine), 16 were 6-AM positive and 37 were negative. Only one of the blood samples, positive for 6-AM by LC-MS/MS at 10.3 ng/mL, was negative by the immunoassay test. Based on the concordance between the results of the 6-AM immunoassay versus the LC-MS/MS, the sensitivity of the 6-AM assay was calculated as 100% and 95% for urine and blood respectively, with a specificity and accuracy of 100% for both biological samples. In addition, the study demonstrated that the 6-AM assay test, originally developed for urine, is also sufficiently sensitive to identify 6-AM in blood. Therefore, it could be applied in cases of vehicle accidents or overdose to distinguish rapidly between very recent heroin use and the intake of other opiates for therapeutic purposes.

A five years study on drug-related deaths in Campania (Italy).

OBJECTIVES: The study regards n. 267 drug related deaths submitted to toxicological analyses for forensic purpose, since 2008 to 2012, at the Laboratory of Forensic Toxicology of the Second University of Napoli (SUN), Italy. Among the cases studied (94% males and 6% females) the 13.1% regards foreign citizens. METHODS: For each case autopsy findings and all drugs detected were recorded. In addition other epidemiological data such as age, race, gender, place and circumstance of death were also supplied. A systematic toxicological analysis (STA) for illicit drug, other pharmaceuticals, new psychotropic substances and ethanol has been applied on the specimens collected at autopsy and all positive results were confirmed by a quantitative analysis (GC/MS or LC-MS/MS). RESULTS: Toxicological results shows that simultaneous use of multiple illicit drugs was responsible of the largest mortality rate (57.7%), in both males and females. In the polydrug use the cocaine was the most detected substance. The association of heroin/cocaine was identified in the 22.5% of deaths. The finding of only one drug was correlated with an opiate drug for the 14.2% and with cocaine for the 4.5% of cases. The distribution by age and gender demonstrates an increase in deaths among males aged over 30 years and among women older than 35 years. The territorial distribution of the deaths in the different surrounding areas and in the residential quarters of the Naples city demonstrates that the 76% of deaths occurred in a place different from the usual residence. CONCLUSION: Results obtained demonstrate that prevails, at present, the need to specifically explore the changing patterns of polydrug use among addicts, because studying the prevalence of mortality subsequent to the consumption of a single illicit drug may be insufficient to guide preventive policies in public health.