Methyl (S)-2-(1-7 (5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (5F-MDMB-PICA) intoxication in a child with identification of two new metabolites (ultra-high-performance liquid chromatography-tandem mass spectrometry).

PURPOSE: Methyl (S)-2-(1-7 (5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (5F-MDMA-PICA) intoxication in 1.5-year-old child was presented, together with diagnostic parameters discussion and 5F-MDMB-PICA determination in biological material. Furthermore, 5F-MDMB-PICA metabolites were identified in a urine sample as markers of exposure in situation when a parent compound is not present in specimens. METHODS: Drugs and metabolites were extracted from serum and urine with ethyl acetate both under alkaline (pH 9) and acidic (pH 3) conditions. Hair, after decontamination and pulverization, were incubated with methanol (16 h, 60 °C). The analysis was carried out using ultra-high-performance liquid chromatography-tandem mass spectrometry. For the identification of 5F-MDMB-PICA metabolites, an urine sample was precipitated with cold acetonitrile. Analysis was performed using ultra-high-performance liquid chromatograph with quadrupole time-of-flight mass spectrometer. RESULTS: 5F-MDMB-PICA was determined only in serum sample at concentration of 298 ng/mL. After 1 year, when analysis was repeated, concentration of synthetic cannabinoid in the same sample was only 17.6 ng/mL which revealed high instability of 5F-MDMB-PICA in serum sample. Eight 5F-MDMB-PICA metabolites were identified in urine sample, including two potentially new ones with m/z 391.18964 and m/z 275.14016. CONCLUSIONS: Toxicological analysis confirmed a 1.5-year-old boy intoxication with 5F-MDMB-PICA. Besides the parent drug, metabolites of 5F-MDMB-PICA were identified, including two potentially new ones, together with possible metabolic reactions which they resulted from. Metabolites determination could serve as a marker of 5F-MDMB-PICA exposure when no parent drug is present in biological material.

A fatal case involving the highest ever reported 4-CMC concentration.

Synthetic cathinones comprise a large amount of substances present on the dark market, which creates an undeniably worldwide problem and still is posing a threat. A 22-year-old man was brought to the Emergency Room from a party, where he had ingested orally 20 g of mephedrone. The man exhibited a disorder of consciousness with no logical verbal contact and dilated pupils. Moreover, a metabolic acidosis was present. The patient died after an hour from an admission to the ER. Blood and vitreous humor collected during an autopsy were analyzed with the use of an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-QqQ-MS/MS) with the use of C18 column in multiple reaction monitoring (MRM) mode. Both biological specimens were prepared using liquid-liquid extraction (LLE) with the use of ethyl acetate and 0.5 M ammonium carbonate water solution (pH 9). The limit of quantification (LOQ) of the method was 0.5 ng/ml in both matrices; precision and accuracy values did not exceed ±15%. Recovery of the method was in the range of 86.1%-102.7%. Determined concentrations of 4-CMC were 8542 and 9874 ng/ml in blood and vitreous humor, respectively. Other substances present in both biological materials were: atropine, diazepam, lidocaine, and its metabolite norlidocaine, as well as methcathinone and ethyl alcohol. The concentration presented in here described case is the highest ever reported 4-CMC concentration. Important aspect is also receiving other NPS by recreational users than intended, which lead to accidental poisoning (in presented case user assumed 4-CMC was 4-MMC).

An ultra-sensitive UHPLC-QqQ-MS/MS method for determination of 54 benzodiazepines (pharmaceutical drugs, NPS and metabolites) and z-drugs in biological samples.

Benzodiazepines exhibit central nervous system depressive activity as well as sedative, hypnotic, and anticonvulsant properties, which enable to use them as medical treatment in anxiety, epilepsy, insomnia and alcohol withdrawal syndrome. However, from 2000s illegal benzodiazepine derivatives have started to emerge on illicit drug market as new psychoactive substances (NPSs) monitored in many countries. Analysis of both pharmaceutical drugs and NPSs from benzodiazepines group could be challenging, as usually very low concentrations need to be determined. Thus, an ultra-sensitive UHPLC-QqQ-MS/MS method was developed for simultaneous determination of 54 benzodiazepines (pharmaceutical drugs, NPS and their metabolites) and 3 z-drugs with one metabolite in biological fluid samples. The lower limit of quantification for most substances was 50 pg/mL, whereas for 17 substances as low as 10 pg/mL was achieved. Together with reduced sample volume to 100 µL it makes the developed method suitable for a sensitive multidrug toxicological analysis. Presented method was applied in routine toxicological practice as well as for the determination of benzodiazepines, z-drugs and their metabolites in 25 authentic biological fluids (blood, urine, vitreous humor and bile), both antemortem and postmortem. 19 different compounds, including benzodiazepines, their metabolites and z-drugs were determined. Antemortem blood concentrations were within 0.2-114.5 ng/mL, whereas concentrations in antemortem urine samples were 0.03-102.6 ng/mL. In postmortem specimens, concentrations ranged within 0.2-473.2 ng/mL, 0.5-94.1 ng/mL, 1.3-208.8 ng/mL and 41.5-42.0 ng/mL in blood, vitreous humor, urine and bile, respectively. The developed method is suitable for a forensic toxicology analysis, as well as clinical toxicology which is evidenced by the positive results of international proficiency tests.

A Case of Amphetamine and Methamphetamine Intoxication in Cat.

Stimulants belonging to the amphetamine group nowadays pose an undeniable worldwide threat to the life and health of users. Intoxications of domestic animals also occur, which can either be accidental or related to intentional human action. This study presents the first ever reported case of a simultaneous amphetamine and methamphetamine intoxication of a cat, along with the results of toxicological studies. Blood, urine, vitreous humor and liver were collected during the cat's autopsy and analyzed by UHPLC─QqQ─MS/MS. The sample preparation technique was based on one-step precipitation of proteins with cold acetonitrile. The determined amphetamine concentrations in the collected biological materials were 93.4 ng/mL in blood, 496.6 ng/mL in urine, 589.2 ng/mL in the vitreous humor and 291.2 ng/g in liver, respectively. Methamphetamine concentrations were 45.5 ng/mL in blood, 263.1 ng/mL in urine, 351.2 ng/mL in vitreous humor, and 97.7 ng/g in liver. Other substances were also found in the biological material, i.e., diazepam, oxazepam and nordiazepam. Cases of intentional or accidental poisoning of pets with psychoactive substances are a serious problem, carrying the risk to the health and life of the animal. Therefore, it is important to increase awareness of the high risk of poisoning of domestic animals, as well as to learn about the incompletely understood mechanisms of pharmacokinetics of various drugs in animals, including cats.

The dark side of social media: Two deaths related with chloroform intoxication.

A suicide pact is an agreement between people to commit suicide together, which usually takes place at the same time, in the same place, by using the same method. Social media serve as a way of communication between people. Thus, they use such platforms to find potential suicide pact partners. Chloroform, although being regarded to as a slightly forgotten poison, is still linked to homicide and suicide cases. Death due to an acute chloroform ingestion may be a result of central nervous system depression. In this paper, we present application of headspace gas chromatographic method using a dual column/dual flame ionization detector (HS-GC-FID/FID) for the determination of chloroform in two fatal intoxication cases, as well as chloroform stability study. Analysis of biological samples revealed chloroform concentrations of 135.8, 16.1, 8.1, and 37.1 µg/ml in blood, urine, vitreous humor, and bile, respectively. Kidney, liver, and muscle specimens contained 119.5, 99.6, and 28.4 µg/g of chloroform, respectively. The results of stability studies indicate the highest decrease of chloroform in room temperature, so it is advised to store samples in a freezer. The addition of sodium fluoride is recommended as in blood samples collected to the test tubes without any preservative agent, the detection of chloroform after 91 days is almost impossible. It is important to emphasize that even old poisons can cause a lot of concerns today, as here described cases are linked to chloroform intoxication, as well as with possible danger which social media bring about nowadays.

Simultaneous poisoning of 48 birds of prey - bendiocarb determination with the use of UHPLC-ESI-MS/MS method in fatal case from Eastern Europe.

Aim: Bendiocarb is used against a wide range of insects but has already been withdrawn from the market in some countries. It poses a high risk to birds as they can accidentally ingest it while searching for food, followed by toxic effects. This paper presents the results of toxicological and histopathological studies of 48 cases of intentional birds of prey poisoning with bendiocarb in Eastern Europe, specifically Poland. Material and methods: A novel ultra-high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for bendiocarb determination in animal liver samples was developed and fully validated. The sample preparation technique was based on one-step precipitation of proteins with cold acetonitrile. The internal standard used was carbaryl-d7. Full time of analysis was less than 10 minutes. The application of the UHPLC-ESI-MS/MS method allowed us to achieve the lowest LOQ (1 ng/g) of bendiocarb in biological samples to date. Results: Necropsies and histopathological examinations of common ravens (Corvus corax), western marsh harriers (Circus aeruginosus), red kites (Milvus milvus), and a white-tailed eagle (Haliaeetus albicilla) revealed multi-organ toxicity manifested as congestion, oedema, or stagnation of blood. An analytical investigation confirmed the presence of bendiocarb in liver in the 1808-7721 ng/g range. Furthermore, the presence of this compound was qualitatively confirmed in the stomach and beak contents and also in the bait located near the deceased animals. Conclusions: A comprehensive forensic examination is crucial to monitor wildlife fatalities, especially applying a combined analytical and histopathological approach to identify and eliminate highly toxic substances which pose a threat to the ecosystem.

Etazene induces developmental toxicity in vivo Danio rerio and in silico studies of new synthetic opioid derivative.

Synthetic opioids are gaining more and more popularity among recreational users as well as regular abusers. One of such novel psychoactive substance, is etazene, which is the most popular opioid drug in the darknet market nowadays. Due to limited information available concerning its activity, we aimed to characterize its developmental toxicity, including cardiotoxicity with the use of in vivo Danio rerio and in silico tools. Moreover, we aimed, for the first time, to characterize the metabolite of etazene, which could become a potential marker of its use for future forensic analysis. The results of our study proved severe dose-dependent developmental toxicity of etazene (applied concentrations 10-300 µM), including an increase in mortality, developmental malformations, and serious cardiotoxic effects, as compared with well-known and used opioid-morphine (applied concentrations 1-50 mM). In silico findings indicate the high toxic potential of etazene which may lead to drug-drug interactions and accumulation of substances. Furthermore, phase I metabolite of etazene resulting from N-dealkylation reaction was identified, and therefore it should be considered as a target for toxicological screening. Nonetheless, the exact mechanism of observed effects in response to etazene should be further examined.

CE-123, a novel dopamine transporter inhibitor, attenuates locomotor hyperactivity and improves cognitive functions in rat model of fetal alcohol spectrum disorders.

Perinatal alcohol exposure can lead to fetal alcohol spectrum disorders (FASD), usually first diagnosed in childhood, that are characterized by hyperactivity, impulsivity and learning and memory disability, among others. To test the hypothesis that dopamine signaling is one of the main factors underlying these impairments, a new atypical dopamine transporter (DAT) inhibitor, CE-123 (1, 3 or 10 mg/kg) was assessed for its potential to overcome the ethanol-induced behavioral effects in a rat model of FASD. In the present study, neonatal rats were exposed to alcohol intubations across the neonatal period (postnatal day (PND)4-9, the third trimester equivalent of human gestation) and, after weaning, the animals (male rats) were assigned randomly to three groups. The first group was tested at PND21 (hyperactivity test). A second group was tested at PND45 (anxiety test), at PND47 (locomotor activity test), at PND49 (spatial cognitive test in the Barnes maze) and PND50 (reversal learning in the Barnes maze). The third group was tested at PND50 (dopamine receptor mRNA expression). Our results support the hypothesis that dopamine signaling is associated with FASD because the dopamine (D1, D2 and D5) receptor mRNA expression was altered in the striatum, hippocampus and prefrontal cortex in adult rats exposed to ethanol during neonatal period. CE-123 (3 and 10 mg/kg) inhibited the hyperactivity and ameliorated (10 mg/kg) the impairment of reversal learning in alcohol-exposed rats. Thus, these findings provide support that CE-123 may be a useful intervention for same of the deficits associated with neonatal ethanol exposure.

Old Poison, New Problem: Cyanide Fatal Intoxications Associated with Internet Shopping.

Widespread access to the Internet has an increasing influence on how suicides are committed. On websites such as eBay® or Amazon.com® highly toxic substances including cyanides are available for purchase. In the last 5 years, a few fatal intoxications associated with Internet shopping and buying "suicide kits" have been reported. Epidemiology of intoxications reported by American Association of Poison Control Centers between 2000-2018 shows that about 10% of all exposures to cyanide were related to suicide attempts and intentional ingestion of this substance. In order to determine the cyanide concentration in four fatal intoxication cases associated with Internet shopping, a headspace gas chromatography with dual column/dual flame ionization detector (HS-GC-FID/FID) method was validated and applied to casework. The method was linear in range, from 1 to 50 µg/mL, with a coefficient of determination of 0.999 (R2). The limit of quantification was 1.0 µg/mL; the detection limit was 0.5 µg/mL. Intra- and inter-day validation precision and accuracy did not exceed 10% and 15%, respectively. Recovery and matrix effect values ranged from 94.8- 103.8% and -5.2─3.8%, respectively. The cyanide concentrations were determined in biological fluids (blood, urine, bile, vitreous humor, gastric content) and postmortem tissue samples (spleen, kidney, liver, brain). The headspace gas chromatographic method, which is routinely used in clinical and forensic toxicology to quantify ethanol with its congeners (methanol, acetone, isopropanol, n-propanol and n-butanol), can be also applied to determine cyanide in intoxication cases. The global problem of a high number of suicides each year, requires increasing and more restrictive control of highly toxic substances available online as well as caution monitoring of human exposure to cyanide. This old and well known poison is being increasingly used nowadays for suicidal purposes, therefore determination of cyanide in biological samples is still important in terms of clinical and forensic toxicology.

Rapid Determination of Sufentanil in Human Plasma by UHPLC-QqQ-MS-MS.

This paper presents a rapid, sensitive and precise method developed and validated for the quantification of sufentanil in biological samples using ultra-performance liquid chromatography coupled with QqQ-MS-MS. Plasma samples were extracted with simple and fast liquid-liquid extraction (ethyl acetate, pH 9). Calibration curve showed linearity in the concentration range of 0.005-30 µg/L. The lower limit of quantification was 0.010 µg/L. The most important method features are low lower limit of quantification value, simple plasma extraction and small sample volume. This method is suitable not only for evaluation of the pharmacokinetics, toxicology, bioavailability and clinical pharmacology of sufentanil but also for the detection and identification of this compound in human plasma samples for forensic purposes.