Giulio Mannocchi

Recreational Use, Analysis and Toxicity of Tryptamines

The definition New psychoactive substances (NPS) refers to emerging drugs whose chemical structures are similar to other psychoactive compounds but not identical, representing a “legal” alternative to internationally controlled drugs. There are many categories of NPS, such as synthetic cannabinoids, synthetic cathinones, phenylethylamines, piperazines, ketamine derivatives and tryptamines. Tryptamines are naturally occurring compounds, which can derive from the amino acid tryptophan by several biosynthetic pathways: their structure is a combination of a benzene ring and a pyrrole ring, with the addition of a 2-carbon side chain. Tryptamines include serotonin and melatonin as well as other compounds known for their hallucinogenic properties, such as psilocybin in ‘Magic mushrooms’ and dimethyltryptamine (DMT) in Ayahuasca brews. Aim: To review the scientific literature regarding tryptamines and their derivatives, providing a summary of all the available information about the structure of these compounds, their effects in relationship with the routes of administration, their pharmacology and toxicity, including articles reporting cases of death related to intake of these substances. Methods: A comprehensive review of the published scientific literature was performed, using also non peer-reviewed information sources, such as books, government publications and drug user web fora. Conclusions: Information from Internet and from published scientific literature, organized in the way we proposed in this review, provides an effective tool for specialists facing the emerging NPS threat to public health and public security, including the personnel working in Emergency Department.

Hepatotoxicity Induced by “the 3Ks”: Kava, Kratom and Khat

The 3Ks (kava, kratom and khat) are herbals that can potentially induce liver injuries. On the one hand, growing controversial data have been reported about the hepatotoxicity of kratom, while, on the other hand, even though kava and khat hepatotoxicity has been investigated, the hepatotoxic effects are still not clear. Chronic recreational use of kratom has been associated with rare instances of acute liver injury. Several studies and case reports have suggested that khat is hepatotoxic, leading to deranged liver enzymes and also histopathological evidence of acute hepatocellular degeneration. Numerous reports of severe hepatotoxicity potentially induced by kava have also been highlighted, both in the USA and Europe. The aim of this review is to focus on the different patterns and the mechanisms of hepatotoxicity induced by “the 3Ks”, while trying to clarify the numerous aspects that still need to be addressed.

Fatal self administration of tramadol and propofol: A case report

We describe a case of unintentional intoxication due to tramadol and propofol self administration, occurred in a middle aged man, healthcare provider, deceased despite advanced medical assistance an hour later the onset of severe and increasing dyspnea. Toxicological analysis performed with gas chromatography-mass spectrometry in blood sample, evidenced a lethal tramadol concentration and therapeutic level of Propofol. Quantitative determination was also performed in other specimens such as bile, tissues (liver, spleen, kidney) and pubic hair, to assess chronic exposure. Toxicological results and autopsy findings, supported by clinical and hematochemical data, suggested a myocardial damage, associated with respiratory failure.

Assessment of the stability of mephedrone in ante-mortem and post-mortem blood specimens

AIMSnThe aim of this work is to test the stability of mephedrone added to whole blood collected from alive and dead mephedrone free-users and stored at three different temperatures (-20, +4 and +20°C) with and without preservatives up to 6 months, trying to establish the best storage condition in order to reduce possible analyte loss/degradation during the storage period.nnnMATERIALS AND METHODSnDifferent sources of blood were obtained as follow: 10 samples of blood came from 10 alive mephedrone free-users (mean age 34±15.8 years old) (Group 1), whereas 10 post mortem blood samples were obtained from 10 cadavers, in which the post mortem interval was between 24 and 36h (Group 2). The cause of death in post mortem cases (mean age 45±14.2 years old) was not drug related. Pools of blood were spiked with mephedrone at the concentration of 1mg/L and 1mL aliquots were transferred in 2mL Eppendorf capped tubes with and without preservatives as follow: with ethylenediaminetetraacetic acid (EDTA) 3%; with sodium fluoride/potassium oxalate (NaF/KOx) 1.67%/0.2%, respectively; without preservatives. All samples were stored at three different temperatures: -20°C, 4°C and 20°C and extracted and analyzed in duplicate by GC-MS according to a previously published method by Dickson et al., every other day during the first month and then weekly up to 6 months.nnnRESULTS AND CONCLUSIONSnour study allow us to affirm that -20°C is the best storage temperature for mephedrone stability in ante-mortem and post-mortem blood samples in comparison to the other two tested temperatures (+4 and +20°C), showing higher values in both groups in samples stored with and without preservatives (p<0.0001). The comparison of Group 1 (samples coming from alive subjects) and Group 2 (post-mortem samples) highlights a better stability of mephedrone in Group 1 (p<0.001) at all tested storage conditions. Finally, the analysis of blood specimens stored with and without preservatives in both groups suggests that specimens stored with NaF/KOx maintain mephedrone stability better than those stored with EDTA (p<0.001) and those stored without preservatives (p<0.0001), therefore, we strongly recommend in order to maintain the highest mephedrone stability in blood, to store specimens at -20°C adding NaF/KOx as preservative.

Determination of GHB levels in breast milk and correlation with blood concentrations.

The sodium salt of GHB or sodium oxybate is approved and registered in some countries as a therapeutic substance (Xyrem(®)) for the treatment of narcolepsy-associated cataplexy. This study was designed to measure the GHB endogenous levels in blood and breast milk of 20 breastfeeding women. In addition, blood and breast milk samples of a 32-year-old narcoleptic nursing mother, who was on sodium oxybate treatment, were simultaneously collected at 0.5, 1, 3, 4 and 5h following a 4.5g GHB dose and analyzed, in order to establish the safety interval of time to breastfeed. A GC-MS method for the detection and quantification of GHB in blood and breast milk was developed and fully validated. The geometric mean of endogenous GHB levels in blood and breast milk detected at time 0 were 0.57mg/L; 95% Reference Interval (RI): 0.21-1.52mg/L and 0.36mg/L; 95% RI: 0.13-1.03mg/L, respectively. The geometric mean of the concentration of GHB in milk was 37% less (95% RI: from 14 to 53%) compared to that found in the blood. The analysis of blood and breast milk samples collected from the 32 years-old female showed the following results: GHB blood concentration 0.5h after medication intake was 80.10mg/L, reaching the peak 1h after the drug administration (108.34mg/L) and it steadily decreased to reach a level of 1.75mg/L, 5h after the medication intake. The GHB concentration found in breast milk followed the same pattern as for the blood, with the highest concentration being 23.19mg/L, 1h after sodium oxybate administration and the lowest 0.99mg/L, 5h after the medications intake. The comparison between blood and breast milk GHB levels in the 32-year-old woman, showed significant lower GHB levels in milk at 0.5, 1 and 3h, ranging from 71 to 80% less. It is interesting to note that only at 4 and 5h the difference between blood and breast milk GHB levels fell within the 95% RI (14-53%) of endogenous levels. Taking into consideration the absence of reference values for endogenous GHB in milk, we suggest the following reference interval: 0.13-1.03mg/L. We would recommend, following these preliminary data, that nursing mothers under sodium oxybate treatment should breastfeed at least 5h after the last GHB administration. However, further studies are necessary in order to confirm these findings.

A Study on the Reliability of an On-Site Oral Fluid Drug Test in a Recreational Context

The reliability of DrugWipe 5A on site test for principal drugs of abuse (cannabis, amphetamines, cocaine, and opiates) detection in oral fluid was assessed by comparing the on-site results with headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS) analysis on samples extracted by the device collection pad. Oral fluid samples were collected at recreational settings (e.g., discos, pubs, and music bars) of Rome metropolitan area. Eighty-three club goers underwent the on-site drug screening test with one device. Independently from the result obtained, a second device was used just to collect another oral fluid sample subsequently extracted and analyzed in the laboratory following HS-SPME procedure, gas chromatographic separation by a capillary column, and MS detection by electron impact ionization. DrugWipe 5A on-site test showed 54 samples (65.1%) positive to one or more drugs of abuse, whereas 75 samples (90.4%) tested positive for one or more substances following GC-MS assay. Comparing the obtained results, the device showed sensitivity, specificity, and accuracy around 80% for amphetamines class. Sensitivity (67 and 50%) was obtained for cocaine and opiates, while both sensitivity and accuracy were unsuccessful (29 and 53%, resp.) for cannabis, underlying the limitation of the device for this latter drug class.

Stability of endogenous GHB in vitreous humor vs peripheral blood in dead bodies

For the first time, the stability of GHB was tested in post-mortem peripheral blood and vitreous humor samples, collected from 22 dead bodies at two different times: at the external body examination at the place of death and then during autopsy. An ad hoc method for the detection and quantification of GHB in vitreous humor by gas chromatography coupled to mass spectrometry (GC-MS) was developed and validated, with a good linearity between 0.1 and 50μg/mL (r2=0.991) and a precision and accuracy always better than 10% and an analytical recovery higher than 90%. The geometric mean of GHB concentration in the 22 peripheral blood samples at t0 was: 3.6μg/mL (95% CI: 2.3-5.9μg/mL) and at t1 it was 7.4μg/mL (95% CI: 5.0-10.9μg/mL); that of GHB in the 22 vitreous humor at t0 was: 2.5μg/mL (95% CI: 1.5-4.1μg/mL) and at t1 it was 3.0μg/mL (95% CI: 1.9-4.8μg/mL). There was no significant difference between the GHB concentrations in vitreous humor and peripheral blood at t0 in all the samples (p>0.10). Conversely at t1, the increase of GHB in the peripheral blood was significantly increased by a 102% (range: 86-120%) (p<0.001 vs t0), while in the vitreous humor only a slight increase by 19% was observed (range: 16-21%) (p>0.05 vs t0). Finally at t1, GHB values in the two matrices were statistically different, being that of peripheral blood higher (p<0.01). This study demonstrated the usefulness of vitreous humor as a more stable alternative matrix in comparison to peripheral blood for the post-mortem determination of endogenous GHB.

Neurotoxicity Induced by Mephedrone: An up-to-date Review

Mephedrone is a β-ketoamphetamine belonging to the family of synthetic cathinones, an emerging class of designer drugs known for their hallucinogenic and psychostimulant properties as well as for their abuse potential. The aim of this review was to examine the emerging scientific literature on the possible mephedrone-induced neurotoxicity, yet not well defined due to the limited number of experimental studies, mainly carried on animal models. Relevant scientific articles were identified from international literature databases (Medline, Scopus, etc.) using the keywords: “Mephedrone”, “4-MMC,” “neurotoxicity,” “neuropharmacology”, “patents”, “monoamine transporters” and “neurochemical effects”. Of the 498 sources initially found, only 36 papers were suitable for the review. Neurotoxic effect of mephedrone on 5-HT and DA systems remains controversial. Although some studies in animal models reported no damage to DA nerve endings in the striatum and no significant changes in brain monoamine levels, some others suggested a rapid reduction in 5-HT and DA transporter function. Persistent serotonergic deficits were observed after binge like treatment in a warm environment and in both serotonergic and dopaminergic nerve endings at high ambient temperature. Oxidative stress cytotoxicity and an increase in frontal cortex lipid peroxidation were also reported. In vitro cytotoxic properties were also observed, suggesting that mephedrone may act as a reductant agent and can also determine changes in mitochondrial respiration. However, due to the differences in the design of the experiments, including temperature and animal model used, the results are difficult to compare. Further studies on toxicology and pharmacology of mephedrone are therefore necessary to establish an appropriate treatment for substance abuse and eventual consequences for public health.

Development and Validation of a GC-MS Method for the Detection and Quantification of Clotiapine in Blood and Urine Specimens and Application to a Postmortem Case.

Introduction. Clotiapine is an atypical antipsychotic of the dibenzothiazepine class introduced in a few European countries since 1970, efficient in treatment-resistant schizophrenic patients. There is little published data on the therapeutic and toxic concentrations of this drug. Aims. The aim of the present study is the development and validation of a method that allows the detection and quantification of clotiapine in blood and urine specimens by gas chromatography-mass spectrometry (GC-MS). Methods. Validation was performed working on spiked postmortem blood and urine samples. Samples were extracted with liquid-liquid extraction (LLE) technique at pH 8.5 with n-hexane/dichloromethane (85/15 v/v) and analysis was followed by GC-MS. Methadone-d9 was used as internal standard. Results. The limit of detection (LOD) was 1.2 and 1.3u2009ng/mL for urine and blood, respectively, while the lower limit of quantification (LLOQ) was 3.9 and 4.3u2009ng/mL, respectively. Linearity, precision, selectivity, accuracy, and recovery were also determined. The method was applied to a postmortem case. The blood and urine clotiapine concentrations were 1.32 and 0.49u2009μg/mL, respectively. Conclusions. A reliable GC-MS method for the detection and quantification of clotiapine in blood and urine samples has been developed and fully validated and then applied to a postmortem case.

A Very Unusual Accidental Mechanical Asphyxia of Choking with a Whole Solea Solea

The case here reported involves a schizophrenic 19‐year‐old girl under treatment with clotiapine, which was well tolerated except for a moderate dry mouth. The woman ingested a whole sole (Solea solea), which caused a very rapid death by choking. A complete autopsy was performed 24 h later, as well as histological and toxicological analysis. At autopsy, the sole was wedged in the esophagus causing a choking ab extrinseco. The fish had a length of 18 cm and a maximum width of 6 cm, weighing 188.7 g. Toxicological analysis detected 0.57 mg/L of clotiapine in blood, which falls within the therapeutic range. The peculiarity of this case is represented by two factors: one is the choking by fish and the second was the adverse affect caused by clotiapine, which induced a condition of dry mouth making the act of swallowing even more difficult, thereby contributing to a very rapid mechanical asphyxia and the death of the young woman.