Study of metabolism and potential toxicity of nine synthetic opioid analogs using the zebrafish larvae model.

The use of novel psychoactive substances (NPSs) has dramatically increased worldwide, and among them, synthetic opioids are one of the fastest growing groups, where cinnamylpiperazines and 2-benzylbenzimidazoles represent two of the most relevant subclasses. However, the data on their toxicity and metabolism are still limited. The aim of the present study was to evaluate the toxicity and metabolic pathways of some compounds belonging to these families, namely, AP-237, 2-methyl AP-237, isotonitazene, flunitazene, etodesnitazene, metonitazene, metodesnitazene, N-pyrrolidino etonitazene, and butonitazene. The study was performed using a zebrafish early life stages model. In fact, zebrafish (Danio rerio) embryos and larvae have recently been recognized as a suitable animal model in alternative to mammals, because they require less time and resources and do not need complex procedures for ethics approval. The cellular toxicity after a single administration was assessed at the fourth day post-fertilization with acridine orange staining. Possible morphological defects were evaluated with a light microscope after 24 h of exposure to 1 µmol/L concentration of each drug. Subsequently, the larvae were euthanized and underwent analysis of drug metabolites using UPLC coupled to an Orbitrap high-resolution mass spectrometer. High rates of morphological defects, as well as of cellular death, were detected, but no significant difference in mortality between treatment and control groups was observed. In addition, several metabolites, mainly produced through monohydroxylation, N-dealkylation, and O-dealkylation, were identified in the larvae extracts.

Hair testing applied to the assessment of in utero exposure to drugs: Critical analysis of 51 cases of the University Hospital of Verona.

The work discusses the results of hair and urine testing performed in 51 cases of suspected in utero drug exposure handled at the University Hospital of Verona from 2016 to 2022. On the day of birth or the day after birth, urine from mother and newborn (UM and UN) and hair from mother (HM), newborn (HN) and father (HF), if possible, were collected. Urine underwent immunoassay and GC-MS analysis, whereas hair underwent LC-MS/MS and GC-MS/MS analysis. In 50 out of 51 cases, HM and/or HN were available. In 92% of them, hair testing resulted in a positive, often (>50% cases) for more than one class of substance. The most detected substances were cocaine, opiates, methadone and cannabinoids. Maternal segmental analysis showed a prevalent decreasing concentration trend during pregnancy in case of positivity for one class of substances, whereas, as expected, a neatly prevalent increasing trend in the case of positivity for more than one class of substances. In nine cases, HF was also available, resulting in all being positive, usually for the same classes of substances identified in HM, thus questioning parental responsibility. In 33 cases, urine samples from the mother or newborn were also collected. Of them, 27 cases (82%) tested positive, showing peri-partum drug consumption and then confirming the severity of the addiction. Hair testing showed to be a reliable diagnostic tool to investigate in utero drug exposure because of the possibility of obtaining a complete picture of maternal addictive behaviour and family background, thanks to segmental maternal hair analysis and father hair testing.

Development and Validation of a Rapid Method for Identification of New Synthetic Cannabinoids in Hair Based on High-Performance Liquid Chromatography-Ion Trap Mass Spectrometry Using a Simplified User Interface.

The penetration of the new psychoactive substances (NPSs) into the market of clandestine drugs is highly dynamic, causing potentially false-negative results using the current analytical instrumentation, particularly in the screening phase. At present, the suggested approach to perform a comprehensive screening requires the use of high-resolution mass spectrometry (MS) with associated high costs of purchase and maintenance and need of skilled and dedicated personnel. Here we describe the development and validation of a simplified approach based on a high-performance liquid chromatography-ion trap MS system with a user-friendly interface dedicated to toxicological analysis. The system, originally intended for a broad toxicological screening, was tuned to identify new synthetic cannabinoids in hair. After a washing step with dichloromethane, hair (about 50 mg) was incubated for 3 h with 1.5 mL ethanol. One milliliter of this solution was then dried, reconstituted with mobile phase and injected. The peak identification was based on the chromatographic retention times and MS2/MS3 data using a database which included up to 158 NPSs. The method was validated according to international guidelines on a selected panel of NPSs, namely methyl 2-[[1-(5-fluoropentyl)indazole-3-carbonyl]amino]-3,3-dimethylbutanoate (5F-ADB), 1-Pentyfluoro-1H-indole-3-carboxylic acid 8-quinolinyl ester (5F-PB 22), N-[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]-1-(5-chloropentyl)indazole-3-carboxamide (5Cl-AB-PINACA), (S)-N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide [5F-APP-PICA (PX-1)],: (R)-N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide [5F-APP-PINACA (PX-2)], N-[(2S)-1-Amino-3-methyl-1-oxobutan-2-yl]-1-(cyclohexylmethyl)indazole-3-carboxamide (AB-CHMINACA), N-[(2S)-1-Amino-3-methyl-1-oxobutan-2-yl]-1-[(4-fluorophenyl)methyl] indazole-3-carboxamid (AB-FUBINACA), methyl (2S)-2-[[1-(cyclohexylmethyl)indole-3-carbonyl]amino]-3,3-dimethylbutanoate (MDMB-CHMICA), (S)-Methyl 2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3-methylbutanoate (MMB-2201) and (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144). The tested analytical method showed detection limits between 0.065 and 0.125 ng/mg. The intraday imprecision of the method showed average values within the range of 7.3-20%. The estimation of the trueness (bias) of method showed average values within the range of 1.5-12.3%. The analytical performance was also successfully assessed by four proficiency test samples containing NPS. No synthetic cannabinoids were detected in application to 82 hair samples from forensic cases previously analyzed with liquid chromatography-MS triple quadrupole.

CDT vs. GGT for the certification of the fitness to hold the driving license. A comparison based on the association of incremented values with the occurrence of alcohol-related road traffic accidents.

BACKGROUND: In the context of fitness certification to hold the driving license, GGT and CDT have been used, sometimes in combination (γ-CDT), to exclude chronic alcohol abuse. The present study was carried out with the aim of comparing the power of these biomarkers as tools for the objective screening of subjects at high risk of alcohol-associated traffic injuries. METHODS: 288 male drivers admitted to hospital after traffic accidents were examined by determination of GGT, CDT and BAC. The degree of association of GGT, CDT and γ-CDT with BAC was analysed using non-parametric statistics. RESULTS: Partitioning the cases using the cut-off concentrations of 0.5 g/L for BAC (the legal limit adopted in most European countries), 55 U/L for GGT and 1.9% for CDT, a highly significant difference was found between the frequency of elevated GGT or CDT in cases where BAC was within the legal limits and those with elevated BAC values (Fisher's exact test: p < 0.001). However, the calculation of the odds ratio showed a much higher increase for CDT (28 times) than for GGT (6 times) in those drivers with a BAC above the Italian legal limit in comparison with those showing a BAC within the cut-off; conversely, γ-CDT does not provide any significant advantage vs. CDT alone. CONCLUSIONS: Both GGT and CDT provide objective evidence of an association with the occurrence of alcohol-related severe traffic accidents, but CDT shows superior association with these events. Therefore, CDT, notwithstanding higher costs, should be preferred in a forensic/certification context.

Fatal, Intentional Overdose of Ranolazine: Post-Mortem Distribution of Parent Drug and its Major Metabolite.

PURPOSE: Ranolazine is a selective inhibitor of the late inward sodium-current, approved for the treatment of chronic angina. Here, we report a case of a possibly suicidal death due to acute ranolazine overdosing. A 41-year-old woman was found unconscious by her son and was urgently admitted to the Intensive Care Unit. She had ingested an unknown amount of ranolazine tablets. Seventeen hours after admission, the patient died. An autopsy was performed 4 days post-mortem. METHODS: A routine screening analysis for drugs of abuse and medicinal drugs performed by liquid chromatography ion trap mass spectrometry on autopsy samples of biological fluids did not detect any relevant presence of toxicologically relevant compounds, but ranolazine. A quantitative analysis was then carried out by liquid chromatography- QqQ mass spectrometry in order to quantify ranolazine and its major metabolite O-desmethyl-ranolazine in biological fluids and organs. RESULTS: Ranolazine concentrations in biological fluids were as follows: cardiac blood, 19.5 µg/mL; femoral blood, 12.3 µg/mL; bile, 0.87 µg/mL and vitreous humor, 15.4 µg/mL. For O-desmethyl-ranolazine the concentrations in cardiac blood, femoral blood, bile and vitreous were 10.7 µg/mL; 9.6 µg/mL; 11,103 µg/mL and 11.4 µg/mL, respectively. CONCLUSIONS: The cause of death was attributed to ranolazine overdosing. To the best of our knowledge, this is the first report of a fatality associated with ranolazine, in which the postmortem distribution of ranolazine and its metabolite has been quantitatively assessed. The present study can therefore provide useful information for interpretation of the causes and mechanisms of death in ranolazine associated fatalities.

Drug screening by using the Toxtyper™ LC-ion trap MS: Optimization of its application on serum samples in a DUID context.

The toxicological approach for monitoring Driving Under Influence of Drugs (DUID) requires analytical techniques with a broad spectrum of identification coupled to a high analytical sensitivity. In this context immunological methods are generally used, while GC or LC-MS are applied for the confirmation step. A different approach for drug screening is represented by the Toxtyper™ instrumentation, an LC-MS platform equipped with a high-speed ion trap mass analyzer, provided with ready-to-use protocols and a database of as many as 4500 therapeutic, toxic/illicit drugs and metabolites. The aim of the present work was to verify its performances in real conditions of drug screening of human serum in the context of DUID. To test and compare its analytical performances, four pooled serum samples were fortified with a selected panel of 47 drugs and metabolites. The agreement between the results from the ToxtyperTM and from the confirmatory techniques currently in use at the University of Verona (GC and LC-MS) was investigated by analyzing 90 real samples chosen from those routinely analyzed. The present study highlights the suitability of the ToxtyperTM for drug screening in serum with a sensitivity compatible with the needs of the DUID for all the tested compounds, with the only exception of cannabinoids.