Fatal N-Ethylhexedrone Intoxication.

N-Ethylhexedrone [2-(ethyloamino)-1-phenylhexan-1-one; α-ethylaminohexanophenone (NEH)] is one of the most recent synthetic cathinones that appeared on the illegal market in late 2015. The majority of information concerning the model of consumption of NEH and its impact on the body originates only from self-reports from gray literature websites and drug forums. There are only limited data associated with the concentrations of NEH in blood samples available in the literature. This article presents a case of fatal NEH intoxication and a method for the determination of this substance in whole blood. A 21-year-old man without any diagnosed diseases was admitted to the hospital due to disorientation, aggression and finally loss of consciousness. Hyperthermia (>41°C), tachycardia (>160 beats per minute), tachypnea (20 breaths per minute), blood pressure (110/60 mmHg) and acute kidney failure were diagnosed. After a few hours of hospitalization, the patient died. A plastic bag with a white powder was found in his underwear. Analysis of the powder by another laboratory revealed cocaine hydrochloride; however, no cocaine or its metabolites were found in the biological material upon testing in our laboratory. Therefore, re-analysis of the powder was performed, and NEH was identified. Liquid-liquid extraction followed by liquid chromatography-triple quadrupole-mass spectrometry (LC-MS/MS) analysis were used for the determination of NEH in blood. The validation parameters were as follows: calibration range 1-250 ng/mL, accuracy 106.5-109.9%, precision 3.5-6.3%, recovery 90.1-96.9%, limit of detection 0.07 ng/mL and limit of quantification 1 ng/mL. NEH was quantified in the blood at a concentration of 145 ng/mL. Additionally, amphetamine at low concentrations and 11-nor-9-karboksy-Δ9-tetrahydrokannabinol (THC-COOH) were detected. Our study provided information on the possible lethal concentration and toxidrome that clinicians can observe for NEH-intoxicated patients and can be helpful during the preparation of toxicology analysis reports for a court of law for proper data interpretation.

Rapid and simple multi-analyte LC-MS/MS method for the determination of benzodiazepines and Z-hypnotic drugs in blood samples: Development, validation and application based on three years of toxicological analyses.

Benzodiazepines (BZDs) and Z-drugs have been particularly important treatments for sleeping and anxiety disorders for many years. However, recently, a number of new benzodiazepines (named designer benzodiazepines, DBZDs) were synthesised, but some of them have never been used in the clinic; they reached the black drug market as new psychoactive substances and are used for recreational purposes. The abuse of these substances has led to many crimes and even deaths. Therefore, it is necessary to develop new methods for their quantification for forensic and clinical toxicology. A liquid chromatography-tandem mass spectrometry-based method was developed for the simultaneous determination of 20 classical BZDS, 4 DBZDs and 3 Z-hypnotic drugs in human whole blood. As a sample preparation step, liquid-liquid extraction requiring the use of only 0.5 mL of blood sample and 1 mL of extraction solvent was applied. The selectivity, linearity, carry-over effects, limits of detection (LOD) and quantification (LOQ), precision, accuracy (both intra- and inter-day assays) and recovery were evaluated for validation. Calibration curves were linear with r values > 0.98. The LODs ranged from 0.01 to 0.33, and the LOQs were assumed to be 1 ng/mL. Inter-day precisions and accuracies were in the ranges of 87.8% - 108.5% and 1.8% - 11.2%, respectively. The recovery values ranged from 81.0% to 106.7%. The developed method proved to be sensitive, specific, simple, and fast and can be quickly modified and expanded for new compounds by the optimization of MRM. The method was applied for analysis of blood samples in 145 toxicological cases over a three-year study (2017 - 2019), which allowed us to obtain information on the prevalence of the use of these substances. The most frequently determined compounds were nordazepam (87 cases; 60%), diazepam (81 cases; 55.9%), temazepam (72 cases; 49.7%), oxazepam (56 cases; 38.7%), and midazolam (36 cases; 24.8%). The ranges of concentrations were wide and are presented as box plots. The results were used for the preparation of medico-legal opinions, which proved the utility of the method for routine toxicology analyses.

Blood concentrations of a new psychoactive substance 4-chloromethcathinone (4-CMC) determined in 15 forensic cases.

PURPOSE: The 4-chloromethcathinone (4-CMC) is a synthetic derivative of cathinone and belongs to new psychoactive substances. Neither data on the effects of 4-CMC on the human body, nor on nontoxic, toxic and lethal concentrations in biological materials have been published in the literature. This paper describes the results of an analysis of the blood concentrations of 4-CMC determined in 15 forensic cases related to nonfatal intoxication including driving under the influence, and fatalities including overdoses, suicide and traffic accidents. METHODS: A new method for the quantification of 4-CMC using gas chromatography-mass spectrometry (GC-MS) was developed. The symptoms of 4-CMC use were also studied based on an analysis of the documents prepared during the collection of samples or at autopsies. RESULTS: The limits of detection and quantification of the method for blood samples were 0.3 and 1 ng/mL, respectively. The calibration curve was linear in the studied concentration range (1-500 ng/mL) with the correlation coefficient at 0.9979. The extraction recoveries varied in the range of 94.3-98.8%. The accuracy and precision were acceptable. The determined concentrations in nonfatal cases ranged from 1.3 to 75.3 ng/mL, and in fatalities from 56.2 to 1870 ng/mL. CONCLUSIONS: Our study can assist in the recognition of the possible effects caused by 4-CMC and can be helpful during the preparation of forensic toxicological opinions for courts of law. The validation parameters indicate the sensitivity and accuracy of the method. This is the first work presenting a validated method for the determination of 4-CMC in blood samples by GC-MS.

[Drug addiction in the medico-legal certification of the Department of Forensic Medicine of Medical University of Gdansk in the years 1996-2005]. / Narkomania w swietle dzialalnosci orzeczniczej Katedry i Zakladu Medycyny Sadowej Akademii Medycznej w Gdansku w latach 1996-2005.

The objective of the study was the presentation of the type and extent of drug addiction in the region covered by the Department of Forensic Medicine of the Medical University of Gdansk based on the results of analyses of biological samples collected from the living and deceased subjects during the decade from 1996 to 2005. The studies were based on 110 cases of lethal intoxication after psychoactive substances overdosing and 63 cases of living people involved in the infringement of the law. Body-fluids (blood, urine) and post-mortem samples (stomach contents, small intestine contents, liver, kidney, brain and lung tissues) were collected and stored at -20 OC. The screening analysis of urine was performed using the Fluorescence Polarization Immunoassay method (TDx, Abbott) and the enzyme immunoassay method (Syva Rapid Test, Dade Behring). The confirmation of positive results was carried out by thin-layer chromatography (TLC), as well as gas and liquid chromatography with various detectors (GC/FID, GC/NPD, GC/MS, LC/UV-DAD). In the last 10 years, there was noted a 3-fold increase in the number of cases that required an analysis of narcotics content, and in the last 4 years, such cases exceeded 10% of the total number of investigated cases. The number of analyses aiming at determination of amphetamine and cannabinol contents in living people (mainly among individuals that caused traffic accidents) increased. Among deceased subjects, deaths were predominantly caused by intoxication with opiates or combinations of opiates and other narcotic substances and medications. The presence of both ethanol and drugs was detected and confirmed mainly in post-mortem biological materials.

[Sampling, storage and transport of biological materials collected from living and deceased subjects for determination of concentration levels of ethyl alcohol and similarly acting substances. A proposal of updating the blood and urine sampling protocol]. / Pobieranie, przechowywanie i transport materialu biologicznego zabezpieczonego przyzyciowo i posmiertnie do badania na zawartosc alkoholu etylowego oraz srodków podobnie dzialajacych do alkoholu. Propozycja aktualizacji instrukcji pobierania próbek krwi i moczu.

The present paper emphasizes the most common mistakes committed at the beginning of an analytical procedure. To shorten the time and decrease the cost of determinations of substances with similar to alcohol activity, it is postulated to introduce mass-scale screening analysis of saliva collected from a living subject at the site of the event, with all positive results confirmed in blood or urine samples. If no saliva sample is collected for toxicology, a urine sample, allowing for a stat fast screening analysis, and a blood sample, to confirm the result, should be ensured. Inappropriate storage of a blood sample in the tube without a preservative can cause sample spilling and its irretrievable loss. The authors propose updating the "Blood/urine sampling protocol", with the updated version to be introduced into practice following consultations and revisions.

[Determination of nicotine and cotinine in human biological materials and their significance in toxicological studies]. / Problematyka oznaczania nikotyny i kotyniny w ludzkim materiale biologicznym w aspekcie badan toksykologicznych.

The aim of this study was the preparation of reliable procedure of the determination of nicotine and cotinine both in classic (serum, urine) and alternative biological materials (hair, saliva) and evaluation of their significance for clinical and forensic toxicology. Biological material samples (blood, urine, saliva) were taken from patients after Percutaneous Trans-luminal Coronary Angioplasty (PTCA). The determination of cotinine and nicotine concentration in the biological material should be optimized depending on the aim of analysis. Liquid-liquid extraction procedure and high performance liquid chromatography HPLC/UV-DAD are reliable, specific and relatively cheap. Serum and saliva are valuable biological materials which allow to determine temporary nicotine and cotinine content on the similar level of concentrations. In the near future it will be able to replace blood with saliva sample because of an easy and non-invasive way of sampling. Evaluation of cotinine concentration in urine allows to distinguish the passive from the active tobacco smokers. Hair analysis allows to control a nicotine abstinence as well as a long-term evaluation of the history of smoking. However usage of hair is limited because of difficulty with sampling. Interpretation of results in analysis of alternative materials (hair, saliva) pose a problem because of lack of sampling standardization and lack of standardization of final analysis method.

[Uncertainty of measurement as an integral part of the result and a tool of quality of work improvement for the chemical-toxicology laboratory]. / Niepewnosc pomiarowa jako integralna czesc wyniku pomiarowego oraz narzedzie doskonalenia jakosci pracy laboratorium chemiczno-toksykologicznego.

An uncertainty in measurements is a numerical defined range that is supposed to find a true value with a given level of probability. The purpose of this study was to estimate a type A and B of uncertainty in the analysis of ethanol using the ADH method and prove it to be useful in lab practice. A large percentage of uncertainty was shown for pipetting of the ADH and perchloric acid solutions. This result formed the basis to make an effort in order to improve the procedure. The results of uncertainty of measurements given by method B are similar to the empiric method A results. The reduction of expenses and time saving of the analysis is an important advantage of the described way of uncertainty.