Karolina Nowak

Bio: Karolina Nowak is an academic researcher from Wrocław Medical University. The author has contributed to research in topics: Urine & Postmortem Diagnosis. The author has an hindex of 4, co-authored 12 publications receiving 42 citations.

Fatal intoxication with N -ethylpentylone: a case report and method for determining N -ethylpentylone in biological material

Abstract: We present a fully validated method for determination of N-ethylpentylone in biological material and a case report of fatal intoxication with N-ethylpentylone. Blood and urine samples were extracted with ethyl acetate from alkaline medium (pH 9). The analysis was carried out using ultra-high-performance liquid chromatography–tandem mass spectrometry. MDMA-d5 was used as the internal standard. Validation criteria were evaluated for blank blood and urine at concentrations of 10 and 100 ng/mL. The validation parameters were as follows: lower limit of quantification: 1 ng/mL for blood and urine, coefficient of determination: blood > 0.9996, urine > 0.9975, precision for 10 and 100 ng/mL, respectively: blood 4.87% and 4.47%, urine 1.93% and 2.43%, accuracy for 10 and 100 ng/mL, respectively: blood 14.7% and −2.95%, urine 19.1% and 2.10%, recovery for 10 and 100 ng/mL, respectively: blood 91.5% and 100.2%, urine 97.4% and 96.7%, matrix effect in blood was 127% and 117% for 10 and 100 ng/mL, respectively, in urine 124% and 117% for 10 and 100 ng/mL, respectively. In the present case of fatal intoxication with N-ethylpentylone, the determined concentration of this substance was 10.6 µg/mL in peripheral blood and 17.6 µg/mL in urine. In both materials, four metabolites of N-ethylpentylone were determined qualitatively. The developed method enables the determination of N-ethylpentylone with high sensitivity and selectivity. The method was used to make determinations in biological material in the case of fatal intoxication with N-ethylpentylone.

Fatal intoxication with U-47700 in combination with other NPS (N-ethylhexedrone, adinazolam, 4-CIC, 4-CMC) confirmed by identification and quantification in autopsy specimens and evidences

Abstract: We present a case of fatal intoxication with U-47700 in combination with other NPS (N-ethylhexedrone, adinazolam, 4-chloro-N-isopropylcathinone (4-CIC), 4-chloromethcathinone (4-CMC) and sertraline) confirmed by identification and quantification in biological materials and evidences found at the scene in 2017 in Poland. Blood and urine samples were extracted with ethyl acetate from alkaline medium (pH 9); powders/crystals were diluted with methanol. The analysis was carried out using ultra-high-performance liquid chromatography–tandem mass spectrometry. Validation criteria were evaluated for blood and urine at the concentrations of 10 and 100 ng/mL. The validation parameters of the method were within acceptable ranges. In the presented case, the determined concentrations of drugs were as follows, in blood: U-47700, 1470 ng/mL; N-ethylhexedrone, 58 ng/mL; adinazolam, 18 ng/mL; 4-CIC, 8.0 ng/mL; 4-CMC, 1.7 ng/mL; in urine: U-47700, 3940 ng/mL; N-ethylhexedrone, 147 ng/mL; adinazolam, 82 ng/mL; 4-CIC, 130 ng/mL; 4-CMC, 417 ng/mL. Sertraline (blood, 89 ng/mL; urine, 32 ng/mL) was also determined in both materials. The same substances were also found in 5 powders/crystals: U-47700 (12% by weight), N-ethylhexedrone (54%), adinazolam (14%), 4-CIC (23%), 4-CMC (26%). After 775 days of storage, biological samples at + 4 °C, the most stable substance was sertraline and the less, synthetic cathinones, especially 4-CIC and 4-CMC. The described case of fatal intoxication with NPS presented postmortem concentrations of U-47700, 4-CMC, N-ethylhexedrone, adinazolam and 4-CIC for the first time in the literature. The paper also showed stability study of these substances stored at + 4 °C for 775 days.

Unstability of 4-CMC in human serum specimen

Abstract: Determination of new psychoactive substances (NPS) and interpretation of the results of research on them is a challenge for both clinical and forensic toxicologists. Among others, scientists undertake to develop methods for rapid determination of NPS in biological material or examine their pharmacokinetic and pharmacodynamic properties. Adequately quick determination of substances in biological material may help in choosing the right treatment method and thus protect patients from serious health consequences or even death. On the other hand, post-mortem toxicological analysis may help to determine the cause of death and prevent similar cases in the future. The results of post-mortem examinations are influenced by thanatochemical processes. Post-mortem changes negatively affect the stability of the substances in biological material, especially when the body is in a state of progressive putrefaction. Researchers [1–5] undertook studies into the stability of NPS in various biological matrices, including synthetic cathinones such as 4-methylmethcathinone (4-MMC), 4-ethylmethcathinone (4-EMC), and 3,4-dimethylmethcathinone (3,4-DMMC). However, none of the abovementioned studies examined the stability of 4-chloromethcathinone (4-CMC) in biological material. The authors of this study noticed the need to carry out research in this area because 4-CMC is highly unstable at 4 °C. The significance of research into this substance is further reinforced by the European Drug Report 2018 [6], according to which in 2016 in Europe 4-CMC was the second most frequently confiscated cathinone and the one seized in the largest amount (890 kg). This letter presents a study of 4-CMC stability in a blood serum sample without any preservatives (obtained from a person taking 4-CMC), stored at 4 °C. The 4-CMC, (1-(4-chlorophenyl)-2-(methylamino)-1propanone), also known as clephedrone, was first introduced to online sales in 2014 [7]. Although this substance has been available on the black market for several years, the results of research on, among others, its pharmacokinetic properties, toxic doses, effects of use, or addictive potential remain insignificant. Information on patterns of use are usually passed between users via online forums [8]. Tomczak et al. [8] determined that the blood concentration of 4-CMC in non-fatal cases ranged from 1.3 to 75.3 ng/mL (n = 9) and in fatal cases from 56.2 to 1870 ng/mL (n = 5). The discussed case concerns a 27-year-old incarcerated man. The reason for ordering toxicological tests is unknown. There is also no information about the medical or drug history of the man. The material submitted for testing in the form of blood serum was screened for numerous drugs. The serum was extracted with ethyl acetate from alkaline medium (pH 9). The analysis was carried out using the technique of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-QqQ-MS/MS). Calibration range 0.5–100 ng/mL; LOQ: 0.5 ng/mL. 4-MMC-d3 was used as an internal standard. Figure 1 shows the chromatogram obtained from first analysis with MRM transitions for 4-CMC and IS. Figure 2 shows mass spectra of 4-CMC. At the time of the first analysis (day 0), the 4-CMC concentration was 11.5 ng/mL. Subsequent analyses were conducted on the days 3, 8, 16, 27, 47, and 113. The sample was stored at 4 °C throughout the testing period. Table 1 shows 4-CMC concentrations on subsequent days. The tests demonstrated that 4-CMC was unstable in a blood serum sample stored at 4 °C. As early as 3 days after the first quantitative measurement, the analyte concentration dropped by 65% (11.5 ng/mL at day 0 vs. 4.0 ng/mL at day 3). The analyte was considered stable if the concentration differences were within ± 20% of the initial concentration. On day 47, the analyte concentration slightly exceeded LOQ * Karolina Nowak karolina_nowak1@wp.pl

The Stability of 4-Chloromethcathinone in Blood and Vitreous Humor.

Abstract: We present results of our study on the stability of 4-chloromethcathinone (4-CMC) in authentic postmortem peripheral blood and vitreous humor samples. The stability of 4-CMC was determined in postmortem blood samples (for a period of 90 days) and vitreous humor (30 days) at three different temperatures: -15°C, +4°C, and + 23°C. The analyses were carried out using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). In both materials, the lowest 4-CMC stability was demonstrated at room temperature. The blood samples stored in a freezer (-15°C) showed stability for the entire study period (90 days), while in the case of the vitreous humor sample stored at the same temperature the concentration of the substance decreased by 53% after 30 days. The study carried out in authentic postmortem blood and vitreous humor samples confirms the previous reports of 4-CMC instability in biological material. Authors suggest that the biological material should be stored frozen until analyses are carried out as soon as possible after collection of the material.

Suicidal deaths due to helium inhalation

Abstract: The authors reviewed the literature paying attention to helium suicide rates, autopsy findings in people exposed to helium, methods of collecting biological material for toxicological tests, and analytical methods used for detection and quantification of helium in the collected specimens. All reviewed information was gathered through a detailed search of Scopus, PubMed and the World-Wide Web using relevant keywords, e.g., helium, asphyxia, and suicide. The use of helium for suicidal purposes is becoming an increasingly common phenomenon around the world. This is undoubtedly influenced by the Internet, which explains in detail the technical aspects of the suicidal use of helium and describes the dying process. Exposure to helium does not lead to poisoning; instead, death is caused by asphyxia. Deaths due to helium asphyxiation are still a problem for modern forensic toxicology. Most current methods for collecting biological specimens and identifying the gas seem to be insufficient to give definite opinions in cases of helium asphyxiation. More attempts should be made to modify these methods in order to improve and facilitate detection and quantitative determination of helium and other inert gases.

Development and validation of a GC–MS/MS method for the determination of 11 amphetamines and 34 synthetic cathinones in whole blood

Abstract: Psychoactive compounds that contain a phenylethylamine structure (such as amphetamine-type stimulants and synthetic cathinones) are one of the major classes of stimulants on the recreational drug market. Approximately 670 new psychoactive substances (NPS) are monitored only in Europe; however, new psychoactive compounds are being developed for illicit trade each year. In this context, the development of new analytical procedures for the determination of such compounds in biological specimens for forensic toxicology is of great importance. Gas chromatography–tandem mass spectrometry (GC–MS/MS) technique was applied for analysis of amphetamines and synthetic cathinones. The volumes of 200 µL of each whole blood sample and 1 mL of liquid-liquid extraction solvent were used for extraction, followed by pentafluoropropionyl derivatization. A high-throughput, robust, rapid, and sensitive procedure involving a simple liquid-liquid extraction for the simultaneous determination of 45 amphetamine-type stimulants and synthetic cathinones in whole blood was developed. The assay was validated based on its recovery (83.2–106%), interday accuracy (89.0–108%), and interday precision (≤ 8.1%). In view of the low limits of detection (ranged between 0.02 and 0.72 ng/mL) and limits of quantification (1 and 2.5 ng/mL), the developed method can serve as a less expensive and more ecologically friendly alternative to the liquid chromatography–tandem mass spectrometric methods. To the best of our knowledge, this is the first work presenting a GC–MS/MS method for the determination of NPS in blood samples. The presented procedure was applied to authentic samples from forensic cases, demonstrating its utility in the quantification of a wide number of psychoactive substances in routine toxicological analyses. The developed procedure can also be easily expanded to additional compounds.

A Review of Synthetic Cathinone-Related Fatalities From 2017 to 2020.

Abstract: Background Synthetic cathinones (SCs) are designer analogs of the natural active principle of khat. Since their appearance on the black market in 2003, their popularity has increased annually, and they have become the most seized class of new psychoactive substances reported to the UNODC Early Warning Advisory system. The constant introduction of newly synthesized molecules makes this issue difficult to monitor. The authors reviewed the most recent SC-related fatalities worldwide to highlight new trends of consumption, reporting acute pharmacological and toxicological symptoms, scene investigations, analytical methods, and reported SC concentrations in diverse biological matrices. Methods A literature search was performed using scientific databases such as PubMed, Scopus, Science Direct, Web of Science, and Research Gate to identify relevant scientific publications from 2017 to 2020. In addition, a search was conducted through the EU EWS. Results From 2017 to 2020, 31 different SCs were identified in 75 reported fatal intoxications in the literature, alone or in combination with other substances. The most abused SCs were N-ethylpentylone, N-ethylhexedrone, and 4-chloromethcathinone. The EU EWS included less detail on 72 additional SC-related fatalities from 2017 to 2020. Conclusions New SCs continuously replace older natural and synthetic stimulant drugs, making determining the cause of death difficult. Analytical methods and high-performance mass spectrometry instruments are essential to detect the low concentrations of these potent new SCs. Little data are available on the pharmacology of these new drugs; the evaluation of toxicological antemortem and postmortem findings provides critical data on the drug's pharmacology and toxicology and for the interpretation of new SC cases.

An updated review on synthetic cathinones

Abstract: Cathinone, the main psychoactive compound found in the plant Catha edulis Forsk. (khat), is a β-keto analogue of amphetamine, sharing not only the phenethylamine structure, but also the amphetamine-like stimulant effects. Synthetic cathinones are derivatives of the naturally occurring cathinone that largely entered the recreational drug market at the end of 2000s. The former “legal status”, impressive marketing strategies and their commercial availability, either in the so-called “smartshops” or via the Internet, prompted their large spread, contributing to their increasing popularity in the following years. As their popularity increased, the risks posed for public health became clear, with several reports of intoxications and deaths involving these substances appearing both in the social media and scientific literature. The regulatory measures introduced thereafter to halt these trending drugs of abuse have proved to be of low impact, as a continuous emergence of new non-controlled derivatives keep appearing to replace those prohibited. Users resort to synthetic cathinones due to their psychostimulant properties but are often unaware of the dangers they may incur when using these substances. Therefore, studies aimed at unveiling the pharmacological and toxicological properties of these substances are imperative, as they will provide increased expertise to the clinicians that face this problem on a daily basis. The present work provides a comprehensive review on history and legal status, chemistry, pharmacokinetics, pharmacodynamics, adverse effects and lethality in humans, as well as on the current knowledge of the neurotoxic mechanisms of synthetic cathinones.

Designer Benzodiazepines: A Review of Toxicology and Public Health Risks

Abstract: The rising use of designer benzodiazepines (DBZD) is a cat-and-mouse game between organized crime and law enforcement. Non-prohibited benzodiazepines are introduced onto the global drug market and scheduled as rapidly as possible by international authorities. In response, DBZD are continuously modified to avoid legal sanctions and drug seizures and generally to increase the abuse potential of the DBZD. This results in an unpredictable fluctuation between the appearance and disappearance of DBZD in the illicit market. Thirty-one DBZD were considered for review after consulting the international early warning database, but only 3-hydroxyphenazepam, adinazolam, clonazolam, etizolam, deschloroetizolam, diclazepam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, phenazepam and pyrazolam had sufficient data to contribute to this scoping review. A total of 49 reports describing 1 drug offense, 2 self-administration studies, 3 outpatient department admissions, 44 emergency department (ED) admissions, 63 driving under the influence of drugs (DUID) and 141 deaths reported between 2008 and 2021 are included in this study. Etizolam, flualprazolam flubromazolam and phenazepam were implicated in the majority of adverse-events, drug offenses and deaths. However, due to a general lack of knowledge of DBZD pharmacokinetics and toxicity, and due to a lack of validated analytical methods, total cases are much likely higher. Between 2019 and April 2020, DBZD were identified in 48% and 83% of postmortem and DUID cases reported to the UNODC, respectively, with flualprazolam, flubromazolam and etizolam as the most frequently detected substances. DBZD toxicology, public health risks and adverse events are reported.