New challenges in toxicology of new psychoactive substances exemplified by fatal cases after UR-144 and UR-144 with pentedrone administration determined by LC-ESI-MS-MS in blood samples.

The topic of this paper relates to the study of cases involving the use of new psychoactive substances (NPS) from the classes of synthetic cannabinoids and cathinones, analyzed from multiple viewpoints including clinical and medico-legal perspectives. The paper investigates three fatal cases in which UR-144 and UR-144 with pentedrone identified in the bodies of victims during post-mortem examinations were responsible for the tragic consequences and proved to be the indirect cause of death. The victims were men aged 16, 22 and 40 years who used drugs, for example they smoked marijuana or its substitutes in the form of synthetic cannabinoids. In addition, all of them had behavioural problems. On account of emotional imbalance attributable probably to the presence of UR-144 (in one case) and a mixture of UR-144 and pentedrone (in the other two cases), two men committed suicide by jumping from a height and hanging, and one man had fatal accidental poisoning with pentedrone which was used to enhance the effect of previously used UR-144. The presence of UR-144 and pentedrone in the post-mortem material was analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS). The results of toxicological tests were analyzed with a focus on possible tragic side effects caused by the presence of UR-144 and UR-144 with pentedrone in the body.

New psychoactive substances in substantive evidence in expert practice of the Department of Forensic Medicine, UJCM in the years 2010-2015.

AIM OF THE STUDY:: Aim of the study was to analyse of 2075 evidences containing new psychoactive substances (NPS). MATERIAL AND METHODS: The prepared samples were identified employing an analytical procedure where the analytes were investigated by gas chromatography-electron impact mass spectrometry (GC-EI-MS) using a created library of mass spectra. RESULTS: The analysis revealed the following substances in the investigated products: piperazine derivatives (including BZP, MPMP, TFMPP), cathinone derivatives (including: pentedrone, 3-MMC, butylone, 4-MEC), pyrovalerone derivatives (MDPV, naphyrone, α-PVP, α-PVT), synthetic cannabinoids (such as AM-2201, UR-144, XLR-11, JWH073, JWH081, PB-22, AB-CHMINACA). Research conducted in 2010-2015 made it possible to track changes in the composition of investigated preparations. CONCLUSIONS: The following relationships has been shown: number of components decreased and in the end of 2015 dominated preparations with single component; introduction of amendments Act on Preventing Drug Addictionn affect the elimination from the market of one compounds and replacing them by their derivatives; since 2011 on the market of 'legal highs' we did not observe occurrence of compounds of piperazine group.

Opium alcaloids in toxicological medico-legal practice of Department of Forensic Medicine, Jagiellonian University Medical College / Alkaloidy opium w toksykologicznej praktyce medyczno-sadowej Zakladu Medycyny Sadowej UJ CM.

Most likely, opium was the first narcotic substance discovered at the dawn of mankind. Contemporary drug abuse predominantly poses a social and clinical problem and encompasses among other aspects emergency procedures in cases of intoxication and treatment of addictions. On the other hand, this is also a problem of the judicial system, which implements the rule of apt punishment in criminal cases (rapes, robberies, drivers, production and trade in narcotic substances) and of the necessity of monitoring drug-associates deaths. In all drug-associated cases, investigative capabilities have increased with the introduction of extremely sensitive and specific analytical methods (GC-MS, LC/MS, HPLC/DAD) allowing for detection and identification of multi-component mixtures of xenobiotics found at low concentration levels in complex biological matrices. The history of the Krakow Department of Forensic Medicine dates back to the year 1877, since archival materials have been kept since that time. Isolated deaths resulting from morphine poisoning, mostly involving individuals employed in the health care sector, constituted the subject of medico-legal expert opinions starting at the beginning of the 20th century, but only the eighties did bring the need for multidirectional toxicological examinations of opiates and their metabolites in diversified biological and non-biological materials. The present report, in addition to the historical background of opiate addiction, discusses selected problems derived from published by Department reports on opiates, including cases of fatal intoxication, hair analysis of drug addicts in its various aspects, interactions in cases of poisoning and others.

On the history of opium / Z dziejów opium.

Most likely, opium was the first narcotic substance discovered at the dawn of humankind. The history of drug addiction is immensely rich and allows for tracing the long way humankind had to travel to reach the contemporary level of consciousness with respect to narcotic substances. A retrospective view of drug addiction that takes into consideration the historical context, while extending our knowledge, also allows for a better understanding of today's problems. The report presents elements of a retrospective view of problems associated with addiction to opium, morphine and heroin over the centuries, what is a subject of scientific interest in contemporary toxicology.

Interpretative problems due to the presence of chloromethcathinone isomers in the biological material from postmortem cases.

While many new psychoactive substances often disappear from the drug market rather quickly, some, such as synthetic cathinones (SCs), still remain due to their popularity among users. The current knowledge of SC concentrations in blood samples is based mainly on the published case reports of intoxications or fatalities caused by SC intake. The aim of the present study was to present and interpret the obtained toxicological analysis results of these cases, in which it was possible to determine or detect the presence of one of the isomers of chloromethcathinone (CMC) along with its intake biomarker-dihydro-CMC. These cases include 27 deaths reported at the Department of Forensic Medicine in Kraków in 2016-2022. CMC constitutes a major toxicological opinion challenge, in terms of toxicological evaluation of poisonings. As presented in this paper, a significant problem is its stability in the biological material and practices in the reporting of the obtained data. It is therefore important to monitor potential intake biomarkers that may show greater stability in the biological material than the parent drug. In the case of CMC isomers, the good biomarker of intake is the dihydro-CMC metabolite, which was detected in the blood sample in every case presented, even with the absence of the parent substance. Interpretation of the results obtained for CMC in terms of assessing their toxicity and possible cause of death is difficult. However, it should be taken into account that in cases of new psychoactive substance poisoning, an in-depth risk assessment is mandatory and the opinion of the unpredictability of the effects is taken as a principle.

Metabolic Evaluation of Synthetic Opioids on the Example of U-47700 with the Use of In Vitro and In Vivo Methods for Forensic Toxicology Application.

Legal highs present a great threat to health, especially in groups of people experimenting with psychoactive substances. The lack of available knowledge on the biotransformation of these substances necessitates symptomatic treatment in the event of intoxication, which, unfortunately, may be ineffective. Opioids, including heroin analogues, such as U-47700, constitute a special group of designer drugs. In this study, a multi-directional approach to trace the biotransformation of U-47700 in living organisms was used. For this purpose, the in silico assessment (ADMET Predictor) was used first and then followed by an in vitro study using human liver microsomes and the S9 fraction. The biotransformation was then followed in an animal model (Wistar rats). Tissues such as blood, brain and liver were collected for analysis. The study was performed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The obtained results were compared to those obtained from the analysis of autopsy materials (cases analysed in the Toxicology Laboratory of the Department of Forensic Medicine, Jagiellonian University Medical College in Krakow).

The Stability of Synthetic Cathinones and the Study of Potential Intake Biomarkers in the Biological Material from a Case of 3-CMC Poisoning.

The objective of the present study was to identify the metabolites of synthetic cathinone (SC), 3-chloromethcathinone (3-CMC), and to select a potential intake biomarker for this compound. The basis of the experiment was the analysis of blood and urine samples from a case of fatal poisoning with this substance. We also evaluated the stability of 3-CMC and the selected potential biomarker, the dihydro-3-CMC metabolite, depending on the time elapsed since the autopsy as well as the storage conditions of the biological material. Studies have shown that 3-CMC shows low stability in the biological material. However, acidification of the biological material or storage at low temperatures has a great impact on maintaining its stability. It is crucial to monitor the presence of the dihydro-3-CMC metabolite in the biological material during analysis, which showed high stability under all of the given storage conditions. SCs might not be detected in the biological material and, if they are, the determined concentrations at the time of testing may not correspond to the actual concentrations at the time of the event or death. Consequently, the interpretation of the results obtained for 3-CMC and dihydro-3-CMC in terms of assessing their toxicity and possible cause of death is difficult. The area of research into the search for other intake biomarkers of unstable halogen derivatives of SCs remains open.

Role of toxicological determinations of amphetamines and cannabinoids in hair of adolescent patients in cardiologic diagnostic management.

BACKGROUND: Parental suspicions that the child may possibly take narcotics may be substantiated by hair analysis, which may not only identify the narcotic abuse profile, but also define the minimum period of narcotic usage. OBJECTIVE: A toxicological hair analysis aiming at detecting the presence of amphetamines and cannabinoids in children with cardiac complaints of unclear aetiology, with a view of establishing a diagnosis. MATERIAL AND METHOD: The authors analysed hair samples collected from the occipital region of three adolescent patients for the presence of substances of abuse--amphetamines and cannabinoids. Hair strands were subjected to segmentation and chemical processing and subsequently analysed by liquid chromatography-mass detection method. RESULTS: Indicating the presence of amphetamines and cannabinoids in hair samples, the toxicological analysis allowed for explaining cardiac complaints and personality changes in adolescent patients, with the said changes being associated with addiction to the above xenobiotics extending over some period.

[Immunoassay methods used to diagnose patients poisoned with psychoactive substances--analytical and interpretation problems]. / Metody immunoenzymatyczne w diagnostyce zatruc substancjami psychoaktywnymi--problemy analityczne i interpretacyjne.

Diagnostics of patients poisoned with psychoactive substances is usually performed by immunoenzymatic methods. The results obtained by the methods are not reliable. A few percent of results can be "false positive" or "false negative". The purpose of the paper was to present cases of "false positive" THC and amphetamine derivatives results obtained in urine of patients treated in Departments of Toxicology in Krakow and in Tarnow. Confirmation tests gave negative results. Interpretation of results obtained by immunoassay methods should include possibility of "false" result. The physician should consider verification of the results and refer confirmation by reference methods.

Post-aggression suicide under the influence of new psychoactive substances AMB-FUBINACA and U-47700.

The objective of the present study was the development and validation of the method for determining AMB-FUBINACA and its metabolite - AMB-FUBINACA O-desmethyl acid - in blood samples, followed by verification of the method in toxicological judicial and forensic medicine practice employing the example of post-aggression suicide. Most likely in consequence of development of adverse effects resulting in psychotic symptoms, a male being under the influence of the synthetic cannabinoid AMB-FUBINACA and the new synthetic opioid U-47700, mortally wounded his female partner and subsequently committed suicide. Identification and determination of the afore-mentioned xenobiotics in blood samples collected from the male and female victims were performed employing high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The analytes were isolated from blood samples using the solid phase extraction (SPE) method. The blood samples collected from the male and female demonstrated respectively 110 and 196 ng/mL of AMB-FUBINACA O-desmethyl acid metabolite, 1935 and 357 ng/mL of U-47700, 250 and 200 ng/mL of N-desmethyl-U-47700, as well as 410 and 200 ng/mL of N,N-didesmethyl-U-47700. The concentration values of new psychoactive substances (NPS's) in blood samples originating from the male and female were within the ranges encountered in cases of poisoning, including these resulting in death. Nevertheless, the evident signs of exsanguination proof that the woman was alive when she sustained lethal injuries. The presented cases illustrate the difficult to be anticipated effect exerted on the users by NPS's.

[State under the influence of drugs or psychotropic agents--a comparison of toxicological and medical examinations in materials of the Department of Forensic Medicine and Toxicology, Silesian University of Medicine, Katowice]. / Stan "pod wplywem substancji odurzajacych lub psychotropowych" porównanie wyników badan toksykologicznych z ocena lekarska w materialach Katedry i Zakladu Medycyny Sadowej i Toksykologii Sadowo-Lekarskiej Slaskiego Uniwersytetu Medycznego w Katowicach.

In the paper, the authors present the results of toxicological examinations of blood samples taken from drivers during road check procedures or from perpetrators of traffic road accidents, which--taking into consideration the kind of the determined agents and their concentrations--were compared with the results of medical examinations from blood sampling protocols studied in the Department of Forensic Medicine and Toxicology, Silesian University of Medicine. All the blood samples were first analyzed using an immunoenzymatic assay (ELISA). Then, the LC-MS method was used. The positive results of screening for the presence of cannabinols were verified by GC-MS. Out of 329 blood samples, 145 were positive. The presence of cannabinols, amphetamine or MDMA was the most predominant finding. Diazepam was determined in 4 cases and opiates in 1 case. Only in 31% cases did positive results of toxicological examinations correspond to deviations found during the medical examinations constituting the basis for the final diagnosis of state "under the influence". In practice, appraisal of drug influence during medical examination seems to be limited and dependent on variable reactions of the examined individuals to a psychoactive agent, time lapse between the traffic road event and the examination or concomitant symptoms associated with ethylene alcohol activity. The final diagnosis of state "under the influence of drugs" or "under the influence of psychotropic agents" given by the physician does not result from the effect of these substances observed during the medical examination, but is very often formulated based on the medical history or police findings. The analysis of the above mentioned cases where Delta9THC or/and amphetamine was detected showed no correlation between the concentration of the psychoactive agent determined in blood and symptoms triggered by its action as described by the physician.

[The importance of the quality of toxicological expertise for the need of the judiciary in the historical and contemporary aspect]. / Znaczenie ekspertyzy toksykologicznej dla wymiaru sprawiedliwosci w aspekcie historycznym i wspólczesnym.

The history of humanity is closely related to the use of various poisons, different in each epoch. They served different purposes for centuries. In addition to being a remedy for various ailments and diseases, they also helped to bear the hard life of a person thanks to the possibility of causing mental elation, making it more bearable. They were also used to kill other people, most often for very low motives. The number of poisons existing in particular stages of civilization has been systematically increasing, reaching the number of 100,000 - 500,000 toxicologically active compounds in modern times, with the generally estimated number of known chemical compounds at the level of about 240 million. The research work of thinkers and people of progress is a counterbalance to the evil deeds of poisoners in antiquity and the Middle Ages. These works appeared in the late Middle Ages and are continued in various forms until the present day. As a result of these works, modern toxicological forensic expertise has been developed. However, before it appeared in its modern shape, it had to go through a very difficult development path, which lasted continuously for several centuries. Modern toxicological expertise, based on a highly specialized instrumental methodology, operating with high methodological standards, is the achievement of many generations. These standards have now become a requirement for expert works of our times, and failure to comply with them is treated as a malpractice. This work is a review of the types of poisons and reports in terms of the development and application of toxicological forensic expertise for the purposes of the judiciary.

[Application of hair analysis of selected psychoactive substances for medico-legal purposes. Part II. Cases of complex fatal poisonings: interactions of heroine - cocaine - amphetamines]. / Zastosowanie analizy wybranych substancji psychoaktywnych we wlosach dla celów opiniowania sadowo-lekarskiego. Czesc II. Przypadki zlozonych zatruc smiertelnych: heroina - kokaina - amfetaminy w mechanizmie interakcji.

The study represents an attempt at employing segmental hair analysis in complex poisonings with xenobiotic mixtures of heroine - cocaine - amphetamines in the context of the cause of death as a consequence of complex interaction mechanisms which occurred prior to death. Two cases of complex poisonings: heroine - cocaine and heroine - cocaine - amphetamines were analyzed and documented with macro- and microscopic examinations and complex toxicological examinations, including the analysis of classic biological material, i.e. samples of selective blood, and alternative material, i.e. hair samples. Determinations of opioids, cocaine and its metabolite and amphetamines in the hair biological matrix were performed using high performance liquid chromatography--atmospheric pressure chemical ionization--tandem mass spectrometry (HPLC-APCI-MS-MS). Segmental hair analysis of the investigated cases indicated a prolonged intake of similar psychoactive substances and a developed adaptation of the addicted to interaction mechanisms, which, however, led gradually to multiorgan anatomopathological changes, and in consequence to death.

Forensic-psychiatric relationships in the context of forensic medical examination of new psychoactive substance-related deaths.

INTRODUCTION: The aim of this study was to find causal relationships between the use of new psychoactive substances (NPS), the so-called "legal highs", and the cause of death, taking into account information indicating the victim's mental disorders before death. MATERIAL AND METHODS: The research material includes the results of toxicological analyses of blood samples collected during autopsies of 40 deceased persons whose cause of death was associated with the ingestion of new psychoactive substances (NPS). The blood samples were subjected to solid phase extraction (SPE), and then analyzed by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) for quantification. Information on individual cases was taken from case files provided by the prosecutor's office for research purposes. RESULTS: As a result of the analyses, 24 types of NPS were detected, comprising synthetic cathinones - 15 cases, synthetic cannabinoids - 10 cases, synthetic opioids - 13 cases, other - 2 cases. In the study group there were 5 women (12.5%) and 35 men (87.5%). Suicide was demonstrated in 10 cases (25%), while the remaining 30 cases (75%) were accidental deaths, including those in which the actual motivation of drug use could not be determined. Drugs used to treat mental disorders were detected in 6 cases, while analgesics were demonstrated in 5 cases. CONCLUSIONS: In the majority of cases, including suicides and accidental deaths where the manner of death was unknown, NPS use and consequent death from various causes are associated with psychiatric disorders.

[Application of hair analysis of selected psychoactive substances for medico-legal purposes. Part I. Segmental hair analysis in cases of fatal opioids and amphetamines poisoning]. / Zastosowanie analizy wybranych substancji psychoaktywnych we wlosach dla celów opiniowania sadowo-lekarskiego Czesc I. Analiza segmentowa wlosów w przypadkach smiertelnych zatruc opioidami i amfetaminami.

The present experimental investigations were inspired by the necessity of standardizing the procedures and analytical methods employed in hair analysis aiming at a retrospective evaluation of ingestion of various xenobiotics. Thus, in keeping with the principal premises, the main objective of the study was development of unique, novel chemico-toxicological procedures for analyzing hair content of psychoactive substances in two basic groups of substances of abuse: opioids (morphine, 6-monoacetylmorphine, codeine) and amphetamines (amphetamine, methamphetamine, MDA, MDMA, MDEA) by HPLC-APCI-MS-MS, followed by verification of the thus worked out procedures in medico-legal practice through opinionating in selected group of patients deceased due to fatal psychoactive substance poisoning (cause of death determination). Determinations of opioids and amphetamines in the hair biological matrix were performed using high performance liquid chromatography - atmospheric pressure chemical ionization - tandem mass spectrometry (HPLC-APCI-MS-MS). In the group of fatal poisonings by ,,Polish heroine", hair segmental analysis confirmed the abuse profile of the opiate or mixed (opiate-amphetamine) type, which to some measure is characteristic of Polish drug addiction, indicating the presence of these xenobiotics in the investigating hair samples in the premortem period.

[Analysis of taxus baccata alkaloids in biological samples with the use of chromatographic methods]. / Analiza alkaloidów cisa pospolitego w materiale biologicznym z zastosowaniem metod chromatograficznych.

The aim of the experiment was to develop a method of the identification of taxines (taxine B and isotaxine B) from Taxus leaves and its application to the analysis of biological samples collected from the deceased girl. The object of the investigation was the analysis of taxines in post-mortem samples collected from a 15-year-old girl, who consumed leaves of Taxus baccata to commit suicide. She died after several hours of hospitalization. Taxus alkaloids, especially taxine B and isotaxine B, are not commercially available. Due to this fact, the authors worked out a preliminary method of isolating taxines from Taxus leaves and their extraction using thin layer chromatography (TLC) and solid-phase extraction (SPE). Identification of the presence of taxine B and isotaxine B in the extract from Taxus leaves was performed by high performance liquid chromatography coupled to tandem mass spectrometry with electrospray ionization (HPLC-ESI-MS-MS). The developed method was applied to determination of taxine B and isotaxine B in the biological samples collected from the girl's body. The samples were prepared using solid-phase extraction (SPE) and analyzed by HPLC-ESI-MS-MS. The results demonstrated that all the samples contained taxine B and isotaxine B. The results of toxicological investigations were confirmed by macroscopic and microscopic histological examinations.

Aspects related to analysis and medico-legal assessment in the context of the presence of synthetic cannabinoids in biological material based on the example of AB-CHMINACA.

AIM OF THE STUDY: The aim of this study was to develop and validate a method for the determination of the synthetic cannabinoid AB-CHMINACA in blood, followed by its verification in forensic toxicological practice. MATERIAL AND METHODS: The case of a 41-year-old man admitted to hospital because of scheduled cardiac surgery was discussed. The man died after 12 hours of hospitalization. Based on collected evidence, AB-CHMINACA poisoning was suspected. The identification and determination of AB-CHMINACA in the man's blood was performed by high-performance liquid chromatography coupled with tandem mass spectrometry using electrospray ionization (HPLC-ESI-MS-MS), after prior solid phase extraction. RESULTS: The concentration of AB-CHMINACA determined in the man's blood sample was 0.5 ng/ml. CONCLUSIONS: In the interpretation of the case, it was concluded that AB-CHMINACA had no direct effect on the patient's death, the cause of which was ascertained as chronic heart failure secondary to aortic valve disease, decompensated by pneumonia. However, an indirect impact of side effects resulting from the use of synthetic cannabinoids cannot be ruled out. They might have exacerbated the man's disease process leading to sudden cardiac arrest caused by asystole.

Usefulness of multi-parameter opiates-amphetamines-cocainics analysis in hair of drug users for the evaluation of an abuse profile by means of LC-APCI-MS-MS.

The report presents a segmental hair analysis for the retrospective multi-parameter evaluation of drugs of abuse including opioids, cocainics and amphetamines. The analysis was carried out with the use of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS). The authors have evaluated the differences in the contents of particular opiates in the hair as related to the origin of a sample taken from Polish drug users taking "Polish heroin", and also from heroin abusers from Western European countries taking "Western heroin". The results indicate distinct differences in the 6-MAM concentration values in the Polish and foreigners, suggesting that the foreigners take products containing high concentrations of heroin and the Polish take the poppy product "compote" characterized by its variable and low heroin content. An additional argument for a different abuse profile in the Polish and Western drug users is found in the presence of cocaine detected in hair samples originating from the latter, while cocaine is much less frequently detected in Polish drug users.

Fatality due to the use of a designer drug MDMA (Ecstasy).

Drugs of abuse belonging to the amphetamine derivatives, which are often taken by adolescents and young adults, pose a serious risk associated with uncontrolled ingestion that sometimes leads to fatal outcomes. The number of deaths, however, related to Ecstasy is small when compared to the frequency of its use. The report presents a fatal poisoning with MDMA--Ecstasy of a 22-year-old male with a documented history of drug abuse. The observations of witnesses to the event made within the period between the exposition and fatal outcome may document the characteristic behavior of a person in the course of progressive poisoning. Toxicological investigations of the autopsy specimens carried out by means of liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC-APCI-MS-MS) demonstrated the presence of MDMA and its metabolite MDA in the blood of the victim, and the concentration level justified the fatal outcome (MDMA--1.42mg/L, MDA--0.17 mg/L), while the detection of high MDMA levels in a 6-cm long strand of hair separated into three segments (11.64 ng/mg in S1; 8.74 ng/mg in the S2; 15.51 ng/mg in the S3) confirmed the history of drug abuse. The report describing the results of macro and microscopic examinations aiming at assessing internal organ damage suggested a mild hepatic damage.

[Opinionating difficulties in cases concerning the use of psychoactive agents by traffic participants]. / Trudnosci opiniodawcze w sprawach dotyczacych substancji psychoaktywnych w ruchu drogowym.

In recent years, there has been observed an increasing number of traffic participants being under the influence of drugs or other than alcohol agents that affect the central nervous system. In the period from 1997 until June 2006, 435 blood samples collected from traffic participants suspected of having ingested psychoactive agents were examined in the Forensic Medicine Department, Medical University of Silesia, Katowice. Eighty-five blood samples were positive.