Toxicity of the New Psychoactive Substance (NPS) Clephedrone (4-Chloromethcathinone, 4-CMC): Prediction of Toxicity Using In Silico Methods for Clinical and Forensic Purposes.

This study reports the first application of in silico methods to assess the toxicity of 4-chloromethcathinone (4-CMC), a novel psychoactive substance (NPS). Employing advanced toxicology in silico tools, it was possible to predict crucial aspects of the toxicological profile of 4-CMC, including acute toxicity (LD50), genotoxicity, cardiotoxicity, and its potential for endocrine disruption. The obtained results indicate significant acute toxicity with species-specific variability, moderate genotoxic potential suggesting the risk of DNA damage, and a notable cardiotoxicity risk associated with hERG channel inhibition. Endocrine disruption assessment revealed a low probability of 4-CMC interacting with estrogen receptor alpha (ER-α), suggesting minimal estrogenic activity. These insights, derived from in silico studies, are critical in advancing the understanding of 4-CMC properties in forensic and clinical toxicology. These initial toxicological findings provide a foundation for future research and aid in the formulation of risk assessment and management strategies in the context of the use and abuse of NPSs.

Silent Bird Poisoning in Poland: Reconfirmation of Bromadiolone and Warfarin as the Proximal Causes Using GC-MS/MS-Based Methodology for Forensic Investigations.

The extensive use of rodenticides poses a severe threat to non-target species, particularly birds of prey and scavengers. In this study, a GC-MS/MS-based method was used to unlock the cause of bird deaths in Poland. Organs (liver, heart, kidney, and lungs) collected during autopsies of two rooks (Corvus frugilegus) and one carrion crow (Corvus corone corone), as well as fecal samples, were analyzed for the presence of anticoagulant coumarin derivatives, i.e., warfarin and bromadiolone. As for warfarin, the highest concentration was found in crow samples overall, with concentrations in the feces and lungs at 5.812 ± 0.368 µg/g and 4.840 ± 0.256 µg/g, respectively. The heart showed the lowest concentration of this compound (0.128 ± 0.01 µg/g). In the case of bromadiolone, the highest concentration was recorded in the liver of a rook (16.659 ± 1.499 µg/g) and this concentration significantly exceeded the levels in the other samples. By revealing the reality of the threat, these discoveries emphasize the need to regulate and monitor the trade in rodenticides.

Hot-Water Immersion (HWI) or Ice-Pack Treatment (IPT) as First Aid for Human Envenomation by Marine Animals? Review of Literature.

Envenomation by marine animals poses a significant health concern globally, affecting both local residents and tourists in coastal regions. The primary objective of this review is to critically evaluate the existing scientific literature to determine the most effective first-aid treatment for envenomations caused by marine animals, specifically whether hot-water immersion (HWI) or ice-pack treatment (IPT) provides the best immediate care. This comprehensive review covers a wide range of marine envenomations, from jellyfish stings to stingray injuries. While our focus is primarily on the efficacy of HWI and IPT, we also explore the role of cold-water treatment as a result of its relevance and similarity to ice-pack applications. In addition, we examine other treatments mentioned in the literature, such as medications or vinegar, and highlight their findings where applicable. To provide a clear and structured overview, we summarised the articles in separate tables. These tables categorise the type of research conducted, the marine species studied, the region of origin of the marine species, and the key findings of each study. Our analysis of the available evidence indicates a general consensus in the scientific community on the effectiveness of HWI or IPT for envenomation by marine animals. However, when treating those injuries, it is crucial to consider all factors since there is no universally superior treatment due to the diverse nature of marine habitats.

The toxicological analysis and assessment of essential elements (Cu, Fe, Mn, Zn) in Food for Special Medical Purposes (FSMP) dedicated to oncological patients available in Polish pharmacies.

Foods for Special Medical Purposes (FSMP) for oncology patients, available in pharmacies, play a crucial role in providing nutrition and supplementation. However, the scientific literature lacks comprehensive research on the safety of essential trace elements in these products. This study aimed to assess Cu, Fe, Mn and Zn levels in commonly prescribed FSMPs (n = 23) from Polish pharmacies. Using ICP-MS after microwave-induced digestion (using concentrated nitric acid and hydrogen peroxide), we evaluated element levels. Our research used three approaches: the raw score for Cu, Fe, Mn, and Zn; single intake per serving; and the daily ration, compared with the reference values of the European Food Safety Authority. Discrepancies were found between the actual and declared product compositions, influenced by the route of administration and the recommended intake. Despite variations, all products were considered safe for oncological patients based on current evidence. However, it is recommended to have clear guidelines for FSMPs in cancer care. This pioneering study evaluates the safety and quality of prescription FSMPs for cancer patients from toxicological and nutritional perspectives, highlighting the need for standardised protocols in pharmacy-dispensed FSMPs.

Gold Nanoparticles (AuNPs)-Toxicity, Safety and Green Synthesis: A Critical Review.

In recent years, the extensive exploration of Gold Nanoparticles (AuNPs) has captivated the scientific community due to their versatile applications across various industries. With sizes typically ranging from 1 to 100 nm, AuNPs have emerged as promising entities for innovative technologies. This article comprehensively reviews recent advancements in AuNPs research, encompassing synthesis methodologies, diverse applications, and crucial insights into their toxicological profiles. Synthesis techniques for AuNPs span physical, chemical, and biological routes, focusing on eco-friendly "green synthesis" approaches. A critical examination of physical and chemical methods reveals their limitations, including high costs and the potential toxicity associated with using chemicals. Moreover, this article investigates the biosafety implications of AuNPs, shedding light on their potential toxic effects on cellular, tissue, and organ levels. By synthesizing key findings, this review underscores the pressing need for a thorough understanding of AuNPs toxicities, providing essential insights for safety assessment and advancing green toxicology principles.

The estimation of acute oral toxicity (LD50) of G-series organophosphorus-based chemical warfare agents using quantitative and qualitative toxicology in silico methods.

The idea of this study was the estimation of the theoretical acute toxicity (t-LD50, rat, oral dose) of organophosphorus-based chemical warfare agents from the G-series (n = 12) using different in silico methods. Initially identified in Germany, the G-type nerve agents include potent compounds such as tabun, sarin, and soman. Despite their historical significance, there is a noticeable gap in acute toxicity data for these agents. This study employs qualitative (STopTox and AdmetSAR) and quantitative (TEST; CATMoS; ProTox-II and QSAR Toolbox) in silico methods to predict LD50 values, offering an ethical alternative to animal testing. Additionally, we conducted quantitative extrapolation from animals, and the results of qualitative tests confirmed the acute toxicity potential of these substances and enabled the identification of toxicophoric groups. According to our estimations, the most lethal agents within this category were GV, soman (GD), sarin (GB), thiosarin (GBS), and chlorosarin (GC), with t-LD50 values (oral administration, extrapolated from rat to human) of 0.05 mg/kg bw, 0.08 mg/kg bw, 0.12 mg/kg bw, 0.15 mg/kg bw, and 0.17 mg/kg bw, respectively. On the contrary, compounds with a cycloalkane attached to the phospho-oxygen linkage, specifically methyl cyclosarin and cyclosarin, were found to be the least toxic, with values of 2.28 mg/kg bw and 3.03 mg/kg bw. The findings aim to fill the knowledge gap regarding the acute toxicity of these agents, highlighting the need for modern toxicological methods that align with ethical considerations, next-generation risk assessment (NGRA) and the 3Rs (replacement, reduction and refinement) principles.

The acute toxicity of Novichok's degradation products using quantitative and qualitative toxicology in silico methods.

The emergence of Novichok agents, potent organophosphorus nerve agents, has spurred the demand for advanced analytical methods and toxicity assessments as a result of their involvement in high-profile incidents. This study focuses on the degradation products of Novichok agents, particularly their potential toxic effects on biological systems. Traditional in vivo methods for toxicity evaluation face ethical and practical constraints, prompting a shift toward in silico toxicology research. In this context, we conducted a comprehensive qualitative and quantitative analysis of acute oral toxicity (AOT) for Novichok degradation products, using various in silico methods, including TEST, CATMoS, ProTox-II, ADMETlab, ACD/Labs Percepta, and QSAR Toolbox. Adopting these methodologies aligns with the 3Rs principle, emphasising Replacement, Reduction, and Refinement in the realm of toxicological studies. Qualitative assessments with STopTox and admetSAR revealed toxic profiles for all degradation products, with predicted toxicophores highlighting structural features responsible for toxicity. Quantitative predictions yielded varied estimates of acute oral toxicity, with the most toxic degradation products being EOPAA, MOPGA, MOPAA, MPGA, EOPGA, and MPAA, respectively. Structural modifications common to all examined hydrolytic degradation products involve substituting the fluorine atom with a hydroxyl group, imparting consequential effects on toxicity. The need for sophisticated analytical techniques for identifying and quantifying Novichok degradation products is underscored due to their inherent reactivity. This study represents a crucial step in unravelling the complexities of Novichok toxicity, highlighting the ongoing need for research into its degradation processes to refine analytical methodologies and fortify readiness against potential threats.

The comprehensive strategy in the human health risk assessment of total chromium impurities in cough syrups with Marshmallow Root (Althaea officinalis) available in Polish pharmacies: regulatory aspects and special emphasis on Cr(VI) mode of action.

Chromium, which can currently only be considered pharmacologically active and not an essential element, is a very intriguing elemental impurity in final pharmaceutical products, especially traditional herbal medicinal products. This kind of traditional herbal medicinal product with Marshmallow root (Althaea officinalis L., radix) registered in the EU is widely used among the European population. The aim of this article is to propose a double regulatory strategy in assessing the human health risk of total chromium impurities in cough syrups with Marshmallow Root (Althaea officinalis) available in Polish pharmacies. We applied the strategy based on the requirements of the ICH Q3D (R1) guideline for the assessment of Cr impurities in final traditional herbal medicinal products with Marshmallow Root registered in the EU. Furthermore, we applied the strategy based on the concept of margin of exposure (MoE) considering Cr(VI) genotoxicity mode of action (MOA) and based on BMD10 for Cr(VI) as a point of departure (PoD). The total Cr content was in the range: 1.12-9.61 µg/L (in comparison with the ICH Q3D R1 guidelines: 1100 µg/g). Total Cr levels in a single dose were relatively high compared to raw results, but were not a threat to patients. Comparison of estimated results with oral PDE value for Cr in final drugs suggested by the ICH Q3D R1 guideline (10,700 µg/day) show that all the products analyzed were below this value (the highest result was 278.40 ng/day). Despite conservative assumptions, the MoE values obtained for Cr in daily dose for each Marshmallow Root cough syrup were above 10,000; therefore, exposure to Cr would not cause a health risk for specific population groups (children and adults). It can be summarized that each of the phytopharmaceuticals analysed with Marshmallow root available in Polish pharmacies does not represent a health hazard to patients. We confirm the safety of Cr impurities by applying a double regulatory strategy without the application of an expansive and demanding HPLC-ICP-MS technique for Cr speciation.

Toxicity of minoxidil - Comprehensive in silico prediction of main toxicity endpoints: Acute toxicity, irritation of skin and eye, genetic toxicity, health effect, cardiotoxicity and endocrine system disruption.

This article focusses on elucidating the toxicological profile of minoxidil, a widely used pharmacological agent for alopecia, through the application of in silico methods (Percepta ACD/Labs software). This research is driven by the need to understand key toxicological endpoints: acute toxicity, skin and eye irritation, genetic toxicity, cardiotoxicity, disruption of the endocrine system, and estimation of various health effects due to the lack of experimental data for this drug. These parameters are critically evaluated to meet the stringent requirements of the pharmaceutical industry's safety assessments. The results obtained for acute toxicity (LD50 for rats and mouse) indicate that minoxidil exhibits a species-dependent acute toxicity profile e.g. 51 mg/kg bw for intravenous administration in mice. The predicted health effects indicate a 93% risk to the gastrointestinal system, 54% for the kidneys, 52% for the liver, 42% for the blood and lungs, and 39% for the cardiovascular system. The prediction of genotoxicity suggests a moderate probability (48%) of inducing a positive Ames test result. Furthermore, moderate inhibition of the hERG channel indicates potential cardiac risks of Minoxidil. Based on the information obtained, we propose subjecting minoxidil to additional toxicological assessments. The successful adoption of these in silico methodologies aligns with the 3 R s principle (replacement, reduction, and refinement) in the field of modern toxicological studies of minoxidil, all without the use of laboratory animals for the novelty of our toxicity assessment.

The prediction of hydrolysis and biodegradation of organophosphorus-based chemical warfare agents (G-series and V-series) using toxicology in silico methods.

Nerve agents (G- and V-series) are a group of extremely toxic organophosphorus chemical warfare agents that we have had the opportunity to encounter many times on a massive scale (Matsumoto City, Tokyo subway and Gulf War). The threat of using nerve agents in terrorist attacks or military operations is still present, even with establishing the Chemical Weapons Convention as the legal framework. Understanding their environmental sustainability and health risks is critical to social security. Due to the risk of contact with dangerous nerve agents and animal welfare considerations, in silico methods were used to assess hydrolysis and biodegradation safely. The environmental fate of the examined nerve agents was elucidated using QSAR models. The results indicate that the investigated compounds released into the environment hydrolyse at a different rate, from extremely fast (<1 day) to very slow (over a year); V-agents undergo slower hydrolysis compared to G-agents. V-agents turned out to be relatively challenging to biodegrade, the ultimate biodegradation time frame of which was predicted as weeks to months, while for G-agents, the overwhelming majority was classified as weeks. In silico methods for predicting various parameters are critical to preparing for the forthcoming application of nerve agents.

Toxicovigilance 2.0 - modern approaches for the hazard identification and risk assessment of toxicants in human beings: A review.

The attempt to define toxicovigilance can be based on defining its fundamental principles: prevention of infections with toxic substances, collecting information on poisonings, both in terms of their sources and side effects, and confirming poisonings, with the aim of improving treatment. Substances referred to include both those originating from animal bites, ingested inadvertently, and those resulting from environmental poisoning in industrial regions of countries, etc. In this review, we provide information about the crucial function of poison control centres in toxicovigilance, the importance of incorporating big data analytics and artificial intelligence to streamline toxicovigilance processes, and examples of toxicovigilance in different countries. In conclusion, we will present the direction that modern toxicovigilance should take, incorporating available artificial intelligence methods to maximise efficiency.

The comprehensive prediction of carcinogenic potency and tumorigenic dose (TD50) for two problematic N-nitrosamines in food: NMAMPA and NMAMBA using toxicology in silico methods.

The identification and toxicological assessment of potential carcinogens is of paramount importance for public health and safety. This study aimed to predict the carcinogenic potency and tumorigenic dose (TD50) for two problematic N-nitrosamines (N-NAs) commonly found in food: N-2-methylpropyl-N-1-methylacetonylnitrosamine (NMAMPA, CAS: 93755-83-0) and N-3-Methylbutyl-N-1-methylacetonylnitrosamine (NMAMBA, CAS: 71016-15-4). To achieve this goal, in silico toxicology methods were employed due to their practical applications and potential in risk assessment. The justification for conducting these studies was incoherent results published by the European Food Safety Authority (EFSA). For this purpose, we applied various in silico tools, including qualitative methods (ToxTree, ProTox II and CarcinoPred-EL) and quantitative methods (QSAR Toolbox and LAZAR) were applied to predict the carcinogenic potency. These tools leverage computational approaches to analyze chemical structures for finding toxicophores and generating predictions, making them efficient alternatives to traditional in vivo experiments. The results obtained indicated that N-NAs are carcinogenic compounds, and quantitative data was obtained in the form of estimated doses of TD50 for the compounds tested.

Hazardous elements in plastic and rubber granules as infill material from sports facilities? Field Portable-XRF spectroscopy as 'white analytical technique' reveals hazardous elements in fall sports facilities in Rzeszów (Podkarpackie, Poland).

Plastic and rubber granules are commonly used as infill material in all-weather sports facilities, providing an ideal activity surface for millions of Europeans on a daily basis. However, concerns have been raised about the presence of hazardous elements in these granules, which can pose risks both to the environment and human health. Our study focusses on the elemental composition of rubber granules used in fall sports facilities in Rzeszów, (Podkarpackie, Poland) using field portable X-ray fluorescence (FP-XRF) as a non-destructive and 'white analytical technique'. The results show the content of Zn, Fe, Cr, Ba, Br, Ti, Cu, Cd, As, Au, Bi, Pb, Ni, Sb, and Sn in the rubber granule samples. This study highlights the need for stringent quality control measures and regulations to ensure the safety of all-weather sports facilities and protect the well-being of sportsman. When modern FP-XRF spectrometry is employed as a "white analytical technique," for the first time it becomes possible to identify the presence of hazardous elements, addressing the pressing concerns highlighted by the ECHA and enabling proactive measures to mitigate potential risks. This approach ensures the protection of the health and sustainability of sports facilities, contributing to the ongoing hot topics in the field.

The toxicological analysis of lead impurities in traditional herbal medicinal product with Thymi herba (Thymus vulgaris L. and Thymus zygis L.) from pharmacies in Poland: the impurity profile and comprehensive toxicological risk assessment.

Traditional herbal medicinal products (THMP) with Thymi herba (Thymus vulgaris L. and Thymus zygis L.) are very popular among the European population. The idea of our study was the toxicological analysis of lead impurities in THMP with Thymi herba obtained from Polish pharmacies. For this purpose, we prepared the impurity profiles and comprehensive toxicological risk assessment. The Pb impurity profiles show lead impurities in all samples investigated (in the range: 2.15-6.99 µg/L). The estimations of Pb impurities in the single dose (32.25-105.01 ng/single dose) and in the daily dose (64.50-210.00 ng/day) were based on the posology recommended by the manufacturers. All the results obtained meet the standards of the ICH Q3D (R1) guideline on elemental impurities according to the levels of Pb. It can be concluded that all investigated THMPs with Thymi herba available in Poland should not represent any health risk to adults.

The Control and Comprehensive Safety Assessment of Heavy Metal Impurities (As, Pb, and Cd) in Green Tea Camellia sinensis (L.) Samples (Infusions) Available in Poland.

Tea is the world's most common drink after water, and the world's annual sales exceed $43 billion, of which more than $11 billion is green tea (Camellia sinensis (L.)). Increasing evidence continues to show that green tea consumption and its ingredients have a variety of potential health benefits. The aim of the study was to control and assess the toxicological risk (TRA) of three heavy metals (As, Pb, and Cd) in infusions of green tea (n = 12) from Polish markets. We applied the method of inductive coupled plasma mass spectrometry (ICP-MS) to determine the selected heavy metal and designed a specific toxicological risk assessment with three crucial tiers. The first tier of our strategy was to determine the elements investigated (heavy metal impurities, µg/L). The second step was to estimate the weekly intake of green tea infusions (µg/week) based on weekly consumption. The third tier was to estimate the weekly intake per weight (µg/L/week/bw), based on the average weekly intake of green tea infusion per adult compared to the provisional weekly intake (PTWI) established by the FAO/WHO Joint Food Additives Expert Committee (JECFA). The levels of the investigated heavy metals occur at different levels in all of the investigated green tea infusions. The heavy metal profile indicated that As (0.0721-10.585 µg/L), Pb (0.386-1.695 µg/L), and Cd (0.126-0.346 µg/L) were present in all samples. Basic analysis of general content shows that As (average = 0.325 µg/L) was at a level similar to Cd (average = 0.214 µg/L). Surprisingly, the Pb content (average 0.891 µg/L) was approximately 2.75-4 times higher than that of As and Cd, respectively. The application of our proposed safety assessment methodology (three tiers) provides satisfactory results for regulatory purposes. The evaluation of the investigated heavy metals in all products analysed from green tea samples (infusions) showed that there were no health hazards to consumers due to weekly exposure. The results indicated that after drinking green tea infusions from Polish markets, the amount of investigated elements in weekly doses does not represent any health risks to consumers.

The prediction of acute toxicity (LD50) for organophosphorus-based chemical warfare agents (V-series) using toxicology in silico methods.

Nerve agents are organophosphate chemical warfare agents that exert their toxic effects by irreversibly inhibiting acetylcholinesterase, affecting the breakdown of the neurotransmitter acetylcholine in the synaptic cleft. Due to the risk of exposure to dangerous nerve agents and for animal welfare reasons, in silico methods have been used to assess acute toxicity safely. The next-generation risk assessment (NGRA) is a new approach for predicting toxicological parameters that can meet modern requirements for toxicological research. The present study explains the acute toxicity of the examined V-series nerve agents (n = 9) using QSAR models. Toxicity Estimation Software Tool (ver. 4.2.1 and ver. 5.1.2), QSAR Toolbox (ver. 4.6), and ProTox-II browser application were used to predict the median lethal dose. The Simplified Molecular Input Line Entry Specification (SMILES) was the input data source. The results indicate that the most deadly V-agents were VX and VM, followed by structural VX analogues: RVX and CVX. The least toxic turned out to be V-sub x and Substance 100A. In silico methods for predicting various parameters are crucial for filling data gaps ahead of experimental research and preparing for the upcoming use of nerve agents.

The comprehensive review about elements accumulation in industrial hemp (Cannabis sativa L.).

Cannabis sativa L., commonly known as industrial hemp, is a versatile plant with applications ranging from medicinal to agricultural and industrial uses. Despite its benefits, there is a notable gap in regulatory toxicology, in understanding the extent of element accumulation in hemp, which is critical due to its ability to absorb various elements from the soil, including heavy metals (Pb, Cd, Hg, and As), uptakes potential toxic elements (e.g., Sb, Sn, Sr, Bi, Tl), problematic elements (Ni, Cr, Co), and essential elements (Zn, Cu, Fe, Mn). The paper aims to enrich current understandings by offering a comprehensive analysis of elements absorption in industrial hemp. This study emphasizes the potential health risks linked with hemp consumption including regulatory toxicology aspects: limits, Permitted Daily Exposures (PDE), recommendations in different countries and from different agencies/bodies (like the WHO and the EU) based on route of administration, jurisdiction and actual literature review. This review contributes significantly to the knowledge base on hemp safety, serving as a valuable resource for researchers, regulatory bodies, and industry stakeholders.

Antidotes in Clinical Toxicology-Critical Review.

Poisoning and overdose are very important aspects in medicine and toxicology. Chemical weapons pose a threat to civilians, and emergency medicine principles must be followed when dealing with patients who have been poisoned or overdosed. Antidotes have been used for centuries and modern research has led to the development of new antidotes that can accelerate the elimination of toxins from the body. Although some antidotes have become less relevant due to modern intensive care techniques, they can still save lives or reduce the severity of toxicity. The availability of antidotes is crucial, especially in developing countries where intensive care facilities may be limited. This article aims to provide information on specific antidotes, their recommended uses, and potential risks and new uses. In the case of poisoning, supportive therapies are most often used; however, in many cases, the administration of an appropriate antidote saves the patient's life. In this review, we reviewed the literature on selected antidotes used in the treatment of poisonings. We also characterised the antidotes (bio)chemically. We described the cases in which they are used together with the dosage recommendations. We also analysed the mechanisms of action. In addition, we described alternative methods of using a given substance as a drug, an example of which is N-acetylcysteine, which can be used in the treatment of COVID-19. This article was written as part of the implementation of the project of the Polish Ministry of Education and Science, "Toxicovigilance, poisoning prevention, and first aid in poisoning with xenobiotics of current clinical importance in Poland", grant number SKN/SP/570184/2023.

The toxicological assessment of hazardous elements (Pb, Cd and Hg) in low-cost jewelry for adults from Chinese E-commerce platforms: In situ analysis by portable X-ray fluorescence measurement.

This article focusses on the environmental implications of low-cost jewelry for adults from Chinese e-commerce platforms ((n = 8) with heavy metal impurities (Pb, Cd and Hg) and their potential impact on human health and the environment. The study highlights the advantages of using portable X-ray fluorescence (pXRF) analysis for rapid, non-destructive, and in situ analysis of heavy metals in jewelry. The results reveal that all products (n = 106) contained heavy metals at varying levels, Hg being the most commonly detected heavy metal. The fact that 71% of the samples exceeded the EU limit for Pb and 51% exceeded the EU limit for Cd is alarming and highlights the need for stricter regulations and monitoring of the jewelry industry to mitigate the risks posed by heavy metals in the environment. The study emphasizes the importance of using pXRF analysis to identify heavy metals in jewelry and address the literature gap in environmental risk assessments of Pb, Cd, and Hg in low-cost jewelry for adults from China. In general, the findings call for urgent action to ensure the safety of consumers and prevent environmental pollution by strengthening regulations and monitoring the jewelry industry.

Development of innovative methodology for determination of 6-thioguanine in whole blood erythrocytes by HPLC-PDA-based technique for medical diagnostics purposes.

6-Thioguanine is an immunosuppressive drug, an analogue of guanine, applied to treat acute leukemia and inflammatory bowel disease. Excessive use of 6-thioguanine during clinical treatment may cause side effects. Moreover, providing a dose too low will be ineffective. Therefore, there is a critical need for a rapid, selective and routine approach to quantifying 6-thioguanine in body fluids to support a clinical application. A fully validated HPLC method has been developed to determine 6-thioguanine in whole blood samples using 5-bromouracil as an internal standard. 6-Thioguanine nucleotides were released from erythrocytes by perchloric acid, and then hydrolysed at 100 °C to the parent thiopurine, 6-thioguanine. The following validation parameters of the method were determined: specificity/selectivity, linearity range (479-17,118 ng/mL, R > 0.992), limits of detection (150 ng/mL) and quantification (479 ng/mL), accuracy (- 5.6 < Bias < 14.7), repeatability (CV 1.30-3.24%), intermediate precision (CV 4.19-5.78%), extraction recovery (79.1-103.6%) and carryover. Furthermore, the stability of the drug in whole blood samples under various storage conditions was investigated. The suggested method is suitable for determining 6-thioguanine in whole blood erythrocyte samples for drug level monitoring, thus correct dosing.