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Recent advances in genetic data generation, through massive parallel sequencing (MPS), storage and analysis have fostered significant progresses in microbial forensics (or forensic microbiology). Initial applications in circumstances of biocrime, bioterrorism and epidemiology are now accompanied by the prospect of using microorganisms (i) as ancillary evidence in criminal cases; (ii) to clarify causes of death (e.g., drownings, toxicology, hospital-acquired infections, sudden infant death and shaken baby syndromes); (iii) to assist human identification (skin, hair and body fluid microbiomes); (iv) for geolocation (soil microbiome); and (v) to estimate postmortem interval (thanatomicrobiome and epinecrotic microbial community). When compared with classical microbiological methods, MPS offers a diverse range of advantages and alternative possibilities. However, prior to its implementation in the forensic context, critical efforts concerning the elaboration of standards and guidelines consolidated by the creation of robust and comprehensive reference databases must be undertaken.

In many countries, adherence testing is used to monitor consumption behavior or to prove abstinence. Urine and hair are most commonly used, although other biological fluids are available. Positive test results are usually associated with serious legal or economic consequences. Therefore, various sample manipulation and adulteration strategies are used to circumvent such a positive result. In these critical review articles on sample adulteration of urine (part A) and hair samples (part B) in the context of clinical and forensic toxicology, recent trends and strategies to improve sample adulteration and manipulation testing published in the past 10 years are described and discussed. Typical manipulation and adulteration strategies include undercutting the limits of detection/cut-off by dilution, substitution, and adulteration. New or alternative strategies for detecting sample manipulation attempts can be generally divided into improved detection of established urine validity markers and direct and indirect techniques or approaches to screening for new adulteration markers. In this part A of the review article, we focused on urine samples, where the focus in recent years has been on new (in)direct substitution markers, particularly for synthetic (fake) urine. Despite various and promising advances in detecting manipulation, it remains a challenge in clinical and forensic toxicology, and simple, reliable, specific, and objective markers/techniques are still lacking, for example, for synthetic urine.

[Display omitted] The analysis of illicit drugs faces many challenges, mainly regarding the production of timely and reliable results and the production of added value from the generated data. It is essential to rethink the way this analysis is operationalised, in order to cope with the trend toward the decentralization of forensic applications. This paper describes the deployment of an ultra–portable near-infrared detector connected to a mobile application. This allows analysis and display of results to end users within 5s. The development of prediction models and their validation, as well as strategies for deployment within law enforcement organizations and forensic laboratories are discussed.

Rapid and reliable detection of illicit drugs such as methamphetamine and Δ9-tetrahydrocannabinol (THC) is critical in forensic toxicology. Immunoassay-based screening methods offer time-efficient alternatives to instrumental techniques, especially for high-throughput forensic applications. This study aims to comparatively evaluate the analytical performance of two widely used DRI and KIMS immunoassay platforms in detecting these drugs in human urine samples, with a focus on forensic applicability. A total of 866 urine samples were analyzed using both DRI and KIMS-based immunoassays. Key performance metrics such as sensitivity, specificity, cross-reactivity, and cut-off concordance were assessed. All results were compared to mass spectrometry-confirmed reference data. Both platforms demonstrated acceptable performance for preliminary drug screening. However, variations were observed in detection sensitivity for THC and in false-positive rates for methamphetamine. In conclusion, both systems are suitable for high-throughput forensic drug screening. And the findings provide practical guidance for forensic laboratories selecting immunoassay platforms for drugs of abuse screening. [Display omitted] ●DRI and KIMS immunoassays were compared for forensic urine drug screening.●Both platforms showed high concordance for methamphetamine and THC.●Moderate agreement for opiates indicated assay-specific variability.●The DRI MDMA assay showed higher specificity and broader screening coverage.●Results support immunoassay platform selection in forensic laboratories.

Dried Blood Spot (DBS) cards are gaining popularity in medicine and toxicology due to their versatility and efficiency. This study aimed to develop and optimize a comprehensive method for the detection of 16 psychoactive substances in post-mortem blood samples using the DBS technique, with comparative analysis against the routinely applied LC-SRM-MS method. The research focused on improving sample preparation proposed in the previous work. Key modifications, such as enhancing the extraction process and eliminating filtration steps, resulted in an increased sample concentration, improving LOD. Key modifications, such as enhancing the extraction process and eliminating filtration steps, resulted in a twelvefold increase in analyte concentration, improving LOD. The DBS/LC-MS method was validated, demonstrating high precision, reproducibility, and sensitivity for a broad range of psychoactive substances. Comparative analysis showed that the DBS/LC-MS method produced results consistent with the LC-SRM-MS method, with the added advantage of lower LOD for certain analytes. The study confirms the reliability of the DBS method in forensic toxicology and highlights its benefits, including reduced sample volume, making it a valuable alternative in toxicological and forensic applications. In Poland, prosecutors decide on toxicological tests and the storage of biological material, often leading to delayed decisions when samples are no longer available. A solution could be the routine use of DBS cards, which are cost-effective to store and could aid investigations. [Display omitted] •The complex method for determining psychotropic substances in post-mortem samples.•The routine use of DBS cards as a good solution.•Simplicity of the sample preparation process in forensic analysis.•The Post-mortem case samples examples in practical analysis.

Metabolome studies in forensic toxicology focus on the search for endogenous biomarkers changed by, e.g., drugs of abuse. However, placebo-controlled studies, the ideal study design, in humans are scarce for ethical reasons. Thus, the idea of using routine samples became popular, although confounding factors cannot be controlled. To systematically evaluate the use of routine samples for metabolomics, a comparison between a placebo-controlled amphetamine study in humans (A, n pos =18, n neg =18) to routine samples either positive or negative for amphetamine, prepared and analyzed over six months (re-evaluated, B, n pos =28, n neg =35) and prepared and analyzed within a single analytical batch (re-extracted, C) was performed. Samples were analyzed using untargeted liquid chromatography-tandem-mass-spectrometry. Comparison was conducted on feature level and based on significance (p- and fold-change-values). Only 3 features were significant in A, B, and C, and 2 were identified as amphetamine-(fragments). All 31 significant features from A were present in B and C; however, only 11 (36%) and 4 (13%) of them were significant mainly because of higher variation. Still, other significant features were found in routine samples (B/C). In conclusion, routine samples are generally suitable for detecting differences in the metabolome, even if they do not match those of a controlled study.

Forensic toxicology plays a crucial role in forensic sciences, focusing on the detection and interpretation of xenobiotics in various biological samples, including bone tissue. This study aimed to compare different extraction techniques for bone tissue and subsequently analyze and validate the best analytical method via liquid chromatography–tandem mass spectrometry. Samples were collected from autopsied cadavers, prepared, and subjected to four different extraction methods (EM-1 to EM-4). Absolute recovery tests indicated EM-1 as the better extraction method, successfully detecting all the target analytes. Method validation for EM-1 demonstrated acceptable bias, precision, interference studies, and stability in various analytes. The validated method was then applied to ten real cases, confirming its efficacy in forensic toxicology for detecting opioids, dissociative anesthetics, stimulants, benzodiazepines, anticonvulsants, antidepressants, antipsychotic medications, and sedatives in bone tissue. This study validated a new extraction method for bone tissue. This technique was applied to forensic real cases. •Comparison of different bone extraction techniques.•The forensic toxicology applied on bone tissue.•Development and validation of bone extraction.•The bone tissue as new and unconventional biological matrix.

For more than four decades, no standardized study design or workflow has been established for systematic entomotoxicological research in forensic casework. This study aimed to develop a universal rearing protocol for fly larvae grown on drug-spiked tissue surrogate and a standardized workflow for the qualitative analysis of pharmacologically active compounds (PACs) in insects of different developmental stages. Larvae of Protophormia terraenovae and Lucilia sericata were reared on minced meat serving as tissue surrogate. First-instar larvae were transferred to minced meat spiked with diazepam, amitriptyline, pipamperone, or lamotrigine, each triturated with placebo tablets. Actively feeding and post-feeding third-instar larvae were soaked for 24 h in ethanol (30 mL, 70:30, v:v), and both the larvae and the soaking solution were analysed using LC-MS/MS. The larval extraction method was optimized by replacing pure acetonitrile with an acetonitrile-methanol mixture and introducing centrifugation at −10°C. PAC detection was achieved in both larvae and soaking solution, confirming the sensitivity and applicability of the method. The workflow was further validated using authentic postmortem case samples. These findings demonstrate that ethanol-soaked larvae can be used effectively for both toxicological and entomological analyses. Overall, this study provides a universal and standardized workflow that simplifies and strengthens the application of entomotoxicology in forensic casework. [Display omitted] •Pharmacologically active compounds detectable in ethanol (70:30, v:v) after soaking larvae.•Similar screening results for ethanol (70:30, v:v) and larvae.•Proof of applicability: analysis by unified workflow successful.•Ethanol-soaked larvae can be used effectively for both toxicological and entomological analyses.

In forensic thanatology, post-mortem blood is often stored for long periods, leading to in vitro variations in drug concentrations. This study evaluated the use of dried blood microsampling supports dried (Dried Blood Spot – DBS and Volumetric Absorptive MicroSampling (VAMS) as an alternative to conventional tubes. 72 bodies were sampled, including 35 analyzed toxicologically. 6 categories of substances (26 molecules) were quantified using LC-MS/MS or LC-HRMS. Heroin, several benzodiazepines, and cocaine showed excellent stability on DBS, while they degraded or disappeared in conventional tubes. In some cases, the absence of a micro-sample would have prevented the diagnosis of intoxication. For other molecules, concentration differences between DBS and conventional tubes did not alter the medico-legal interpretation, with equivalent values for morphine, codeine, and tramadol. THC and its metabolites remained difficult to extract from DBS. Microsampling has improved the qualitative detection of unstable drugs in post-mortem blood in routine forensic thanalogic sampling practice. [Display omitted] •Post-mortem blood preservation stage causes in vitro changes in drug concentrations.•Dried micro-samples (VAMS, DBS) limit post-mortem degradation of many substances.•Unstable drugs degrade in tubes but remain stable on VAMS and DBS.•The lack of micro-samples can hinder the diagnosis of certain types of poisoning.

In forensic toxicology, the rapid and reliable detection of emerging synthetic opioids and hallucinogens is crucial for case investigations and public health monitoring. This work describes the development, optimization and validation of a simple, fast and sensitive methodology for the simultaneous analysis of 6 new synthetic opioids (carfentanil, fentanyl, isotonitazene, metonitazene, norfentanyl, and sufentanil) and 2 hallucinogens (lysergide [LSD] and mescaline), together with the main LSD metabolite 2-oxo-3-hydroxy-lysergide [LSD-OH], in whole blood samples by liquid chromatography coupled to tandem mass spectrometry. Under optimized experimental conditions, linearity was verified between 0.1 and 20 ng/mL for all analytes except mescaline (2.5–500 ng/mL), with r2 > 0.99 for 1/x weighting, and no significant carryover or matrix effects were observed. Good precision (% RSD < 13 %) and trueness (% Bias within ± 20 %) values were achieved. The estimated limit of quantification (LOQ) was 0.1 ng/mL for all compounds except mescaline (2.5 ng/mL). Authentic forensic samples were also analyzed, and positive samples for fentanyl, norfentanyl, and sufentanil were identified. The proposed methodology allows the simultaneous analysis of compounds from different families of psychoactive substances, in both postmortem and in vivo samples, using only 50 µL of whole blood. The demonstrated speed, simplicity, and effectiveness make it particularly advantageous for routine implementation in forensic toxicology laboratories. [Display omitted] •First known UHPLC-MS/MS method for nitazenes and hallucinogens analysis.•Validation of fast method for in vivo and postmortem whole blood samples analysis.•Low sample volume required is advantageous for forensic toxicology laboratories.•Sensitive and extensive linear range for a broad spectrum of concentrations.•Applied to suspected intoxication cases and feasible to routine forensic work.