Application In Forensic Toxicology Research Articles

Metabolism study of 3-chloromethcathinone (3-CMC) by dried blood spot (DBS) sampling after controlled administration using a murine model.

The metabolism of 3-chloromethcathinone (3-CMC) was studied after controlled administration in a murine model using the dried blood spot (DBS) technique for the sampling, storage and purification of blood samples. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was used for the identification of metabolites and investigation of their fragmentation pattern. Subsequently, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for their identification and 3-CMC quantification in routine workload. The main metabolites identified were two stereoisomers of dihydro-CMC, N-demethyl-CMC, and dihydro-N-demethyl-CMC. The stability of 3-CMC and of its metabolites deposited on DBS was evaluated by replicate analyses after 30, 50, and 90 days, demonstrating a decrease in concentration. It was more pronounced for 3-CMC, with -67% and -82% percentage deviation from the initial concentrations, and for N-demethyl 3-CMC (decrease comprised between -48% and -88%) than for the di-hydro metabolites, ranging from -5% to -37%. Regardless, all of them were detectable till 90 days after deposition as DBS. The possibility of identifying 3-CMC and its metabolites with high sensitivity is an invaluable tool for the diagnosis of exposure to the substance, also in low doses or after some hours, and for various applications in clinical and forensic toxicology, such as driving under the influence, drug-facilitated crimes, and addiction to intoxications. DBS demonstrated to be a reliable technique for the sampling, storage, and purification of the blood specimen for 3-CMC and metabolite detection.

Dried blood spot (DBS) analysis of synthetic cathinones by different liquid chromatography-mass spectrometry techniques and interlaboratory validation for application in forensic toxicology

Dried Blood Spot (DBS) technique was employed as sample pretreatment for the detection of synthetic cathinones in blood samples, including from postmortem caseworks, by Liquid Chromatography-tandem Mass spectrometry (LC-MS/MS) techniques. The study involved the interlaboratory validation to evaluate the applicability of the method with different instruments, to assess the applicability and the ruggedness of the method for its wide application. The stability of cathinones in DBS was also evaluated.Authentic casework from drug-related deaths (DRD) involving synthetic cathinones were analysed and results compared in the two laboratories, using the developed methods. Metabolites were studied by two different LC-HRMS methods, orbitrap and Q-TOF.Validation results obtained in both laboratories demonstrated the suitability of DBS as sample storage and pre-treatment technique, allowing good sensitivities, repeatability, matrix effect and %bias. Limits of detection LOD and of quantification LOQ ranged between 0.3 and 1 ng/ml and between 0.5 and 10 ng/mL, respectively, for the studied cathinones in both laboratories. Stability studies demonstrated the high percent of degradation of cathinones, depending on their molecular structure, not attributable to DBS. The analysis of forensic caseworks containing α-PHP and MDPHP by DBS allowed their identification and quantification and the detection of various metabolites in postmortem blood samples. α-PHP degraded over time, while MDPHP concentration did not show significant differences in a three-months period, probably due to the presence of the metylendioxy moiety.These results demonstrate DBS to be a rapid and efficient technique for the analysis of cathinones, and that quantitative results for these analytes should be carefully interpreted due to their possible degradation.

Detection of 3-4 Methylenedioxyamphetamine from Drug Abuser’s Fingers and Toenails using Liquid Chromatography with Mass Spectroscopy

information regarding drug abuse and pharmaceutical use. In recent years, drug analysis in human nail clippings has proven its significant value in forensic toxicological applications, identification of in utero drug exposure, monitoring of drug treatment programmes, and therapeutic drug monitoring. Nails have various advantages over conventional matrices (blood and urine), which include a longer detection window (months to years), non-invasive sample collection, and easy storage and transportation. These aspects make nails a very significant matrix for forensic toxicology and therapeutic drug monitoring. Because of the low concentrations of drugs of abuse and pharmaceuticals present in nails and the complexity of the keratinized matrix, analytical techniques need to be more sensitive, and sample preparation is crucial. The aim of the present study is to develop a simple, high-performance liquid chromatography-mass spectroscopy (LC-MS) method for the identification and quantitation of 3,4-methylenedioxyamphetamine (MDA) in fingernail and toenail clippings. Finger and toenail clippings were collected from six users undergoing treatment at a rehab center in Ujjain, M.P., India. Nail clippings were initially decontaminated, then hydrolyzed in 1 M NaOH at 370°C, extracted with ethyl acetate, diluted with methanol, and then subjected to LC-MS analysis. The calibration curve was constructed over the 0.5 to 30 ng/mL concentration range using the MDA reference standard. The limit of detection was calculated at 1.10 ng/mL and the limit of quantification was recorded at 3.67 ng/mL in standard solutions, whereas the respective values in spiked nail clippings were 1.21 and 4.6 ng/mg. The developed method has obtained significant results in original nail clippings with mean concentration ranges of 0.12 ng/mg in fingernails and 0.08 ng/mg in toenails in six abuser samples. The new method developed has been found to be capable of detecting the 3,4-methylendioxyamphetamine MDA drug in nail clippings even after 90 days of drug intake.

Determination of cannabinoids in 50 μL whole blood samples by online extraction using turbulent flow chromatography and LC-HRAM-Orbitrap-MS: Application on driving under the influence of drugs cases.

The analysis of cannabinoids in whole blood is usually done by traditional mass spectrometry (MS) techniques, after offline cleanup or derivatization steps which can be lengthy, laborious, and expensive. We present a simple, fast, highly specific, and sensitive method for the determination of Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-hydroxy-Δ9 -tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in 50 μL whole blood samples. After the addition of deuterated internal standards (IS) and a simple protein precipitation step, an online extraction of sample supernatants using turbulent flow chromatography (TurboFlow-Thermo Scientific) was carried out. Analytes were separated on a C18 analytical column and detected by LC-HRAM-Orbitrap-MS using a Thermo Scientific Q Exactive Focus MS system. MS detection was performed in polarity switching and selected ion monitoring (SIM) modes using five specific acquisition windows, at a resolution of 70,000 (FWHM). Total run time was about 10min including preanalytical steps. Method validation was carried out by determining limit of detection (LOD), lower limit of quantitation (LLOQ), linearity range, analytical accuracy, intra-assay and interassay precision, carry-over, matrix effect, extraction recovery, and selectivity, for all analytes. Measurement uncertainties were also evaluated, and a decision rule was set with confidence for forensic purposes. The method may become suitable for clinical and forensic toxicology applications, taking advantage of the small matrix volume required, the simple and cost-effective sample preparation procedure, and the fast analytical run time. Performances were monitored over a long-term period and tested on 7620 driving under the influence of drugs (DUID) samples, including 641 positive samples.

The Integration of Artificial Intelligence in the Forensic Medicine and its Applications in Medico-Legal Autopsy, Forensic Toxicology, and Disaster Victim Identification

Artificial Intelligence (AI) is revolutionizing various fields of medicine and science. In the realm of forensic medicine, AI is playing an increasingly significant role, not only in enhancing traditional forensic practices but also in improving the teaching of forensic medicine. This article explores the integration of AI in the teaching of forensic medicine and its applications in medico-legal autopsy, forensic toxicology, and disaster victim identification. By examining current AI technologies and their potential impacts, this article sheds light on the future of forensic medicine education and practice, emphasizing the need for continued research and development in this dynamic field.

Development of a new green microextraction technique, switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction, to analyze synthetic cannabinoids in plasma

Synthetic cannabinoid receptor agonists (SCRA) are currently the most popular class of new psychoactive substances (NPS) in several countries, including Brazil. Notwithstanding that, they pose a threat to public health, as little of their toxicological properties is known. Moreover, these SCRAs have different presentations compared to classic cannabinoids and are not usually detected by conventional analytical methods due to their chemical variety. Thus, the development of new methodologies fit to analyze SCRAs is extremely warranted. In that regard, the use of environmentally benign or green approaches has been a trend in the last decades and has gained attention in Forensic Toxicology – so much so that guidelines have been established for that purpose, e.g. green analytical toxicology (GAT). With that in mind, the aim of this work was to develop and validate a quantitative method to analyze SCRAs in plasma by a novel green technique that agrees with the principles of GAT. A switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction (SHS-HLLME) was developed and statistical tools were used to optimize it. Analysis of Variance (ANOVA) was chosen to find the best switchable hydrophilicity solvent (SHS) and Full Factorial and Central Composite design to study the following variables: volume of SHS:HCl 6 M mixture (200–500 μL) and NaOH 10 M (200–500 μL), salting-out effect, and time of extraction (1–5 min). The ANSI/ASB Standard 036, 1st edition 2019 guidelines were used for method validation. All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring approach. Ethical Committee approval No. 46404121.8.3001.0067. Funded by CNPq 142056/2020-0, CAPES/INSPEQT 16/2020, and FAPESP 21/09857-8. ANOVA showed that between two SHS tested, N,N-dimethylcyclohexylamine and dipropylamine, the latter yielded higher analyte recovery ( P < 0.05). Moreover, the Design of Experiment tools herein used showed that the salting-out effect and time of extraction had no major impact on analyte recovery; in contrast, 500 μL of both SHS:HCl mixture and NaOH yielded higher analyte recovery ( P < 0.05). Once optimized, the proposed technique was successfully validated according to international guidelines with the following parameters: limits of detection: 0.01–0.08 ng/mL; limit of quantitation: 0.1 ng/mL; linearity: 0.1–10 ng/mL; bias and precision < 15%; no carryover was observed and the method proved to be selective for the aimed SCRAs. The matrix effect, recovery efficiency, and process efficiency studies are being currently carried out. The microextraction technique herein developed is based on an acid-base neutralization between HCl and NaOH, making it an approach significantly more sustainable when compared to conventional choices that rely entirely on organic solvents. In that regard, only 250 μL of dipropylamine was required–an organic solvent considerably less toxic than those used in common extractions, organochlorides for example. In addition, a small volume was used compared to more conventional techniques in forensic analyses, such as solid-phase extraction (> 500 μL). Hence, we were able to successfully detect and quantitate 31 SCRAs at extremely low concentrations (0.01–0.1 ng/mL, respectively) using only 300 μL of plasma, showing that the SHS-HLLME can be used for the analysis of multiple SCRAs in a single run, thus could be a valuable tool in forensic cases. We demonstrated for the first time the application of the novel SHS-HLLME for synthetic cannabinoids. The microextraction technique herein developed has promising applications in Forensic Toxicology, as it consists of a simple and fast procedure that yields high sensitivity with only a few microliters of a biological sample. Also, it avoids the use of highly toxic chemicals and generates less residue, thus it fits the growing trend of using sustainable analytical methodologies, established by GAT and other similar guidelines.

Qualitative screening analysis of urine samples by GC-MS method: Application in forensic toxicology

Toxicological screening is used to identify the ingested toxic in order to confirm or to refute a possible cause of toxic death. A gas chromatography–mass spectrometric (GC-MS) method allows toxicological screening of drugs and pesticides when the toxic is unidentified. The purpose of this study is to describe the data and the results of the screening analyses in forensic toxicology carried out by GC-MS at the toxicology laboratory of Annaba University Hospital. Toxicological screening has been realized following the suspicion of a toxic death, in the context of chemical submission (CS) and for drivers involved in traffic accidents. Analyses were performed on urine samples by GC-MS (a GC-Perkin Elmer, Clarus® 680 coupled to a Perkin Elmer mass spectrometer, Clarus® SQ8T) after a liquid-liquid extraction using tetrazepam as internal standard. Between May 2018 and March 2019, sixteen cases were included. The average age of the cases was 31.62 ± 12.55 years. The sex ratio (M/F) was 07. The requests were mainly from the forensic medicine service of Annaba University Hospital (31.25%) followed by forensic medicine service of SOUK AHRAS, EL KALA and EL BESBES with a rate of 18.75% and last and in the same rank the forensic medicine service of Guelma and the police department with a rate of 6.25%. Among these sixteen cases, the number of positive cases was 14 (87.50%), represented mainly by tobacco markers, and at a rate of 71, 42% followed by drugs, caffeine and cocaine, with a rate of 25%, 23.81% and 4.76%, respectively. The toxicological screening confirmed and identified the presence of urinary drugs and different molecules belonging to various drug classes in a forensic toxicology context.

Case studies in automation of forensic toxicology practices with R

The last decade has seen a considerable growth in data generated by scientific instrumentation. Accountability requirements have grown similarly, requiring forensic toxicology laboratories to generate summary of findings to funders, stakeholders and the general public. This work aims to show how the programming language R and its integrated development environment (IDE) RStudio can be harnessed to speed up data analysis and produce summary reports in three different forensic toxicology applications. R version 3.6.1 or above and RStudio® version 1.2.5001 or above were used in developing and implementing all applications. A wide array of packages were employed, including readr, readtext and docxtractr to import data; tidyr and dplyr for data preparation (“wrangling”); stringr for text manipulation; ggplot2 and plotly for data visualization; rmarkdown to generate HTML and PDF reports; and shiny for the creation of interactive web applications. Automation can begin with the instrumental output analysis. The authors have already published two such examples (Desharnais et al. Journal of Analytical Toxicology 2017;41:261–268 and Desharnais et al. Journal of Analytical Toxicology 2019;43:512–519). A new example presented here is the implementation of trend monitoring, as required for the ISO 17025 accreditation. An interactive application allows for importation of raw instrument output into the database and periodic creation of PDF reports assessing a number of fixed criteria for the wide-scope LC-MS/MS method under analysis. Furthermore, a diagnostic tool allows interactive creation of scatterplots where a variable of interest (area, area ratio, concentration) is plotted for selected time frame, instrument(s), sample type(s) (e.g., standards, QC) and analytes. This allows to not only identify concerns via the presence of warning and action lines, but to explore potential causes of instrument issues with a single tool, which decreases troubleshooting time. This type of interactive data exploration is also possible for casework. Indeed, an application allowing inquiry into basically all variables related to driving under the influence of drugs or alcohol cases was built. The user can obtain summary statistics (e.g., total number of cases) and graphs (e.g., bar plots) by selecting and/or modulating one or more of the following: biological matrix analyzed, drug(s) detected, concentration, administrative region, date and time of the event, etc. As an example, with a few clicks, the user can find out how many DUI cases included a combination of THC and flubromazolam between June 2020 and September 2021 in a given city. This allows for seamless production of yearly reports (within 30 minutes instead of 3 days) as well as quick answers to punctual inquiries by other stakeholders such as police officers, prosecutors and deputy ministers. Annual summary reports can also be extracted from atypical datasets, such as toxicology reports in .docx or PDF format. Using this tool, a periodic update of xenobiotics concentrations found in postmortem casework can be quickly performed. Toxicologists can therefore inform their interpretation with recent data from within the lab–without a time-consuming manual search. It also allows for the production of an annual prospectus designed for the general public, summarizing the caseload and findings. When a suitable Laboratory Information Management System (LIMS) is not available, or mis-adapted to accomplish the desired task, R and its IDE RStudio offer unparalleled power and flexibility to achieve data importation, analysis and presentation. Since they are both available at no cost, the online documentation and community to support the user is vast, which is an advantage for laboratories with scant resources. In order to add another pillar to this network, the creation of an R User Group in Forensic Toxicology is first announced here.

Quantification of phosphatidylethanol combining dried blood spots sampling with Orbitrap-based LC-HRMS platform: Method validation and proficiency testing

Phosphatidylethanols (PEth) are phospholipids specifically synthetised by an enzymatic reaction in presence of ethanol. PEth is a mid-term direct biomarker for alcohol consumption or abstinence, leading to a growing interest for forensic toxicology purposes. Blood deposition onto a filter paper card as dried blood spots (DBS) allows PEth stabilisation which is otherwise easily degraded at room temperature. The goal of this study was to develop and validate a procedure for the quantification of PEth 16:0/18:1, the most abundant analogue, in DBS samples benefiting from the selectivity brought by Orbitrap-based high-resolution mass spectrometry (HRMS). Calibration samples and quality controls (QCs) are volumetrically spotted (10 μL) onto filter paper card (Whatman 903 protein saver) either using HemaXis DB10™ device, or using laboratory micropipette. The whole DBS is manually punched out and the PEth is then directly extracted using an in vial approach with 100 μL of PEth 16:0/18:1-D5 in methanol. Analyses are then performed by LC-HRMS in PRM mode using a QExactive Orbitrap platform. Validation was carried out on with respect to the Food and Drug Administration (FDA) criteria, using three different QC concentrations (39, 87 and 350 ng/mL) over three non-consecutive days. To assess the robustness of our method, an interlaboratory proficiency testing was carried out using 3 different commercial standards at 4 levels (50, 100, 200, and 300 ng/mL) of quality controls (QCs) provided by ACQ Science (Germany). The method was fully validated according to guidelines set forth by the FDA. Calibration curve was built using a weighting factor of 1/x. Trueness was measured between 100.1 and 103.6% for the tested concentrations. Repeatability and intermediate precision were found to be lower than 5%. Linearity was evaluated between the lower limit of quantification of 20 ng/mL and the upper limit of quantification of 2000 ng/mL with an R2 greater than 0.998. Recovery (RE) and matrix effect (ME) evaluated at 2 concentrations ranged from 76 to 99% and 8 to 10%, respectively. During the proficiency test, precision was evaluated below 8.0%, while the accuracy ranged from 93.8% to 105.9% and from 101 to 104.5% when compared with the expected concentration and the mean measured value of all the labs participating respectively. The validation results demonstrate that the use of Orbitrap-based PRM quantification present an interesting alternative over triple quadrupole platforms considered as the gold standard for LC-MS/MS quantitative approaches. Indeed, multiple reaction monitoring (MRM) and PRM provide comparable performance in terms of sensitivity, linearity, dynamic range, precision and repeatability. Moreover, the use of PRM provides access to the full fragmentation spectra without any preanalytical considerations. The proficiency testing results confirm the suitability of PRM mode showing great quantification performances. In addition, those results and especially the repeatability exhibit the precision of the volume control provided by the use of HemaXis™ device. This tool enables the standardisation of the DBS collection at the fingertip in a non-hospital environment either by minimally-trained medical staff or by the patient himself. A rapid, sensitive, and robust method was developed and fully validated for the detection and quantification of PEth 16:0/18:1 in DBS samples. The results of the validation process and the proficiency testing confirm that the use of DBS associated with HRMS PRM-based analyses does not hinder the analytical performances, while providing full information over the MS-MS spectra. The development of analytical tools enabling the use of DBS microsampling that can be performed in almost every environment opens new opportunities toward clinical and forensic toxicological applications.

Hyphenated Techniques in Liquid Chromatography and their Applications in Forensic Toxicology

Conventional analytical methods, such as gas chromatography, high performance liquid chromatography (LC), ultra-violet, and others, are ineffective in addressing the increasing number of problems in forensic toxicology. Hyphenated analytical methods, wherein the separation method are coupled or combined with spectral methods, with the help of a proper interface, are the available alternative options. The key benefits of these methods are the requisites of low limits for detection, shorter analytical time, the possibility of automation, better reproducibility, and high precision and repeatability. This review discusses on some of the hyphenated analytical methods that involve LC as the separation tool, for their most recent applications in the area of forensic toxicology focusing on the screening of drugs of abuse, the usage of alternative matrices for monitoring drug abuse, analysis of chemical warfare agents, determination of doping agents and related substances, natural toxins, environmental poisons, and examination of food produce adulteration. The incorporation of the more user-friendly LC-interfaces, such as atmospheric pressure chemical ionization, and electrospray ionization in the LC- mass spectrometry has increased the popularity of this technique tremendously among scientists of different disciplines. Hyphenated approaches have extremely low constraints regarding the identification and quantification, and offer high reproducibility, with unparalleled potential.

Mass Spectrometry Imaging and Its Application in Forensic Toxicology.

Mass spectrometry imaging （MSI） is a new imaging technology that can simultaneously detect and record the spatial distribution information of multiple molecules on the sample surface without labeling. The main principle of MSI is to combine mass spectrometry with imaging technology and irradiate the sample slice with ion beam or laser to ionize the molecules on its surface, obtain the mass spectrometry signal through the detector, convert the obtained data into pixel points by the imaging software, and then construct the spatial distribution image of the target compound on the tissue surface. The sample preparation for MSI include： sample collection and storage, tissue section, tissue pretreatment, selection and application of matrix. At present, this technology has been widely used in the fields of biomedicine, new drug development and proteomics, and its application in the field of forensic toxicology has also gradually attracted attention. This article reviews the principles and sample preparation process of MSI, describes the application of MSI in abused substances and metabolites of various material matrices, herbal mixtures, latent fingerprints, hair and animal and plant tissues, and discusses the prospects of the application of this technology in forensic toxicology, in order to provide ideas and references for the application of MSI technology in forensic toxicology.

Application of microextraction techniques in alternative biological matrices with focus on forensic toxicology: a review.

The interest in alternative biological matrices (e.g., hair and saliva) for forensic toxicology analysis has increased, and recent developments in sample preparation have targeted rapid, cheap, efficient and eco-friendly methods, including microextraction techniques. For this review, we have gathered information about these two hot topics. We discuss the composition, incorporation of analytes and advantages and disadvantages of different biological matrices, and also present the operation principles of the most reported microextraction procedures and their application in forensic toxicology. The outcome of this review may encourage future forensic researches into alternative samples and microextraction techniques.

Human hair tests to document drug environmental contamination: Application in a family law case involving N,N‐dimethyltryptamine

For 40 years, hair tests have been presented as the best approach to document long-term consumption of a drug. This unique property has found numerous applications in clinical, forensic, and occupational toxicology. However, since the beginning of its implementation in biology, external contamination, with an associated risk of false positive result, has been presented as the key in the final interpretation. Evidence of environmental contamination and subsequent health issues can be the task of any toxicologist. Because of recent progress of analytical equipment, it is now possible to quantify drugs in hair with high level of accuracy and specificity at the pg/mg range. Therefore, segmental hair tests can be used to document environmental contamination and are the objective of this publication. In a family law case, N,N-dimethyltryptamine (DMT), a powerful hallucinogen, has been found in the hair of the partner of a repetitive DMT smoker at 4 to 13 pg/mg in 6 × 1 cm segments, with a regular increase of concentrations from the proximal to the distal hair end. The low measured concentrations and the particular pattern of DMT distribution along the hair shaft seem to be typical of environmental contamination, the older hair (those of the distal part) being for a longer time in contact with the drug. Despite strong decontamination, drugs from the environment can remain bound to the hair matrix and therefore be able to be used to document environmental contamination.

Evaluation of Forensic Data Using Logistic Regression-Based Classification Methods and an R Shiny Implementation.

We demonstrate the use of classification methods that are well-suited for forensic toxicology applications. The methods are based on penalized logistic regression, can be employed when separation occurs in a two-class classification setting, and allow for the calculation of likelihood ratios. A case study of this framework is demonstrated on alcohol biomarker data for classifying chronic alcohol drinkers. The approach can be extended to applications in the fields of analytical and forensic chemistry, where it is a common feature to have a large number of biomarkers, and allows for flexibility in model assumptions such as multivariate normality. While some penalized regression methods have been introduced previously in forensic applications, our study is meant to encourage practitioners to use these powerful methods more widely. As such, based upon our proof-of-concept studies, we also introduce an R Shiny online tool with an intuitive interface able to perform several classification methods. We anticipate that this open-source and free-of-charge application will provide a powerful and dynamic tool to infer the LR value in case of classification tasks.

Development and validation of a simple and rapid thin-layer chromatography–UV densitometry method for the determination of triazophos in human whole blood for forensic toxicological applications

No published studies reported triazophos (TZP) analysis in human whole blood following fatal intoxication. In this study, a method for the determination of TZP in human whole blood using liquid–liquid extraction (LLE) and thin-layer chromatography (TLC)–ultraviolet (UV) densitometry is described. Chromatography was performed on silica gel 60F254 TLC plates using mobile phase n-hexane:acetone (8:2, v/v) and UV densitometric detection at 248 nm. Better extractions were achieved by using ethyl acetate solvent at pH 5 with a recovery of 92.17%. Calibration curve for TZP in blood was linear from 2 to 100 μg/mL (0.04 to 2 μg/spot) and the sensitivity of the method (LLOQ) was 2 μg/mL (0.04 μg/spot). The method showed excellent within-day precision (0.37 to 0.82%, RSD) and between-day precision (0.39 to 1.47%, RSD) for spiked blood samples at 2, 10, and 50 μg/mL. The measured concentrations of TZP in blood did not deviate more than 2.69% under different storage conditions. TZP concentrations in two fatal cases of poisoning were reported. To the best of our knowledge, this is the first validated TLC–UV densitometric method developed to determine TZP in human whole blood.

Identifying and Quantifying Cannabinoids in Biological Matrices in the Medical and Legal Cannabis Era.

Cannabinoid analyses generally included, until recently, the primary psychoactive cannabis compound, Δ9-tetrahydrocannabinol (THC), and/or its inactive metabolite, 11-nor-9-carboxy-THC, in blood, plasma, and urine. Technological advances revolutionized the analyses of major and minor phytocannabinoids in diverse biological fluids and tissues. An extensive literature search was conducted in PubMed for articles on cannabinoid analyses from 2000 through 2019. References in acquired manuscripts were also searched for additional articles. This article summarizes analytical methodologies for identification and quantification of multiple phytocannabinoids (including THC, cannabidiol, cannabigerol, and cannabichromene) and their precursors and/or metabolites in blood, plasma, serum, urine, oral fluid, hair, breath, sweat, dried blood spots, postmortem matrices, breast milk, meconium, and umbilical cord since the year 2000. Tables of nearly 200 studies outline parameters including analytes, specimen volume, instrumentation, and limits of quantification. Important diagnostic and interpretative challenges of cannabinoid analyses are also described. Medicalization and legalization of cannabis and the 2018 Agricultural Improvement Act increased demand for cannabinoid analyses for therapeutic drug monitoring, emergency toxicology, workplace and pain-management drug testing programs, and clinical and forensic toxicology applications. This demand is expected to intensify in the near future, with advances in instrumentation performance, increasing LC-MS/MS availability in clinical and forensic toxicology laboratories, and the ever-expanding knowledge of the potential therapeutic use and toxicity of phytocannabinoids. Cannabinoid analyses and data interpretation are complex; however, major and minor phytocannabinoid detection windows and expected concentration ranges in diverse biological matrices improve the interpretation of cannabinoid test results.

Development and validation of the methods of phenytoin determination in urine by the method of gas–liquid chromatography

Development of the methods of strong medicines determination in human biological liquids for application in forensic and clinical toxicology is one of actual problems of pharmaceutical science, but validation of the analytical methods becomes much more vital and widely discussed problem in analytical toxicology in the last decade.&#x0D; The purpose of the paper was to develop the methods of phenytoin quantitative determination in urine based on the offered before procedure of the analyte determination by the method of gas–liquid chromatography and to carry out validation of the developed methods in the variant of the method of calibration curve according to the approaches offered by authors for choosing the optimal procedure of phenytoin isolation from the mentioned biological matrix.&#x0D; The set of GLC-methods of phenytoin quantitative determination in urine using different procedures of sample preparation – by extraction with chloroform in the acid medium and by maceration with acetonitrile without acidifying and at pH = 2 with subsequent separation of organic layer under the conditions of aqueous phase saturation by ammonium sulphate – has been developed.&#x0D; Validation of the developed methods has been carried out and it has been set that acetonitrile application in the acid medium (рН = 2) is optimal for phenytoin determination in urine – extraction efficiency is maximal and equal to ~97%, and performance of linearity, accuracy and precision is optimal.&#x0D; Application possibility of the offered approaches to validation of quantitative determination methods for forensic and toxicological analysis in the variant of the method of calibration curve for validation of procedure using the method of gas–liquid chromatography has been shown.

Oznaczenie fentanylu i remifentanylu we krwi metodą chromatografii cieczowej sprzężonej z tandemową spektrometrią mas LC-MS/MS

ABSTRAKTWstęp: Fentanyl i remifentanyl to syntetyczne związki należące do grupy opioidów działające agonistycznie na receptory opioidowe. Są powszechnie używane w neuroleptanalgezji w różnych zabiegowych dyscyplinach medycznych, jak: ginekologia, kardiochirurgia, ortopedia oraz onkologia. Ze względu na szerokie zastosowanie i efektywne biologicznie działanie w niskich stężeniach coraz częściej zachodzi potrzeba zarówno jakościowej identyfikacji, jak i jednoczesnego ustalenia stężeń fentanylu i remifentanylu w materiale biologicznym. Dlatego też opracowanie czułych i zarazem precyzyjnych oraz pewnych procedur analityczno-toksykologicznych jest istotnym problemem dla diagnostów pracujących w specjalistycznych laboratoriach toksykologicznych.Celem pracy było opracowanie diagnostycznej procedury jednoczesnego jakościowego i ilościowego oznaczenia fentanylu oraz remifentanylu we krwi pełnej i ustalenie optymalnych parametrów analitycznych. Materiały i metody: Materiałem biologicznym była krew pełna zakupiona w Wojewódzkiej Stacji Krwiodawstwa w Szczecinie. Próby poddawane badaniom uzyskano poprzez obciążenie krwi pełnej wzorcami: fentanylu, remifentanylu i fentanylu-d5 o znanych stężeniach. Izolację analitów z matrycy przeprowadzono techniką ciecz–ciecz, stosując jako czynnik ekstrahujący octan etylu. Uzyskane próbki analizowano z wykorzystaniem wysokosprawnej chromatografii cieczowej sprzężonej z tandemowym spektrometrem mas (LC-MS/MS). Na podstawie uzyskanych wyników wyznaczono średnią, odchylenie standardowe, względne odchylenie standardowe i współczynnik zmienności. W celu zwiększenia czułości metody LC-MS/MS na podstawie analizy napięcia rozkładu cząsteczki w zakresie 0–300 V dokonano wyboru napięcia rozkładu cząsteczki DP = 80. Ustalono odpowiednią energię kolizyjną (35 V). Wysoka rozdzielczość aparatu pozwoliła na wybranie wąskich przedziałów mas dla każdego związku: fentanyl-d5 – 188,143 ±0,0047 Da, fentanyl – 188,1445 ±0,0043 Da i remifentanyl – 317,186 ±0,007 Da. Wyniki: Wyniki analizy fentanylu i remifentanylu pozwoliły na uzyskanie krzywych kalibracyjnych w zakresie 1–200 ng/mL, które wykazały wymaganą liniowość.Wniosek: Otrzymane wyniki umożliwiły identyfikację oraz ocenę ilościową fentanylu i remifentanylu we krwi pełnej. Metoda LC-MS/MS znajduje zastosowanie zarówno w diagnostyce ostrych zatruć, monitorowaniu terapii, jak i praktyce orzeczniczo-sądowej.

Development and validation of an EI-GC-MS method for the determination of 11 antihistamines in whole blood. Applications in clinical and forensic toxicology

Antihistamines are used widely in clinical practice to alleviate symptoms associated with allergic reactions, or to treat insomnia and motion sickness.

Dried blood spots combined to an UPLC–MS/MS method for the simultaneous determination of drugs of abuse in forensic toxicology

A method for the simultaneous determination of 11 illicit drugs, using the dried blood spot (DBS) sampling technique combined with the UPLC–MS/MS technology was developed to study its applicability within the forensic toxicology. The DBS samples, prepared from a blood volume of 50μL and using the Whatman® BFC 180 bloodstain cards, were extracted with a methanol/acetonitrile mixture. The chromatographic separation was performed using an Acquity UPLC® HSS T3 column (100mm×2.1mm, 1.8μm) and an acetonitrile/2mM ammonium formate (0.1% formic acid) gradient. The detection was accomplished with a TQ Detector, operating in the ESI+ and MRM modes. The method was validated in terms of selectivity, matrix effect, extraction recovery (42%–91%), carryover, LOD and LOQ (0.5–1ng/mL and 1–5ng/mL, respectively), linearity (LOQ to 500ng/mL), intraday and interday precision (3.8–14% and 5.3–13%, respectively), accuracy (−9.3% to 7.9%) and dilution integrity. An eight months stability study at room temperature, 2–8°C and −10°C, was also performed, with the best results obtained at −10°C. The procedure was applied to 64 real samples (92 positive results for substances included in this study). The results were compared with the methodologies routinely applied in the laboratory and the statistical analysis allowed to establish an acceptable correlation. This study permitted to determine that the DBS can represent an alternative or a complement to conventional analytical and sampling techniques, responding to some of the present issues concerning the different forensic toxicology applications.