Forensic Toxicology Research Articles

Rapid Analytical Method for Quantification of Gamma-Hydroxybutyrate (GHB) in Hair by UPLC-MS/MS.

Gamma-hydroxybutyrate (GHB), an endogenous compound related to the neurotransmitter gamma-aminobutyric acid (GABA), is used as a therapeutic and recreational drug and as a "weapon" in drug-facilitated crimes. The very short window of detection of GHB in conventional matrices (blood and urine) makes necessary the use of alternative matrices like hair. Hair has a long window of detection and the possibility to perform segmental analysis, which makes it very useful for proving GHB intake. In the present work, a method for quantification of GHB in hair was developed and validated. Hair (10 mg) was washed twice with dichloromethane and then incubated at room temperature with Milli-Q water in an ultrasound bath for 30 min. Analysis was performed by UPLC-MS/MS using a CORTECS UPLC HILIC (1.6 μm), 2.1 × 100-mm column, and a gradient with acetonitrile and ammonium acetate (10 mM) at pH 6.0, with a total run-time of 10 min. For detection, a triple quadrupole mass spectrometer in ESI negative mode was used. The method was validated, following the criteria established in the "AAFS Standard Practices for Method Validation in Forensic Toxicology" guideline, obtaining satisfactory results for linearity (0.5-50 ng/mg), accuracy (95.0%-103.2%), imprecision (< 10.2%), limit of detection (0.1 ng/mg) and quantification (0.5 ng/mg), exogenous selectivity (no interferences), matrix effect (less than -44.2%), extraction efficiency (> 86.4%), process efficiency (> 46.1%), and autosampler stability (< 4.3%). The method was used for the analysis of 26 authentic hair samples, 25 from non-drug users, obtaining values between < LOQ and 6.25 ng/mg of endogenous GHB and 1 from a former GHB chronic user to prove abstinence.

Evaluation of Spontaneous Overtime Methemoglobin Formation in Post-Mortem Blood Samples from Real Cases in Critical Storage Conditions.

Nitrite/nitrate poisoning is an emerging problem, with an ongoing escalation of reported self-administration with suicidal intent in several countries. Nitrites toxicity mainly consists of their interaction with hemoglobin (Hb), causing its oxidization to methemoglobin (MetHb). In order to give support to the correct procedures for the analysis of these cases, this study aims to evaluate spontaneous sample degradation and consequent MetHb formation in the typical storage conditions of a forensic toxicology laboratory. Two different types of samples have been used in this study: the first stage of our study consisted of a retrospective analysis of blood samples obtained by judicial autopsies already stored in the toxicology laboratory, collected over four years (2018-2021), while the samples used for the second stage were appositely collected during judicial autopsies. The data obtained by the application of a derivative spectrophotometry method on these samples suggest that there seems not to be a maximum threshold for MetHb formation within which it is possible to state with a sufficient grade of certainty that the concentration of MetHb found is consistent with an ante-mortem formation and is not the result of an artifact due to sample degradation and storage conditions. On the other hand, the results suggest that MetHb formation depends on the time passed between sample collection and analysis, so that a tempestive sample processing, performed as soon as the samples are received in the laboratory, is crucial to obtain the maximum reliability and diagnostic values from the data when MetHb quantitation is necessary.

Identification of 5-aminometonitazene and 5-acetamidometonitazene in a post-mortem case: Are nitro-nitazenes unstable?

In recent years the use of 2-benzylbenzimidazole opioids ('nitazenes') has increased with them becoming one of the most prominent synthetic opioid subclasses of novel psychoactive substances. With the increased prevalence there is also a concern of the dangers to public health with the use of nitazenes due to their high potency especially with polypharmacy. To aid in the detection of such compounds it is important that forensic toxicology laboratories maintain up-to-date compound libraries for drug screening methods and that sensitive analytical instrumentation is available to detect the low blood/plasma concentrations of more potent drugs. This includes not only the compounds themselves but also potential metabolites and/or degradation products. Metonitazene is a 'nitro-nitazene' with a nitro group at position 5 of the benzimidazole ring. As a nitro-nitazene there is a potential for bacterial degradation of metonitazene to 5-aminometonitazene, as occurs with nitro-benzodiazepines. In this study we provide evidence from a post-mortem case of degradation of metonitazene in unpreserved post-mortem blood using LC-QQQ-MS, and putative identification of the degradation/ metabolic products 5-aminometonitazene and 5-acetamidometonitazene by LC-QTOF-MS. The results from LC-QQQ-MS analysis indicated that there did not appear to be such degradation in preserved (fluoride/oxalate) blood. These results suggest that nitro-nitazenes may be subject to similar in vitro stability/degradation issues as nitro-benzodiazepines. These breakdown products should be added to instrument libraries to aid in the detection of the use of nitro-nitazenes, and nitro-nitazenes should be quantified in preserved blood samples where available.

Application of a molecular networking approach using LC-HRMS combined with the MetWork webserver for clinical and forensic toxicology

Backgrounds and aimsIn toxicology, LC-HRMS for untargeted screening yields a great deal of high quality spectral data. However, there we lack tools to visualize/organize the MS data. We applied molecular networking (MN) to untargeted screening interpretation. Our aims were to compare theoretical MS libraries obtained in silico with our experimental dataset in patients to broaden its application, and to use the MetWork web application for metabolite identification. MethodsSamples were analyzed using an LC-HRMS system. For MN, data was generated using MZmine, and analyzed and visualized using MetGem. MetWork annotations were filtered and this file was used for annotation of the previously obtained MN. Results155 compounds including drugs found in patients were recorded. Using this dataset, we confirmed in 60 patients intake of tramadol, amitriptyline bromazepam, and cocaine. The results obtained by the reference methods were confirmed by MN approaches. Eighty percent of the compounds were common to both conventional and MN approaches. Using MetWork, metabolites and parent drugs such as amitriptyline, its metabolite nortriptyline and amitriptyline glucuronide phase 2 metabolites were anticipated and proposed as putative annotations. ConclusionThe workflow increases confidence in toxicological screening by highlighting putative structures in biological matrices in combination with CFM-ID (Competitive Fragmentation Modeling for Metabolite Identification) and MetWork to extend the annotation of potential drugs even without a reference standard.

Detection of N-desethyl etonitazene in a drug checking sample: Chemical analysis and pharmacological characterization of a recent member of the 2-benzylbenzimidazole “nitazene” class

The emergence of 2-benzylbenzimidazole “nitazene” opioids is stirring up the recreational synthetic opioid market. Many nitazene analogues act as potent agonists at the µ‑opioid receptor (MOR), as demonstrated in various in vitro and in vivo studies. Severe intoxication and overdose deaths associated with nitazene analogues are increasingly being reported. Nitazene opioids are classified as a public health threat, stressing the need for close monitoring of new developments on the recreational drug market. This study reports on the detection of N-desethyl etonitazene in a sample handed in by a recreational drug user at a Swiss drug checking service in August 2023. The person bought the sample through an internet source where it was stated to contain isotonitazene. Chemical analyses were conducted to characterize the sample, i.e. nuclear magnetic resonance (NMR), capillary electrophoresis (CE), and high-resolution mass spectrometry (HRMS). The sample was additionally investigated using two different in vitro MOR activation assays. NMR and high-performance liquid chromatography (HPLC) coupled to HRMS confirmed the presence of N-desethyl etonitazene at a high purity and in the absence of isotonitazene and etonitazene. N-Desethyl nitazene analogues have been detected before as metabolites of isotonitazene and etonitazene. However, as first seen with N-desethyl isotonitazene, they are now emerging as standalone drugs. The applied bioassays demonstrated increased efficacy and approximately 6–9-fold higher potency of N-desethyl etonitazene at MOR compared to fentanyl. N-Desethyl etonitazene showed EC50 values of 3.35 nM and 0.500 nM in the β-arrestin 2 recruitment and Aequoscreen® assays, respectively. The opioid activity present in the collected sample was additionally evaluated using the bioassays and showed good overlap with the reference standard, in line with the analytical purity assessment. This demonstrates the potential of these bioassays to provide a rapid opioid activity assessment of authentic samples. The emergence of other N-desethyl nitazene analogues must be considered during forensic and clinical toxicology casework, to avoid misclassification of intake of such analogues as metabolites. Finally, drug checking services enable the close monitoring of market developments and trends and are of great value for early warning and harm reduction purposes.

Potential of high-resolution mass spectrometry for identification and structural elucidation of scopolamine metabolomic biomarkers in a confirmed case of Brugmansia intoxication. Specially application in drug-facilitated crimes

In forensic toxicology, scopolamine remains as one of the most challenging alkaloid in terms of analytical detection. Given its rapid elimination, the detection window in common matrices is short. Taking advantage of a real case of Brugmansia intoxication, a metabolic study was carried out. We report the real case of a 16-year-old boy admitted to an Emergency Unit after consumption at high school of a beverage made of Brugmansia dried flowers. The medical staff noticed agitation, mydriasis and tachycardia. Scopolamine and atropine were positively detected in biological fluids using liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS/MS). To better characterize the intake and identify metabolic biomarkers, we developed a data mining workflow specific to tropane alkaloids and applied it to the urine sample. This metabolic profile may be useful in providing analytical methods with a wider detection window particularly in drug-facilitated crimes (DFC). Scopolamine and atropine metabolites were predicted in silico with GLORYx freeware to assist in metabolite identification. The previously published metabolic pathways for scopolamine and atropine in mammals were studied as well. A total of fifteen phase I and II metabolites were tentatively identified for scopolamine, while one metabolite was detected for atropine. In addition, we identified some tropane alkaloids from the plant that were also metabolized. These metabolites can be used as biomarkers of exposure to Solanaceae plants and may also be useful to distinguish between natural product use and clinical therapy.

Society of Forensic Toxicologists, Inc.

Society of Forensic Toxicologists, Inc.

Direct and selective alkylation of indazole-3-carboxylic acid for the preparation of synthetic cannabinoids and metabolites

The most common method for the synthesis of synthetic cannabinoids with an indazole core utilizes methyl indazole-3-carboxylate as a starting material. However, this method commonly suffers from poor selectivity and low yield. In the current work, a method using indazole-3-carboxylic acid as the starting material was developed and successfully applied in the synthesis of nine synthetic cannabinoids and six of their metabolites. The method provided selective alkylation at the N1-position and resulted in overall yields in the range of 51–96 %. Five of the synthetic cannabinoids have not been reported in the literature and were synthesized in a proactive attempt to predict future SCs. All of the synthesized metabolites have previously been encountered in either in vitro studies or authentic urine samples. Hence, the method proved to be useful for production of SC metabolites, which are relevant for forensic toxicology. All synthesized compounds were characterized with NMR and LC-QTOF-HRMS.

Structural elucidation of tramadol, its derivatives, and metabolites using chemical derivatization and liquid chromatography-high-resolution tandem mass spectrometry.

Tramadol (T) is a strong painkiller drug that belongs to the opioid analgesic group. Several accidental intoxication cases after oral administration of T have been reported in the past decade. Tramadol, its derivatives, and metabolites present information-limited mass spectra with one prominent peak representing the amine-containing residue; therefore, their structural determination based on both electron impact mass spectrometry (EI-MS) and ESI-MS/MS spectra could be misleading. A novel analytical method for the structural elucidation of tramadol, its four homologs, and its two main phase I metabolites (N-desmethyltramadol and O-desmethyltramadol) was developed using chemical modification and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) with Orbitrap technology. After chemical derivatization, each of the investigated T series exhibited informative mass spectra that enabled better exposition of their structures. The developed method was successfully implemented to explicitly identify the structures of tramadol and its N-desmethyltramadol metabolite in urine samples at low ng/mL levels. An efficient derivatization-aided strategy was developed for rapidly elucidating the structure of tramadol-like compounds. The method is intended to assist forensic chemists in better diagnosing T and its analogs and metabolites in clinical or forensic toxicology laboratories.

Interlaboratory comparison of phosphatidylethanol in dried blood spots using different sampling devices.

Phosphatidylethanol (PEth) has become an important marker to assess drinking behaviour and monitor abstinence. Despite its increasing use, knowledge on robustness and standardization and comparability of methods and results are still limited. In 2022, the first international consensus for the use of PEth and its interpretation was published. To establish an experience-based foundation for further harmonization, three rounds of interlaboratory comparison using microsamples were conducted. Participating laboratories sent their sampling devices to the laboratory of Forensic Toxicology at the University of Bern, where for each round, four different authentic blood samples were applied to the devices and sent back. The PEth 16:0/18:1 target concentrations covered a range between 16 and 474 ng/mL (0.023 and 0.676 μmol/L, respectively) and included sample concentrations close to the decision limits of 20 and 200 ng/mL (0.025 and 0.28 μmol/L, respectively). Evaluation of the results based on guidelines by Horwitz and the Society of Toxicological and Forensic Chemistry (GTFCh) showed that 73% of all participating laboratories quantified and reported all samples (N = 4 for each round) within the acceptable limits. More than 90% quantified and reported at least one sample within the acceptable limits.

Standardization via Post Column Infusion-A Novel and Convenient Quantification Approach for LC-MS/MS.

Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach-quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques.

Toxicity of Bromo-DragonFLY as a New Psychoactive Substance: Application of In Silico Methods for the Prediction of Key Toxicological Parameters Important to Clinical and Forensic Toxicology.

Bromo-DragonFLY is a synthetic new psychoactive substance (NPS) that has gained attention due to its powerful and long-lasting hallucinogenic effects, legal status, and widespread availability. This study aimed to use various in silico toxicology methods to predict key toxicological parameters for Bromo-DragonFLY, including acute toxicity (LD50), genotoxicity, cardiotoxicity, health effects, and the potential for endocrine disruption. The results indicate significant acute toxicity with noticeable variations across different species, a low likelihood of genotoxic potential suggesting potential DNA damage, and a notable risk of cardiotoxicity associated with inhibition of the hERG channel. Evaluation of endocrine disruption suggests a low probability of Bromo-DragonFLY interacting with the estrogen receptor α (ER-α), indicating minimal estrogenic activity. These insights from in silico investigations are important for advancing our understanding of this NPS in forensic and clinical toxicology. These initial toxicological examinations establish a foundation for future research efforts and contribute to developing risk assessment and management strategies for using and misusing NPS.

Development of a GC-MS Method for the Analysis of Selected Opioids in Human Hair Samples

Background: Hair samples are recognized as alternative biological specimens in forensic and clinical toxicology for detecting drug abuse and poisoning. Forensic testing for opioids in hair has become a useful diagnostic measure for assessing chronic drug usage through segmental analysis. However, accurate, sensitive and specific analytical methods are needed. The aim of this study was to introduce a simple, sensitive and specific GC-MS method for the identification and quantitation of selected and commonly abused opioids (tramadol, methadone, morphine, and codeine) in hair samples. Methods: After external decontamination, a 50 mg portion of powdered hair sample was combined with hydrochloric acid (0.1 M) and incubated on a magnetic stirrer at 56°C for 16 hours. Then, 1 mL of sodium hydroxide (0.1 M) and 2 mL of phosphate buffer (1 M, pH=8.4) were added. Chloroform-isopropanol (ratio: 80:20 V/V) was utilized as the extracting solvent and the sample was homogenized and centrifuged for 5 minutes (at 3500 rpm). After centrifugation, the organic phase was dried using dry nitrogen gas. The sample was derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The blank, standards, and real samples were subsequently analyzed by GC-MS.Results: The limits of detection in the linearity experiments ranged from 0.12 to 0.21 ng/mg. According to the validation results, the method exhibited linearity in the concentration range of 0.1-2.5 ng/mg for all analytes, with calibration curve slopes ranging from R2=0.98 to 0.99. Good inter and intra-day precision relative standard deviations (RSDs) were observed to be &lt;3.5% for all compounds. Extraction efficiency varied from 91.8 to 102.4%. Conclusion: The validation and analysis of actual samples indicate that this method is straightforward, sensitive, and specific for the analysis of opioids in routine hair analysis.

Forensic toxicology backdates the use of coca plant (Erythroxylum spp.) in Europe to the early 1600s

Cocaine hydrochloride salts are one of the most commonly used drugs of our days, yet there is very little hard evidence regarding when people started consuming such an extensively popular drug in Europe. In this paper, we report the exceptional finding of Erythroxylum spp. in human remains dated to the 1600's in Milan, Italy.Toxicological analyses were performed on preserved human brains revealing the first evidence of Erythroxylum spp. use in Europe before the 19th century, backdating our understanding of the presence of the plant by almost two centuries. Specifically, the alkaloid of cocaine was detected in two separate biological samples and can be associated to Erythroxylum spp. consumption. Given that the plant was not listed inside the detailed hospital pharmacopeia, it may not have been given as a medicinal remedy but may have been used for other purposes. This study demonstrates the importance and the potential of the application of toxicological analyses to archaeological contexts and allows to backdate the arrival of the Erythroxylum spp. in Europe by almost two hundred years.

Assessment of Δ9-THC and Δ9-THCCOOH bias, precision, and ionization suppression/enhancement between solid tissue homogenate and supernatant by LC-MS-MS.

Liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS) assays are frequently utilized for screening and confirmatory purposes in the forensic toxicology laboratory. While these techniques are excellent for the targeted identification and quantitation of a wide variety of drug classes, validation and determining fit-for-purpose is a significant requirement for each method. In the USA, the American National Standards Institute and Academy Standards Board first edition of Standard 036 currently serves as a primary resource in forensic toxicology method validation and mandates that laboratories evaluate critical performance characteristics to help ensure the production of forensically defensible results. Due to the variability of specimen quality frequently encountered in the discipline of postmortem toxicology, the State of Maryland Office of the Chief Medical Examiner Forensic Toxicology Laboratory routinely analyzes solid tissue specimens as part of the medicolegal death investigation process and evaluates liver as a representative solid tissue matrix during method validation. Authentic postmortem specimens (e.g. liver, kidney, skeletal muscle, and spleen) were used to investigate the effects of analyzing solid tissue homogenate versus solid tissue supernatant on bias, precision, and ionization suppression/enhancement of Δ9-THC and Δ9-THCCOOH. Bias was <20% for Δ9-THC and Δ9-THCCOOH in liver homogenate and supernatant with a single exception of the low QC concentration for Δ9-THC in liver homogenate (-29%). Within-run and between-run CV was <20% for Δ9-THC and Δ9-THCCOOH in liver homogenate and supernatant. Δ9-THC and Δ9-THC-d3 exhibited significant ion suppression in both liver homogenate and supernatant, while Δ9-THCCOOH and Δ9-THCCOOH-d3 showed both ion suppression and enhancement in these matrices. Noticeable quantitative differences were observed in authentic postmortem solid tissue homogenate and supernatant specimens despite evaluating identical tissue samplings. A brief discussion of the results is presented using a validated LC-MS-MS method for the confirmation and quantitation of Δ9-THC and Δ9-THCCOOH in postmortem casework.

Ethanol concentrations in various biological specimens: Living and postmortem forensic toxicology analysis and comprehensive literature review

Alcohol use upsurges the risk for many chronic ill-health consequences such as hepatitis, malignancies, and disastrous outcomes like road traffic accidents ending in fatal injuries. Biochemical and toxicological analysis of different body fluids is crucial for identifying the cause of death and postmortem interval in many forensic cases. Blood, urine, and vitreous fluid are the most valuable body fluids for detecting alcohol during any toxicological analysis. Alcohol is responsible for widespread morbidity and mortality worldwide. Blood alcohol concentration (BAC) is a necessary toxicological test to investigate various crime and accident scenes. This study comprehensively explores the demographic characteristics, BAC distribution, and correlations of alcohol concentrations in postmortem and living cases. Postmortem cases (N = 166) reveal intriguing demographic patterns, with notable variations in year distribution, nationality, sex, age groups, occupation, smoking habits, place of death, and psychiatric history. Living cases (N = 483) exhibit distinct demographic profiles, emphasizing differences in year distribution, nationality, sex, age groups, and smoking habits.Analysis of BAC distribution reveals diverse patterns in both postmortem and living cases, providing valuable insights into the prevalence of different BAC levels in each group. Correlation analyses unveil strong associations between alcohol concentrations in various biological samples in postmortem cases, highlighting the interdependence of blood, vitreous, and urine alcohol concentrations. Conversely, living cases display a moderate positive correlation between blood and urine alcohol concentrations.Comparative analyses showcase significant differences in mean alcohol concentrations between postmortem and living cases, suggesting variations in alcohol metabolism and distribution. These findings underscore the importance of considering temporal factors in interpreting alcohol concentrations in forensic and clinical contexts.In conclusion, this study enhances our understanding of alcohol-related incidents by delineating demographic profiles, BAC distributions, and correlations between different biological samples. Such insights are crucial for refining investigative and clinical approaches, contributing to the broader fields of forensic science and public health.

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.

Synthetic cannabinoid identification in cases associated with blue lotus and valerian root vaping products.

Synthetic cannabinoids emerged in the early 21st century and have continued to evolve and flourish to present day. Like other novel psychoactive substances (NPS), synthetic cannabinoids have been sold under the guise of legitimate products. Some examples include "potpourri," "incense," and herbal material. Between May 2020 and December 2023, the United States Army Criminal Investigation Laboratory, Drug Chemistry Division (USACIL) received 29 seized drug cases mentioning "blue lotus" or "valerian root." In 90% of these cases, at least one exhibit contained one or more synthetic cannabinoids. During the same timeframe, the Armed Forces Medical Examiner System, Division of Forensic Toxicology received 65 toxicology cases that contained synthetic cannabinoids and/or their corresponding metabolites where case history mentioned "blue lotus." The most frequently observed synthetic cannabinoids between laboratories were 5F-MDMB-PICA, ADB-BUTINACA, and MDMB-4en-PINACA. Innocuous branding and marketing may deceive law enforcement, investigators, and healthcare providers into believing that the adverse effects of erratic behavior, sedation, slurred speech, and hallucinations are a result of toxicity from botanical extracts (e.g. apomorphine and nuciferine in blue lotus). Due to the dangerous nature of these NPS, synthetic cannabinoid screening is recommended for all cases where there is suspected use of vaping products suggested to contain "blue lotus" or "valerian root" as vendors continue to conceal the presence of these compounds.

Multi-drug extraction using octanol supported rotating cellulose paper disc (RPD) device from complex biological matrices: Fabrication and application in forensic case work

For the first time, a novel rotating paper disk (RPD) device to simplify the sample preparation process for the analysis of a broad spectrum of analytes with varying log Kow (i.e., 0.65–5.4) values is introduced.These analytes encompass diverse classes, including benzodiazepines (such as diazepam, chlordiazepoxide, flurazepam), barbiturates (such as barbital, allobarbital, phenobarbital), antihistamines (i.e., pheniramine, chlorpheniramine), antidepressants (such as fluoxetine, amitriptyline, venlafaxine), antipsychotics (like clozapine, chlorpromazine), opioids (i.e., tramadol) and z-drugs (such as zolpidem). RPD synergistically utilizes the adsorption capabilities of cellulose paper (solid substrate) in conjunction with 1-octanol supported onto the paper. In order to fabricate the RPD device, a magnetic bar was sandwiched between octanol supported cellulose paper layers. RPD was then directly immersed into the diluted biological samples (postmortem biological matrices i.e blood and urine) and allowed to rotate at 400 rpm for 30 min for optimum adsorption of target analytes. Subsequently, adsorbed analytes were back-extracted into 1 mL of methanol without any external diffusion process and finally determined by gas chromatography-mass spectrometry (GC-MS). Design of experiment-based optimization was performed using Box-Behnken design (BBD) for the optimization of significant factors of sample preparation such as pH of the sample, stirring speed, extraction time and ionic strength. Under the optimized conditions, the method was found to be linear in the range of 0.1–10 μg mL−1 for blood and urine samples. Limit of quantification (LOQ) for blood and urine samples were determined within the range of 0.084–0.099 and 0.060–0.078 μg mL−1, respectively. Intra-day and inter-day precisions were less than 6 and 10%, respectively. Good relative recoveries in the range of 50.4–98.2%, were obtained for the proposed procedure. Furthermore, the proposed method has been effectively applied in forensic toxicology case work. Additionally, the proposed method underwent evaluation for its whiteness and blueness using the RGB12 algorithm and BAGI software, respectively.

Detection and quantification of selected cannabinoids in oral fluid samples by protein precipitation and LC-MS/MS

Cannabis is the most widely consumed illicit drug worldwide. As consumption rates increase, partially due to the decriminalization of its use for medicinal and recreational purposes, analytical methods for monitoring different cannabinoids in several biological matrices have been developed. Herein, a simple and fast extraction procedure to extract natural cannabinoids from oral fluid (OF) samples was developed and fully validated according to the ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Using only 0.2 mL of neat OF, the analytes [Δ9-tetrahidrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD)] were extracted by protein precipitation with a mixture of methanol:acetonitrile (80:20, v/v); the extracts were centrifuged, evaporated to dryness and reconstituted in 100 µL of methanol. Analysis was performed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The developed methodology produced linear results for all compounds, with working ranges of 0.1–50 ng/mL for THC, 0.5–50 ng/mL for THC-OH, CBN and CBD, and 0.05–1 ng/mL for THC-COOH. Ion suppression was observed for THC, CBN and CBD, which did not impair sensitivity considering the low limits of quantification (LOQs) and limits of detection (LODs) obtained (which varied between 0.05 and 0.5 ng/mL). The extraction procedure produced great recoveries, and the compounds were stable. No interferences were found, and the method proved to be extremely fast, selective, precise, and accurate for use in routine analysis. The method was successfully applied to authentic samples.