Toxicology Laboratory Research Articles

In vitro evaluation of maize varieties on the development and reproduction of Chilo partellus (Lepidoptera: Crambidae)

In vitro experiments were conducted to evaluate the developmental and reproductive responses of Chilo partellus on different maize varieties in the Pesticide Toxicology and Application Technology Laboratory, Sindh Agriculture University, Tandojam. The maize stem borer, C. partellus, is a highly destructive insect pest that severely affects both fodder and cultivated crops worldwide. Although pesticides provide limited control of early infestations, they are ineffective against internal feeders and pose environmental risks. In contrast, utilizing resistant maize cultivars offers a sustainable, ecologically safe, and socially acceptable pest management strategy. The study revealed significant differences in the survival rates and developmental durations of C. partellus between hybrid and conventional maize under laboratory conditions. On conventional maize, the total developmental period ranged from 46.22 to 69.34 days (mean: 57.78 ± 11.56 days), whereas on hybrid maize, it ranged from 26.87 to 40.99 days (mean: 33.93 ± 7.06 days). Each developmental stage was shorter on hybrid maize. Females survived longer than males on both maize varieties, but their lifespan was notably shorter on hybrid maize. Regarding reproductive traits, C. partellus showed higher mean fecundity on conventional maize (604 ± 104.67 eggs, range: 500.11-709.44) compared to hybrid maize (302 ± 95.06 eggs, range: 207.44-397.56). Egg fertility was also significantly higher on conventional maize (mean: 55.44 ± 1.81%) compared to hybrid maize (mean: 24.61 ± 1.77%). Statistical analysis confirmed significant differences in developmental durations and reproductive traits between the two maize varieties. These findings highlight the potential of hybrid maize as a resistant cultivar against C. partellus.

Quantitative analysis of cannabinoids and metabolites in oral fluid by volumetric absorptive microsampling combined with UHPLC-HRMS.

With recent evolution of cannabis legalization around the world and multiplication of cannabis derived products, identifying and qualifying cannabis consumption has a proven interest. Although blood, plasma, and urine are common matrices widely used in toxicology laboratories, oral fluid presents specific advantages. In the context of doping tests, addiction consultation or roadside checks, where other matrices are impractical to collect or can be adulterated, oral fluid is a promising matrix that allows a non-invasive, rapid, and monitored self-sampling. However, available devices required a consequent volume of oral fluid,more than 250 µL, sometimes difficult to collect. We present here a fully optimized quantitative method for seven cannabinoids, including four metabolites, in oral fluid, Δ9-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; cannabidiol, 7-hydroxy and 7-carboxycannabidiol; and cannabinol. After self-collection of 20 µL using an accurate and precise volumetric absorptive microsampling device (VAMS®), cannabinoids were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate to increase sensitivity. The successive steps of the proposed method, including biosampling, 1 h sample preparation with derivatization, and acquisition by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry, were fully optimized. A limit of quantification of 0.5 ng/mL (≈10 pg per sampling) was thus targeted, adapted to the legal threshold required by the authorities and to clinical monitoring. Applied to six cannabis consumers, the proposed method made it possible to quantify in 20 µL oral fluid samples, Δ9-tetrahydrocannabinol ranging from 0.5 to 6236 ng/mL, cannabidiol from 0.6 to 190 ng/mL and cannabinol from 0.5 to 118 ng/mL.

Enhancement and Validation of a Quantitative GC-MS Method for the Detection of ∆9-THC and THC-COOH in Postmortem Blood and Urine Samples

Cannabis is frequently detected in forensic investigations and is associated with an increased risk of fatal car crashes. This study aims to develop a method to detect and measure ∆9-tetrahydrocannabinol (∆9-THC) in blood and its metabolite, 11-nor-9-tetrahydrocannabinol-carboxylic acid (∆9-THC-COOH), in urine. The procedure employs two liquid-liquid extraction methods in conjunction with GC-MS in SIM mode. Both compounds were successfully processed, demonstrating the method's ease of use and efficiency. The method was validated for selectivity, identification capability, linearity, precision, limit of detection (LOD), limit of quantification (LOQ), and accuracy. Its effectiveness was further demonstrated by applying it to 30 authentic urine and blood samples from cannabis-related cases, establishing it as a valuable option for routine cannabinoid analysis in forensic toxicology labs.•The linearity range was 25–300 ng/mL for ∆9-THC in blood, and 50–300 ng/mL for ∆9-THC-COOH in urine, and calibration curves for both analytes showed R² values consistently above 0.989, confirming their linearity.•The LOD and LOQ for THC-COOH in hydrolyzed urine were 25 ng/mL and 50 ng/mL, respectively, and for THC in blood, they were 15 ng/mL and 25 ng/mL, respectively.•The variation coefficients were below 14%, and recoveries exceeded 81% for both compounds.

Analysis of new psychoactive substances (5F-EMB PICA, MDMB-4-en PINACA, isotonitazene, 3-fluoroamphetamine, 2-methylamphetamine, flualprazolam) in narcotic cases at the Silk Road crossing point in Turkey

ABSTRACT Turkey’s pivotal position bridging Asia and Europe, amid ongoing civil unrest in the south, has turned it into a crucial hub for drug trafficking along the Silk Road. Yet, there is a notable dearth of research on the influx of new psychoactive substances along this route. The aim of this study was to determine the frequency of use of 5F-EMB PICA, MDMB-4-en PINACA, isotonitazene, 3-fluoroamphetamine, 2-methylamphetamine, and flualprazolam. A quantitative analysis of urine and blood samples of 250 participants was performed which were sent to our Forensic Toxicology Laboratory for drug/stimulant investigation. The analysis was carried out using liquid chromatography sequential mass spectrometry. The average age of starting substance use was 19.51 ± 7.25 years, and the mean duration of substance use was 6.34 ± 5.77 years. The MDMB-4-en PINACA substance was detected in 18.4% of 250 participants, while no other NPS group substances were found. The MDMB-4-en PINACA was detected in 18% of all urine samples and 12.4% of blood samples. The frequency of use of MDMB-4-en PINACA has increased significantly in our region, albeit in a short period of time. MDMB-4-en PINACA should be added to the systematics of toxicology laboratories.

Brewer’s spent grain as a potential sorbent for toxicology methods: Application to antidepressant analysis in urine

The use of antidepressants is well-documented for several health conditions. The determination of these drugs in biological fluids is often important in intoxication cases. However, appropriate sample preparation needs to be employed, such as dispersive liquid phase microextraction (DSPME). Therefore, this study aimed to develop a method for the determination of antidepressants in urine using Brewer’s spent grain (BSG) as sorbent in a DSPME procedure, followed by GC-MS analysis. In this methodology, only 500 µL of urine was required, alongside 15 mg of BSG as the sorbent for the DSPME technique. Desorption step was performed with 500 µL of ethyl acetate:MTBE solution (1:1, v/v), followed by evaporation of the organic layer, reconstitution in acetonitrile and injection into the analytical system. BSG was further characterized by several analytical techniques. The DSPME procedure was optimized using multivariate strategies, and the method was fully validated according to proper guidelines. Lower limits of quantitation (LLOQ) were set between 50 and 200 ng/mL, while linearity was achieved over the specified range of LLOQ to 5000 ng/mL, with R2 ≥ 0.99. Additionally, the method was applied to the analyses of 109 urine samples. Of these, 76 were positive for at least one antidepressant, with the most prevalent being nortriptyline, amitriptyline, and fluoxetine. This study is the first to report the use of BSG as a sorbent for DSPME, demonstrating good efficiency as indicated by the analytical figures of merit. Moreover, the method proved to be applicable in real poisoning case samples. The analytical performance, combined with advantages such as high throughput and a green profile, suggests this method as a valuable alternative for toxicological laboratories.

Establishment of an evaluation method of a chemical-susceptibility for wildlife using counter-QSAR and machine learning

Certain environmental chemicals are harmful to lifeforms. Determining the extent to which they are damaging to animals is challenging as it is ethically reprehensible to expose animals to lethal doses of chemicals in the lab and, besides, not every species of animal can be housed in a laboratory. So, how can experiments involving animals in their natural habitat, and without causing harm, be conducted? A team of researchers at the Laboratory of Toxicology in the School of Veterinary Medicine at Kitasato University in Japan, which includes Junior Associate Professor Kazuki Takeda, is working to develop a way to determine the susceptibility of animal species to given environmental chemicals. Takeda is applying his background in environmental toxicology to the research. The focus for the team is on wild animals and using non-invasive methods. As there is such great interspecies variability in chemical sensitivity, it is difficult to extrapolate toxicity test results obtained from laboratory animals to wild animals. Therefore, the researchers are using genetic information and protein-chemical binding simulations to evaluate interspecies differences in chemical sensitivity. The goal is to develop a model that can predict chemical sensitivity differences between species using simulation methods and machine learning using DNA information obtained from hair.

Assessment of Clinical Toxicological Resources in Countries Belonging to the World Health Organization's Regional Office of the Eastern Mediterranean.

Poison Centers are vital to prevent and manage exposures to chemicals, toxins, and ionizing radiation. However, many countries still lack sufficient poison information center services. Within the World Health Organization's Eastern Mediterranean Regional Office (EMRO) region, we were not able to identify a formal assessment of its toxicological resources. We sought to assess the clinical toxicological resources in the EMRO region and the attitudes, needs, andperspective of the workforce in thatregion. METHODS: This was a two part survey study. First, wedeveloped a poison center questionnaire and scoring system based on the World Health Organization's Guidelines for Establishing a Poison Center; thisinstrument wasadministeredby phone to poison center directors and faculty in the EMROregion. Questions assessed for the presence or absence of important poison center capabilities listed in the WHO guidelines, and points were assigned based on response. Secondly, we administered an electronicworkforce survey to clinical toxicology professionals in the region; participanteligibility for the workforce survey was based on themembership roster ofMENATOX. Out of 22 EMRO countries, 16 countries have an established poison center, and 14 of 16 participated in the poison center survey. The results reflected a wide variability in capabilities amongcountries. Areas such as call centers, clinical toxicology units, laboratory services, and data management were relatively lacking while some other areas including toxicovigilance, chemical incidents and emergencies, antidotes/antivenoms, poisoning information sources, and the availability of free services were relatively stronger.Twenty-seven out of 32 workforce respondents were physicians. Results from that survey highlighted a desire for more support from government and better compensation. Our studyidentified areas of need for the EMRO region.The poison center survey results reflect large disparities amongcountries with some areas being stronger than others. The workforce survey highlighted issues such as compensation and government recognition. Overall, a formal assessment of individual poison centers can provide benchmarks to drive improvement of services, but further research is needed to identify and characterize country-specific needs.

Double Designers: Detection of Bromazolam and Metonitazene in Postmortem Casework.

The identification of novel psychoactive substances (NPSs) in casework at the Los Angeles County Department of Medical Examiner (LACDME) is constantly evolving. The case detailed herein marks the first detection of metonitazene in forensic casework at the LACDME which occurred in August 2023. Furthermore, bromazolam was found in the decedent's system and both substances were identified in drug evidence collected at the death scene. No other drugs were detected and the manner and cause of death were determined as accidental due to effects of bromazolam and metonitazene. The concentrations detected were 1.6 ng/mL and 4.4 ng/mL of metonitazene in the jugular blood and femoral blood, respectively, and 93 ng/mL of bromazolam in the femoral blood. Constraints in screening techniques conducted by toxicology laboratories create challenges in which numerous NPSs available on the illicit drug market can go undetected. Even if labs detect an NPS in their screening methodology, confirmation methods might not cover every NPS, given the impracticality of labs keeping pace with validations as new NPSs emerge in casework. The significance of testing medical evidence collected at death scenes by drug chemistry analysis becomes crucial when initial toxicology results are negative. In cases where there is evidence of potential drug paraphernalia this is especially true as it can be pivotal in determining the cause and manner of death.

B-274 Determining Novel Biomarker Candidates Associated With Heroin and Cocaine Exposure Using Urine Metabolomics

Abstract Background Substance abuse poses an escalating health risk in the United States. While acute drug exposure can be tracked using drugs and their metabolites as biomarkers in clinical settings, there is a notable absence of biomarkers for chronic drug exposure or addiction. The development and availability of such biomarkers could significantly enhance clinicians’ ability to identify patients at high risk of addiction, and more accurately predict disease progression and treatment responses. Methods Our study aimed to discover novel biomarkers associated with substance abuse by analyzing raw mass spectrometry (MS) data from urine comprehensive drug screening (UCDS) conducted at the University of Pittsburgh Medical Center (UPMC). The MS data, acquired through untargeted data collection using LC-qToF-MS at the UPMC Clinical Toxicology Laboratory, was initially preprocessed (including peak identification, alignment, and putative annotation) using MS-DIAL to generate a data matrix. This matrix, consisting of objects (cases) and features (m/z and retention time), was further normalized and analyzed using MetaboAnalyst. A combination of statistical methods, including point biserial correlation analysis, volcano plot, significance analysis of microarrays and metabolites (SAM), empirical Bayesian analysis of microarrays and metabolites (EBAM), partial least squares discriminant analysis (PLS-DA), and random forest (RF), were employed to select features significantly associated with the EIA-results. These features were subsequently evaluated for analyte identification using MS-FINDER. Results Our analysis identified nine significant features associated with 6MAM-EIA for heroin abuse, including 6-monoacetylmorphine itself and norfentanyl. For features associated with cocaine metabolite-EIA, 37 significant features were selected, including multiple cocaine metabolites, nicotine metabolites, and norfentanyl. Some of these selected features are likely products of in-source fragmentation or endogenous metabolites. Conclusions Our study has identified a set of potential biomarker candidates for illicit drug exposure. Notably, norfentanyl was found to be significantly associated with both 6MAM-EIA and cocaine metabolite-EIA, reflecting current trends in illicit drug use. Further chemical identification of these biomarkers is planned for future work.

Development of two ultra-sensitive UHPLC-QqQ-MS/MS methods for the simultaneous determination of hydroxyzine and its active metabolite (cetirizine) in human blood: applications to real cases of forensic toxicology.

Both postmortem toxicological and medical-forensic examinations are very important in the case of analyzing various types of chemical substances. Hydroxyzine (HZ) is a first-generation antihistamine drug with a sedative effect that disrupts cognitive function and affects the ability to drive motor vehicles. Enzymatic oxidation of the hydroxy-methyl group to the carboxyl group leads to the formation of its main metabolite-cetirizine (CZ). CZ is the active substance of antiallergic drugs. Because it does not cross the BBB (blood-brain barrier) easily, it is less likely to cause drowsiness or affect memory and impair cognitive function. Therefore, in criminal studies, it is often important what medication had been taken by a person involved, e.g., in a car accident, HZ or CZ. The analysis of both antihistamine drugs is challenging, as usually very low concentrations of the compound of interest need to be determined. Thus, an ultra-sensitive UHPLC-QqQ-MS/MS method was developed for simultaneous determination of HZ and CZ in biological fluid samples. The lower limit of quantification (LOQ) for HZ and CZ was calculated as 0.345 and 0.3696ng/mL, respectively. Together with a reduced sample volume to 200 μL, it makes the developed method suitable for a sensitive multidrug forensic toxicological analysis. Samples were extracted with simple and fast liquid-liquid extraction (ethyl acetate, pH 9). The present method for the determination of HZ and CZ in human blood proved to be simple, fast, selective, and sensitive. The quantification by LC-MS/MS was successfully applied to the samples coming from 28 authentic biological fluids (blood, urine, vitreous humor, bile and stomach content), both antemortem and postmortem. The performed studies confirm that the developed method is characterized by a high extraction efficiency. Its accuracy, reproducibility, simplicity, and selectivity suggest its application in clinical, toxicological, and forensic laboratories.

Development and validation of multiresidue analysis method for biomonitoring of pesticides and metabolites in human blood and urine by LC-QToF-MS

The widespread use of pesticides and their consequential presence in the environment is a growing concern due to the harmful health effects associated with pesticide exposure. For clinical and toxicology laboratories, a method for simultaneously determining these compounds and their metabolic products in body fluids, such as blood and urine, is important. In the present study, a rapid, sensitive and simultaneous LC-QToF-MS method for detecting multiclass pesticides and metabolites in blood and urine samples has been developed and validated. Four sample preparation procedures, protein precipitation and three different variants of QuEChERS-based extraction were evaluated to find a suitable, simple, and effective sample pretreatment technique. The final optimized sample preparation method (acetonitrile; 400 μl, MgSO4; 40 mg and NaCl; 10 mg) was validated for accuracy, precision, matrix effect, recovery, stability, carryover, and dilution integrity. Analyte recoveries ranged from 75.40 to 113.54 % while accuracy was evaluated in the range of 71.41–108.26 % and precision (%RSD) in the range of 0.01 %–16.85 %. The limit of quantification (LOQ) for all compounds was established in the range of 0.82–7.05 ng mL-1. The developed reliable, robust, and sensitive method was successfully applied for the quantification of target pesticides and metabolites in human blood and urine samples. Evaluated samples resulted in detection of eleven analytes (seven pesticides and four metabolites).

In vitro β-hydroxybutyrate stability evaluation in femoral blood and vitreous humor for integration into forensic toxicology practices

Thanatological biochemistry has gained prominence in determining causes of death, especially when suspected fatal pathologies do not exhibit clear postmortem macroscopic and/or microscopic features, such as in cases of ketoacidosis. Indeed, in these cases, the measurement of β-hydroxybutyrate (BHB) in femoral blood and/or vitreous humor is of particular importance. However, data on its in vitro stability remain scarce, especially in vitreous humor. In this context, the study reported here aims to assess the in vitro stability of BHB. BHB quantification was performed using a liquid chromatography coupled with tandem mass spectrometry method. To investigate BHB stability, two different postmortem matrices were considered: femoral blood and vitreous humor. These matrices were pooled, aliquoted and spiked with BHB at three different concentrations (50 mg/L, 100 mg/L, and 200 mg/L; n = 3). Initial BHB concentrations were established on day 1. Each sample was then divided into two aliquots for storage under two conditions: 20 °C and 4 °C. Analyses were performed on Day 3, 7, 14, and 28. The study revealed no significant degradation of BHB in femoral blood or vitreous humor over time (days 1–28), confirming the robustness and reliability of BHB measurement in these matrices as a postmortem biomarker of ketoacidosis under the tested temperature conditions (+4 °C or −20 °C). These results support a systematic integration of BHB measurement into the routine workflow of forensic toxicology laboratories.

Paraquat Poisoning: Insights from Autopsy, Histology, and Liquid Chromatography with Tandem Mass Spectrometry in Multidisciplinary Forensic Toxicology Practice.

The herbicide paraquat (PQ) is responsible for a significant number of fatalities resulting from self-poisoning. Nevertheless, only a limited number of comprehensive studies focusing on fatal PQ poisoning, which include examination of autopsy findings, histopathology, and quantitative analysis of post-mortem samples, have been published. This study aimed to evaluate autopsy findings, histopathology, and quantitative analysis of PQ in post-mortem human serum samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a simple, sensitive, and specific method. Autopsies were performed on all deaths due to PQ poisoning, and serum samples were sent to the toxicology laboratory for chemical analysis. The method was successfully applied to seven human serum samples, and the results indicate its reliability for detecting PQ. The study reports fatal serum PQ levels ranging from 0.5 to 372.0 µg/mL. The comprehensive data presented in this study can be useful for further research and practical applications.

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.

Exploiting the triple quadrupole mass analyzer for the open detection and tentative identification of synthetic cannabinoid receptor agonists based on common fragmentation pathways

BackgroundThe use of new psychoactive substances (NPS) has emerged as a significant public health concern globally, due to their unknown and unpredictable effects on both physical and mental health. Among them, synthetic cannabinoids receptor agonists (SCRAs) currently stand as the most widely consumed NPS family in Europe. Since the detection of JWH-018 in 2008, the structures of these compounds have evolved to circumvent legislation and/or enhance their effects, consequently increasing the number of reported SCRAs to be monitored. Therefore, new strategies are needed to identify these compounds, whether in seized products or in biological samples. ResultsThis study presents the development of an open method for detecting SCRAs employing a “pseudo-target” screening approach, a strategy previously developed and used in our laboratory for synthetic cathinones identification. The methodology involves monitoring the main product ions and neutral losses derived from 179 SCRAs of the third and fourth generations, based on their fragmentation pathways. This approach allows for the tentative identification of the SCRAs, supported also by the created database. The versatility of the developed methodology is highlighted, extending its utility beyond seizure products or ‘legal highs’, to biological samples. In this sense, it has been successfully applied not only to the detection of SCRAs in research chemicals but also in authentic urine from an anonymous SCRAs consumer, through the identification of a metabolite. SignificanceThis strategy will be particularly useful for the rapid detection of SCRAs in forensic and toxicological laboratories equipped with low-resolution MS/MS instrumentation. This is a valuable tool for the identification and monitoring of SCRAs across various contexts, significantly contributing to public health and forensic security efforts. It is especially beneficial for healthcare providers, enabling them to make informed treatment decisions.

Identification of 5-aminometonitazene and 5-acetamidometonitazene in a postmortem 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 postmortem (PM) case of degradation of metonitazene in unpreserved PM blood using liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS), and putative identification of the degradation/metabolic products 5-aminometonitazene and 5-acetamidometonitazene by liquid chromatography-quadrupole time-of-flight mass spectrometry. 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.

Nephrotoxicity related to the use of high-dose methotrexate: a case report

Introduction. Methotrexate is one of the main chemotherapeutic choices for various types of cancer, the use of the latter at high doses can cause renal toxicity, we report here the case of a patient with acute lymphoblastic leukemia, treated with high-dose methotrexate and which complicated with acute nephrotoxicity and a remarkable delay in the elimination of MTX despite the use of nephroprotection means. Observation and discussion. A 25-year-old woman, admitted to the University Hospital Center of Constantine-Algeria for the treatment of acute lymphoblastic leukemia (ALL) in March 2024. She received a dose of 8500 mg of MTX in 24-hour infusion and folinic acid, hydration with glucose serum and alkalinization with bicarbonates. An MTX dosage was carried out at the toxicology laboratory by an immunoenzymatic method. The samples received at the laboratory correspond to sampling times of 48h, 120h, 192h and 288h from the start of the infusion. This clinical case highlights a remarkable delay in the elimination of MTX; the dosage gave concentrations higher than the standards used for the interpretation of the results of therapeutic monitoring and an increase in creatinine levels. Based on the thesaurus of drug interactions, no interactions were reported. Conclusion. Nephroprotective measures are recommended and monitoring of the blood concentration of MTX is necessary from the start of treatment.

High-performance liquid chromatography coupled to Orbitrap mass spectrometry for screening of common new psychoactive substances and other drugs in biological samples.

The complexity of the drug market and the constant updating of drugs have been challenging issues for drug regulatory authorities. With the emergence of new psychoactive substances (NPS) and the nonmedical use of prescription drugs, forensic and toxicology laboratories have had to adopt new drug screening methods and advanced instrumentation. Using high-performance liquid chromatography coupled with Orbitrap mass spectrometry, we developed a screening method for common NPS and other drugs. Two milliliters of mixed solvent of n-hexane and ethyl acetate (1:1, v:v) were added to 500 μL of blood or urine sample for liquid-liquid extraction, and methanol extraction was used for hair samples. The developed method was applied to 3897 samples (including 332 blood samples, 885 urine samples, and 2680 hair samples) taken from drug addicts in a province of China during 2019-2021. For urine and blood samples, the limits of detection (LODs) ranged from 1.68 pg/mL to 10.7 ng/mL. For hair samples, the LODs ranged from 3.30 × 10-5 to 4.21 × 10-3 ng/mg. The matrix effects of urine, blood, and hair samples were in the range of 47.6%-121%, 39.8%-139%, and 6.35%-118%, respectively. And the intra-day precision was 3.5%-6.0% and the inter-day precision was 4.18%-9.90%. Analysis of the actual samples showed an overall positive detection rate of 58.9%, with 5.32% of the samples indicating the use of multiple drugs.

Analysis of 132 submandibular salivary glands using the Randox Evidence Investigator and Randox DOA ULTRA WB array.

Occasionally, obtaining an adequate or acceptable postmortem blood specimen for drug analysis is not possible due to factors such as decomposition, exsanguination, or embalming. Submandibular salivary gland tissue, one of three major types of salivary gland tissue in the oral cavity of humans, has been reported to be a viable alternative postmortem specimen for toxicological testing. In this study, we evaluated the performance of the Randox Evidence Investigator instrument and Randox DOA (Drugs of Abuse) Ultra Whole Blood Array for the semi-quantitative determination of 21 immunoassays in an alternative matrix, submandibular salivary gland tissue. We analyzed 132 submandibular salivary gland tissue specimens and compared the generated results to concomitantly collected postmortem whole blood specimen results. Oxycodone 2, meprobamate, barbiturate, benzodiazepine assay 1, zolpidem, and buprenorphine all showed perfect agreement (Cohen's kappa score = 1.00) between the submandibular salivary gland tissue results and the postmortem whole blood results; dextromethorphan, fentanyl, benzoylecgonine, methamphetamine, tricyclic antidepressants, oxycodone 1, and opiate showed an almost perfect agreement (Cohen's kappa score = 0.81-0.99); methadone, generic opioids, and amphetamine exhibited substantial agreement (Cohen's kappa score = 0.61-0.80). Tramadol demonstrated fair agreement (Cohen's kappa score = 0.41-0.60). The lowest measure of agreement was observed with cannabinoids, meeting criteria for slight agreement (Cohen's kappa score = 0.01-0.20). An application of the techniques described in this study could be implemented in postmortem toxicology laboratories as well as medical examiners offices to provide preliminary drugs of abuse test results that can be used to direct additional testing. This study highlights the successful integration of a novel specimen matrix and an "off-label" use of an established analytical technique.

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.