Journal of analytical toxicology最新文献

Δ9-tetrahydrocannabinol (Δ9-THC)-dominant cannabis use can cause impairment and risks to workplace safety, which makes the detection of Δ9-THC in oral fluid (OF) important for workplace drug testing. However, cannabidiol (CBD)-dominant cannabis sold as legal hemp products (≤0.3% Δ9-THC) often contain some Δ9-THC. In the present study, participants self-administered 1.5 mL medium-chain triglyceride (MCT) oil containing 100 mg CBD and either 0, 0.5, 1.0, 2.0, 2.8 or 3.7 mg Δ9-THC twice daily for 14 days (n = 10/Δ9-THC dose condition), followed by a 7-day washout period. OF CBD, 7-hydroxy-cannabidiol (7-OH-CBD), 7-carboxy-cannabidiol (7-COOH-CBD), Δ9-THC, 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ9-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-COOH) were measured by LC-MS/MS (cutoff 0.025 ng/mL). Median CBD peaked at 2,198 ng/mL 0.5 h after dosing, which likely reflects a high amount of direct oral cavity deposition, followed by a rapid decline. CBD pharmacokinetics were unaffected by the co-administration of Δ9-THC. CBD and Δ9-THC metabolite concentrations were low (<2 ng/mL), with some accumulation observed for 7-COOH-CBD with twice-daily exposure. After dosing with 100 mg CBD + 0.5 mg Δ9-THC, 1/10 participants had a positive OF test (≥2 ng/mL Δ9-THC) 1.5-6 h after a single acute dose. The rate of positive test results increased as Δ9-THC doses increased to 8/10 participants testing positive after acute doses of 100 mg CBD + 2.8 or 3.7 mg Δ9-THC. A consumer of hemp products might be unaware of the risk of a positive drug test as many products do not specify that they contain Δ9-THC. One positive sample was obtained at baseline, possibly due to direct oral cavity deposition of environmental contamination. Five samples in the CBD alone group, collected 0.5 h after dosing, were positive, likely due to minimal (0.02-0.15%) conversion of CBD to Δ9-THC during analysis. Laboratories are advised to take action to identify specimens where OF Δ9-THC results could be influenced by these factors.

Synthetic cathinones are a class of New Psychoactive Substances (NPS) with 3-chloromethcathinone (3-CMC) accounting for over 46% of NPS-related seizures in 2023. Sold as a racemate, 3-CMC exhibits enantioselective metabolism and pharmacological effects, making enantioselectivity a critical factor in evaluating its toxicokinetics and toxicodynamics. This study aimed to evaluate the enantiomeric biodistribution, metabolic profile, and toxicity of 3-CMC racemate in Wistar rats following acute exposure. For this purpose, a gas chromatography-mass spectrometry (GC-MS) method was validated for quantifying 3-CMC in biological matrices and for characterizing its biodistribution in vivo. Rats were intraperitoneally administered with saline (control) or 3-CMC (10 or 20 mg kg-1, b.w.). Animals were sacrificed 24 h after administration, and plasma, urine, and tissues were collected for biodistribution, biochemical, and histopathological analyses. 3-CMC was exclusively detected in the urine, along with three additional pairs of enantiomeric metabolites. Both 3-CMC and its metabolites exhibit enantiomeric fractions (EF) different from 0.5, indicating enantiomeric enrichment. Administration of 3-CMC significantly decreased plasma levels of creatine kinase-MB, alkaline phosphatase, and aspartate aminotransferase, along with increased levels of glucose and urea. In the urine, decreased levels of albumin were observed. Oxidative stress and energy biomarkers were altered in the brain, lungs, and kidneys. Histopathological analysis revealed morphological alterations in the brain, liver, and lungs at both doses, and in the kidneys at the highest dose. However, no significant alterations were observed in the other tissues. Taken together, our findings suggest enantioselective metabolism and indicate that, although rapidly eliminated by the kidneys, 3-CMC still causes significant toxicity in target organs, such as the brain, liver, lungs, and kidneys. This highlights the high toxicity of the drug or its metabolites, even over short-term exposure.

Etizolam (EZM), a benzodiazepine drug, is a derivative of thienodiazepine. EZM displays an array of biological activities, including as an amnesic, anxiolytic, hypnotic, and muscle relaxant. Given that EZM is associated with instances of lethal intoxication and suicide, it is crucial to establish its exact levels in postmortem (PM) blood. However, EZM concentration at autopsy often diverges from that at the point of death. Here, we demonstrate EZM undergoes hydroxylation and/or oxidation in a mixture of hemoglobin (Hb) and hydrogen peroxide (H2O2) at temperatures between 4 to 45 °C. Mass spectrometry combined with liquid chromatography analysis showed the formation of 1-(4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1, 2, 4]triazolo[4,3-a][1, 4]diazepin-2-yl)ethan-1-ol (α-hydroxyetizolam, M1), 4-(2-chlorophenyl)-2-ethyl-9-methyl-6H-thieno[3,2-f][1, 2, 4]triazolo[4,3-a][1, 4]diazepin-6-ol (M2) and 1-(4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1, 2, 4]triazolo[4,3-a][1, 4]diazepin-2-yl)ethan-1-one when EZM was incubated with Hb/H2O2. M1 and M2 were detected in the PM blood of individuals who had died after ingestion of drug, carbon monoxide poisoning, heart attack or choking, following deliberate ingestion of EZM. Our results show that M1 and M2, formed by Hb/H2O2-mediated PM EZM decomposition, are potential biomarkers that can be used to correct the EZM concentration in PM blood.

Ensuring analyte stability is essential for accurate forensic and clinical detection of sedative-type drugs. This study systematically evaluated the stability of 22 sedative-type drugs and metabolites in human urine under controlled conditions varying by pH (4.0, 7.0), temperature (25 °C, 4 °C, -20 °C), and freeze-thaw cycles (5 cycles), using a fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. While compounds such as midazolam, clobazam, and zolpidem remained highly stable, others-including alprazolam, triazolam, and lorazepam-exhibited notable degradation, particularly under acidic pH and elevated temperature. Flunitrazepam and clonazepam showed distinct degradation with the formation of 7-amino metabolites at neutral pH. Notably, this transformation occurred only in urine and not in phosphate-buffered saline, suggesting a urine-specific mechanism. These findings highlight the importance of compound-specific preservation strategies. In scenarios where analyte identity or sample pH cannot be verified promptly, immediate refrigeration or freezing (ideally at -20 °C), along with minimizing freeze-thaw cycles, is strongly recommended to preserve sample integrity and ensure reliable toxicological interpretation.

Cannabidiol (CBD) is a non-intoxicating cannabinoid found in cannabis and often used for its purported therapeutic benefits. In the form of Epidiolex®, CBD is an FDA-approved treatment for seizure disorders in children. After the 2018 Farm Bill removed hemp (cannabis with <0.3% THC) from the Controlled Substance Act in the United States, non-pharmaceutical CBD became widely available on the retail market. With increased use of CBD, it is important to measure CBD in various biological matrices. In urine, previous studies have measured 7-hydroxy-CBD and 7-carboxy-CBD, analogous to the major metabolites of Δ9-tetrahydrocannabinol (THC). The aim of this study was to identify metabolites of CBD and verify if 7-hydroxy-CBD and 7-carboxy-CBD are the major metabolites. To identify CBD metabolites, 34 urine samples collected after controlled dosing of 100 mg CBD, representing a wide range of time points (1.5-22 hours), and formulations (Epidiolex, syrup, and vaporized administration) were analyzed by liquid-chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) with and without hydrolysis and compared to 11 samples collected after placebo dosing. Thirteen CBD metabolites were identified, including hydroxylation, carboxylic acid formation, alkyl loss, and dihydrodiol formation. The most abundant metabolites included 7-hydroxy-CBD, 6α-hydroxy-CBD, and a novel metabolite indicating hydroxylation on the pentyl sidechain. Most metabolites were >90% conjugated demonstrating that hydrolysis is required for detection in urine. After oral dosing, metabolite concentrations were higher in urine samples collected 4 and 6 h after dosing compared to 1.5 and 11-22 h. CBD concentrations were higher when CBD was administered as Epidiolex compared to synthetically derived CBD in oral syrup or vaping. In conclusion, the results support the use of 7-hydroxy-CBD as a marker of CBD exposure in hydrolyzed urine, but also identified several novel metabolites that might further our understanding of CBD pharmacokinetics.

The increase of Δ9-tetrahydrocannabinol (THC) in breath after cannabis inhalation has been well-documented in the forensic field, but the trends after ingestion of cannabis-infused edibles have not yet been investigated. In this study, participants ingested a cannabis-infused edible and provided breath samples before and at three timepoints after ingestion. Participants were assigned to one of two breath sampling devices. THC was found in most pre-use breath samples, and THC concentration had variable trends after ingestion. Nineteen participants exhibited a maximum in their THC concentration at 47, 92, or 180 min after ingestion, while six participants had their highest THC concentration before the observed ingestion, and four participants had no significant change in THC concentration over the four samples. Five additional cannabinoids were detected in breath. While cannabidiol (CBD) trends followed THC trends for some participants, diverging trends in other participants suggest different biological processing of CBD derived from edibles. This proof-of-concept study shows that THC concentration in breath can increase after the ingestion of cannabis-infused edibles, but the uncertainty of breath measurements and a longer time window need to be further explored.

In postmortem forensic toxicology, the accuracy and reliability of toxicological results are critical to the medicolegal death investigation process. ANSI/ASB Standard 056: Standard for Evaluation of Measurement Uncertainty in Forensic Toxicology establishes the minimum requirements for evaluating measurement uncertainty (MU) in quantitative methods utilized in forensic toxicology. Accurate evaluation of MU increases confidence in results, supports scientific rigor, enables inter-laboratory comparability, and ensures legal defensibility. Using the National Institute of Standards and Technology (NIST) 8-step procedure described in ANSI/ASB Standard 056, postmortem forensic toxicology laboratories can develop customized, flexible MU budget templates that accommodate a variety of analytical workflows and sample preparation techniques commonly used in the field. This manuscript highlights the use of a template that is adaptable to both routine quantitative workflows and those employing method of standard addition, providing example MU calculations for each. By aligning laboratory practices with the NIST 8-step procedure, as well as integrating accreditation requirements and published ANSI/ASB Standards into their quality management system, laboratories enhance the accuracy and reliability of their toxicological results. Adhering to ANSI/ASB Standard 056 ensures that the inherent variability in postmortem toxicological analyses is appropriately assessed and managed in a manner consistent with best practices.

Hair testing is often employed by court-ordered mandatory drug testing (COMDT) programs; however, as of December 2024, many of these programs still do not include fentanyl in their testing panels. Further, testing panels including fentanyl for purposes of workplace testing are rare, and concentrations of fentanyl in hair of people who have used drugs are needed to validate future testing cutoffs. In this study, we analyzed 1025 hair specimens, originally collected for COMDT purposes, for 26 substances, including 13 fentanyl-related compounds. Methamphetamine was the most frequently detected compound (n = 266, 26%), followed by hydrocodone (n = 157, 15%). Fentanyl was the most detected fentanyl-related compound, followed by 4-ANPP. Fentanyl was detected in 151 (15%) hair specimens. 12 specimens contained a fentanyl-related compound with no detectable fentanyl. Of the 163 specimens in which fentanyl or a fentanyl-related compound was detected 31 (19%) had no other analytes detected. Using a cutoff of 1 pg/mg the detection rate for fentanyl was 14.7%. Conversely, most commercial testing laboratories utilize cutoffs between 20 and 100 pg/mg. For the 98 specimens with fentanyl concentrations in the quantifiable range (5-2000 pg/mg), the maximum, mean, and median concentrations were 1946, 223, and 55 pg/mg, respectively. An additional 7 specimens had concentrations greater than the upper limit of quantification of 2000 pg/mg with an estimated maximum fentanyl concentration of 9246 pg/mg. Forty-four specimens contained detectable norfentanyl. The norfentanyl: fentanyl ratios ranged from 0.02 to 0.46 with a mean of 0.09. COMDT programs that do not include fentanyl or employ common commercial cutoffs in their testing protocols for fentanyl are potentially missing drug positive specimens.

The purpose of this study was to develop and validate an analytical method to chromatographically separate, identify, and quantify ortho-methylfentanyl (o-methylfentanyl) in postmortem blood. A combination of simple protein precipitation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized to facilitate chromatographic separation of similar fentanyl analogs, including both meta (m-) and para (p-) methylfentanyl. The analytical range was 1 to 200 ng/mL; the method was validated in accordance with ANSI/ASB Standard 036. In addition to providing details of the validated analytical method, this study details the results of the analysis of 112 case samples (101 postmortem case samples and 11 antemortem case samples) from drug-toxicity related death investigations completed by the toxicology laboratory of the Office of the Chief Medical Examiner, Edmonton, Alberta, Canada. Analytical data is presented which compares concentrations of ortho-methylfentanyl in paired postmortem blood collected from both a visualized, ligated femoral vein together with postmortem blood collected directly from the heart, that is, visualized. Median blood ortho-methylfentanyl concentrations were found to be 5.94 ng/mL (femoral) and 8.04 ng/mL (cardiac). The median cardiac-to-femoral blood concentration ratio across the entire data set was 1.19. The study highlights the varied distribution in the body based on the median concentration of these drugs in postmortem blood.