Journal of analytical toxicology最新文献

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.

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.

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.

Tobacco cigarette smoking is the leading cause of preventable diseases and death in the USA. Exposure to secondhand smoke (SHS) can also cause heart disease, lung cancer, and respiratory illness. Cotinine (COT) and trans-3'-hydroxycotinine (HCT) are the primary metabolites of nicotine, the main addictive alkaloid in tobacco products. For many years, we have measured serum levels of COT and HCT in National Health and Nutritional Examination Survey (NHANES) participants to monitor exposure of the US population to active smoking and SHS. As exposure to SHS is decreasing, a more sensitive analytical method is needed to detect the lower levels of these biomarkers for SHS assessment. We developed and validated a new automated method for the detection of COT and HCT in human serum. We implemented a new liquid handling automation system to aliquot and prepare samples using supported liquid extraction. Samples were analyzed by liquid chromatography-tandem mass spectrometry. The new automated sample preparation method increases sample throughput by reducing sample cleanup time to 2 hours for preparing a 96-well plate. The method has excellent sensitivity, specificity, precision (<10%), and accuracy (±15%). We were able to lower the estimated limit of detection (LOD) for COT by 33% and HCT by 73% from our previous LOD. The new LODs for COT and HCT are 0.010 and 0.004 ng/mL, respectively. These lower LODs would enable better detection of SHS in future NHANES surveys.

Oral fluid is considered a favorable matrix for the identification of drug intake mainly because of its simple, observed, non-invasive collection. Fentanyl and fentanyl analog use, misuse, overdose, and deaths are currently occurring at an alarming rate in the USA. The law enforcement community, the Food and Drug Administration (FDA) and the Centers for Disease Control (CDC) are all keenly aware of the urgency in addressing an unmet public health need to identify opioid overdose in individuals as rapidly as possible. As part of a National Institute of Justice grant, the present study was intended to develop and validate an environmentally friendly, rapid, sensitive quantitative method using liquid chromatography coupled to tandem mass spectral detection (LC-MS/MS) for fentanyl and fentanyl analogs in oral fluid collected using the nform rapid test device. Oral fluid samples were subjected to liquid-liquid extraction incorporating bio-renewable solvents where possible, reducing the environmental footprint of the assay. A buffer/salt free mobile phase was employed consisting of 0.1% formic acid in water (95%): 0.1% formic acid in methanol (5%) at a flow rate of 0.8 mL/min; the run time was 4.5 minutes, again reducing environmental impact in terms of salt and solvent usage. The method included fentanyl, 4-anilino-N-phenethylpiperidine; (4-ANPP; desproprionyl fentanyl), acetyl fentanyl, carfentanil, p-fluorofentanyl, valeryl fentanyl, p-fluorobutyrylfentanyl, furanyl fentanyl and benzoyl fentanyl as well as xylazine, which is often detected with fentanyl. The method was validated according to ANSI/ASB 036 (2019) Standard Practices for Method Validation in Forensic Toxicology.

Traditional immunoassay (IA)-based drug screens are limited in their scope of analysis and specificity. Their reliance on the cross-reactivity of antibody reagents is a limiting factor, particularly in light of the emergence of new therapeutics, emerging drugs, and new psychoactive substances (NPS). High resolution mass spectrometry (HRMS)-based techniques can offer improved versatility and specificity and increase the scope of analytical testing. In this study, a validated HRMS screening procedure was used to reanalyze adjudicated blood samples previously tested by immunoassay. IA methods employed enzyme-linked immunosorbent assay (ELISA) and enzyme multiplied immunotechnique (EMIT®). The comprehensive HRMS screen utilized supported liquid extraction (SLE) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Specimens previously tested by IA were reanalyzed using the HRMS screen following long term storage. The LC-QTOF-MS toxicology screen produced an additional 709 positive drug findings (67 compounds) among a population of 919 previously analyzed blood specimens. This study highlights the analytical benefits of MS-based toxicological screening and the advantages of data acquisition modalities that permit retrospective data interrogation.