Journal of analytical toxicology最新文献

The aim of this study was to develop and validate a quantitative method for the analysis of designer benzodiazepines in postmortem blood samples using micro-QuEChERS extraction and liquid chromatography tandem mass spectrometry (LC-MS/MS). A comprehensive optimization of the method was performed using a multivariate statistical approach, incorporating validation criteria in line with established practices for method validation in forensic toxicology. The method showed linearity between 1 and 200 ng/mL (r2>0.990), with good imprecision (<9.8%) and inaccuracy (<11.1%) evaluated at three different quality control concentrations. Matrix effects and recovery rates were found to be better than 58% and 77.5%, respectively. No carryover or interferences were detected during the analysis. The method was effectively utilized on two real forensic postmortem blood samples, both of which tested positive for bromazolam, showing concentrations of 31 ng/mL and 40 ng/mL. The micro-QuEChERS extraction method demonstrated satisfactory analytical performance and is an environmentally sustainable option, minimizing the use of solvents and reagents, with potential for application in both clinical and forensic analyses, aligning with green analytical toxicology principles.

Immunoassay-based urine drug screens are widely employed in clinical toxicology due to their speed, low cost, and ease of automation. However, these assays are inherently limited by antibody cross-reactivity, which can result in false-positive findings and incorrect interpretations with major implications for patient care, employment, and legal outcomes. This review updates prior literature by analyzing reported false-positive interferences published between 2013 and 2024 across commonly screened drug classes, including opioids, amphetamines, benzodiazepines, cannabinoids, barbiturates, phencyclidine (PCP), cocaine, ethanol, and ethyl glucuronide. A total of 61 studies met inclusion criteria from 569 unique publications retrieved via PubMed. Each report was categorized by level of evidence, ranging from single case reports to controlled spiking experiments. Despite advances in antibody specificity, immunoassay drug screens remain presumptive and require confirmation by orthogonal techniques such as gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS/MS). This review provides updated reference data on known interferents, emphasizes the need for laboratorian-clinician communication, and supports continued education on assay limitations. Reliable interpretation of presumptive immunoassay drug screen results remains essential to prevent inappropriate clinical care decisions.

2,4-Dinitrophenol (2,4-DNP) was discovered and popularized in the 1930s as a weight-loss drug. Due to multiple adverse effects, including death, its use as a prescription drug was banned in 1938. However, toxicity cases have risen over the past two decades as 2,4-DNP is easily obtained illegally online. In a fatal case at our hospital involving rapid, unexplained deterioration, 2,4-DNP was identified through a full toxicological analysis of blood and urine. An LC-MS/MS method for detecting and quantifying 2,4-DNP in plasma and urine was developed and validated according to European Medicine Agency (EMA) criteria. It also allowed quantification of its main metabolites 2-amino-4-nitrophenol (2A-4NP) and 4-amino-2-nitrophenol (4A-2NP) in urine. In plasma, 2A-4NP was only semi-quantifiable; 4A-2NP was undetected, likely due to matrix effects reducing sensitivity. Results obtained with and without enzymatic hydrolysis showed that 2,4-DNP-glucuronide and 2A-4NP plus its glucuronide are the primary metabolites, while 4A-2NP and its glucuronide are less prominent. No sulfate conjugates were detected. This study is the first to compare sample preparation with and without enzymatic hydrolysis, offering new insights into 2,4-DNP metabolism and the relative importance of its major metabolites and their glucuronides.

Recently developed dried blood analysis methods for cannabinoid quantitation utilize small blood volumes, making them microsampling-compatible, but are limited by hematocrit-related bias for dried blood spots (DBS) and higher consumable costs for volumetric absorptive microsampling (VAMS®). To address these issues, we developed a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying cannabinoids in 50 µL of liquid whole blood, providing a practical microsampling alternative to dried blood approaches. Using liquid-liquid extraction (LLE) with sodium hydroxide alkalinization and acetonitrile precipitation, followed by quantitative analysis on an Agilent 6495 liquid chromatography-triple quadrupole (LC-TQ) mass spectrometer, we achieved lower limits of quantitation (LLOQs) of 0.10 ng/mL for Δ9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabigerol (CBG), 0.20 ng/mL for cannabidiol (CBD), 0.50 ng/mL for 11-hydroxy-THC (11-OH-THC), and 1.0 ng/mL for 11-nor-9-carboxy-THC (THC-COOH). Calibration was linear from the LLOQ to 300 ng/mL for all analytes. To our knowledge, this is one of the first validated LLE approaches for cannabinoid quantitation in less than 100 µL of liquid whole blood. Further, it achieves sub-ng/mL sensitivity, exceeding the LLOQs of most published methods which require ≥100 µL of whole blood. We anticipate particular utility for our method in obtaining evidence from suspected impaired drivers at the roadside when paired with capillary microsampling, such as via finger prick. This approach enables measurement of THC levels at the time of driving, thereby overcoming current limitations, including the decrease in THC levels that occurs with delayed blood sampling, requirement for larger sample volumes (≥100 µL), and dependence on trained phlebotomists for venipuncture.

Rocuronium bromide is a non-depolarizing neuromuscular blocking agent (NMBA) commonly used in anesthesia for its rapid onset and intermediate duration of action. While its therapeutic use has been well-documented in humans and experimentally in dogs, this report describes the first known case of its malicious use in the fatal intoxication of a canine. Herein we describe the case of a six-year-old male Plott Hound found deceased under suspicious circumstances. Postmortem investigation revealed focal hemorrhages near potential injection sites and toxicological analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the presence of rocuronium in muscle tissue, heart blood and urine. The dog had no surgical history or indication for anesthesia. However, rocuronium concentrations mirrored those seen in anesthetized human patients under ventilatory support. The owner, a licensed pharmacist, was suspected of drug theft and aggravated animal abuse. This case highlights the lethality of NMBA's when misused outside of the clinical setting and further underscores a collaborative, multi-institutional, multidisciplinary approach to veterinary forensic investigations.

The ability to distinguish illicit fentanyl use is becoming increasingly critical in toxicological investigations. 4-Anilino-N-phenylethylpiperidine (4-ANPP), also known as despropionylfentanyl, is both a precursor in illicit fentanyl production and a minor metabolite frequently detected alongside fentanyl in forensic toxicology. Its presence may assist in distinguishing medical and illicit fentanyl sources. This study evaluated 4-ANPP concentrations and 4-ANPP: fentanyl (4-ANPP to fentanyl) ratios in clinical (presumed medicinal) and postmortem (forensic) submissions to ascertain trends that may aid source attribution and toxicological interpretation. Blood and serum/plasma (s/p) samples were analyzed via liquid-liquid extraction followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) throughout 2023. A total of 32,723 forensic and 1,015 clinical cases positive for fentanyl were included in the analysis. Most clinical 4-ANPP concentrations (69%) were below 0.50 ng/mL, compared to 20% of forensic cases. Forensic blood samples with reportable 4-ANPP concentrations (N = 29,701) had a median of 2.1 ng/mL (mean ± std dev: 6.5 ± 42 ng/mL, range: 0.20-4100 ng/mL). In clinical serum/plasma samples with reportable 4-ANPP (N = 451), the median was 0.96 ng/mL (mean ± std dev: 3.9 ± 17 ng/mL, range: 0.20-306 ng/mL). In cases with reportable 4-ANPP concentrations, the median 4-ANPP: fentanyl ratio was 0.141 (mean ± std dev: 0.22 ± 0.95; range: 0.000078-140) for forensic, while the clinical median was 0.105 (mean ± std dev: 0.44 ± 3.0; range: 0.005-60). Notably, 91% of forensic cases had reportable 4-ANPP concentrations (≥0.2 ng/mL) compared to 44% of clinical cases, excluding more than half of the clinical cases from ratio calculations. Although overlapping 4-ANPP: fentanyl ratios limit its utility as a clear indicator of illicit fentanyl use, elevated 4-ANPP concentrations are more strongly associated with non-pharmaceutical sources and may serve as valuable support in forensic interpretation.

The concentrations of some drugs in biofluids can be affected by different storage conditions, including the type of sample collection tube. This phenomenon has been observed in gel separation tubes, where drug adsorption to the gel separator can lead to the underestimation of the drug`s concentration, thus potentially affecting the interpretation of analytical results. The purpose of this study was to determine if concentrations of tetrahydrocannabinol (THC), its metabolites, and related cannabinoids decrease over time when stored in plasma separation tubes (PSTs) as compared to non-PSTs. Plasma samples with a high concentration [HP-24 ng/mL THC, 150 ng/mL carboxy-THC (THC-COOH), 48 ng/mL hydroxy-THC (THC-OH), 15 ng/mL cannabidiol (CBD), and 15 ng/mL cannabinol (CBN)], and low concentration [LP-5.0 ng/mL THC, 31 ng/mL THC-COOH, 10 ng/mL THC-OH, 3.1 ng/mL CBD, and 3.1 ng/mL CBN] of cannabinoids were stored in PSTs and non-PSTs for analysis by liquid chromatography-tandem mass spectrometry at one-hour, three-day, one-week, two-week, three-week, one-month, two-month, and three-month intervals. Statistically significant differences in cannabinoid concentrations (p < 0.05) were observed between non-PSTs and PSTs. All cannabinoids except THC-COOH showed a greater reduction in concentration when stored in PSTs compared to non-PSTs. In contrast, THC-COOH showed an increase in concentration when stored in PSTs compared to non-PSTs. Over a three-month period, concentrations in PSTs decreased for THC by 84% and 81%, THC-OH by 66% and 63%, CBD by 69% and 62%, and CBN by 75% and 70%, in LP and HP samples, respectively. In conclusion, for forensic cases involving cannabinoids, the adsorption of these compounds should be considered in the toxicological interpretation of samples collected in PSTs.

This study measured the concentrations of blood ethanol (EtOH) and acetaldehyde (AcH) in mice to examine the roles of aldehyde dehydrogenase 2 (ALDH2) and sex following intragastric administration of EtOH. The experiment utilized males and females of two mouse strains: C57BL/6N (wild-type, WT) and Aldh2-knockout (Aldh2-KO) mice. Aldh2-KO mice lack the ALDH2 enzyme, leading to the accumulation of high levels of AcH in the blood. The mice were fasted for approximately six hours before EtOH administration. EtOH (1.0, 2.0, and 3.0 g/kg) was administered intragastrically, and blood samples were collected at 30, 60, 120, 180, 240, and 300 minutes post-EtOH administration through retro-orbital puncture. The samples were then analyzed using headspace gas chromatography. The results for both male and female WT mice showed that EtOH and AcH levels increased in a dose-dependent manner, peaked at 60 min post-ingestion, and then gradually decreased. While there were no significant differences in blood EtOH concentrations between males and females, the concentrations of AcH were significantly higher in female mice than in male mice, indicating potential sex-related differences in EtOH metabolism. In Aldh2-KO mice, the EtOH and AcH levels increased initially and peaked at 30-60 minutes post-ingestion, with no significant differences in EtOH or AcH concentrations between the sexes. While the concentrations of EtOH in both male and female Aldh2-KO mice gradually decreased, the concentration of AcH remained elevated until six hours post-ingestion due to the ALDH2 deficiency inhibiting AcH oxidation. Our findings emphasize the importance of considering the influences of sex and ALDH2 when researching the effects of alcohol, particularly in relation to the EtOH byproduct AcH.