Journal of analytical toxicology最新文献

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.

The establishment of stringent identification criteria is essential for accurate reporting of toxicological drug testing, particularly in forensic settings involving medico-legal cases. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is widely employed for its broad analyte coverage and high mass accuracy, yet limited published and validated identification criteria pose significant challenges for its use beyond presumptive screening in low case volume settings. This study characterized, optimized and selected LC-QTOF-MS identification criteria, assessing the influence of concentration, matrix and drug class on their performance. In addition to standard identification parameters, an effective combined weight score (CWS) threshold that emphasized library score and mass error was established. Higher analyte concentrations improved spectral reproducibility, while urine matrices introduced variability in isotope ratios and library scores. Authentic casework demonstrated 99.9% efficiency, 98.9% sensitivity, and 100% specificity, indicating a highly reliable method that achieves excellent accuracy, minimizes false positives as required for confirmatory techniques, and maintains sufficient sensitivity for effective screening of casework, thereby supporting robust and defensible forensic toxicology workflows. These findings also highlight the importance of refining LC-QTOF-MS specific identification criteria to enhance consistency and reliability in forensic toxicology reporting, and allows for reproducibility across other instrumentation, workflows, and fields.

This review is intended for forensic toxicologists and cheminformaticians seeking an understanding of the past implementations and future directions of artificial intelligence (AI) and machine learning (ML) for high-resolution mass spectrometry (HRMS) data interrogation in forensic toxicology. It provides a comprehensive overview of the data processing steps required to generate valid ML inputs, including molecular representation, augmentation, tokenization, embedding, and spectral deconvolution. We examine the advantages and disadvantages of different modeling strategies and summarize existing models from forensic toxicology and related domains. Applications are grouped into spectra-to-compound, compound-to-spectra, and classification models, with attention to recent advances and the practical challenges of limited data, polysubstance use, and validation. By leveraging advances from related fields, ML can enhance forensic HRMS workflows, enabling more efficient unknown screening, structural elucidation, and classification of emerging substances. This review aims to bridge disciplinary perspectives and support the practical integration of ML into routine forensic toxicology.

Nitrous oxide (N2O), the colorless, odorless gas known as "laughing gas", has gained recent attention for its misuse as a recreational drug. As an anesthetic, N2O produces sedation, euphoric and possible hallucinogenic effects. Adverse effects may include disorientation, psychomotor retardation, hypoxia, and asphyxia. N2O misuse has grown due to its ease of availability, rapid onset of effects, and increased social media attention, leading to anticipated increases in forensic testing needs. Due to its short half-life and volatility, analytical detection can be challenging. From January 2022 through September 2025, over 1700 cases were analyzed for N2O using headspace-gas chromatography-mass spectrometry (HS-GC-MS) over a calibration range of 1.8-180 mcg/mL. Total test requests and percent positivity increased during this timeframe for both driving (DUID) and postmortem (PM)/clinical casework. Blood, brain, liver, lung, and urine yielded positive detections. Attempts at repeat testing indicate significant losses in analyte concentrations. Consideration of pre-analytical and analytical factors are critical for suspected inhalant casework. Overwhelmingly, both DUID and PM casework noted N2O canisters present at the scene. Common driver behaviors included disorientation, slow reaction times, struggling with speech, inability to follow directions, and difficulty maintaining balance. DUID blood draws should be collected as close as possible to the suspected incident. Further review of case histories and testing practices generated handling recommendations for suspected inhalant case samples: fill containers to limit headspace; glass containers and tight-fitted closures are preferred; avoid transferring volume to alternate containers; limit container ingresses; and avoid repeat testing within the same container. Multiple matrices/containers should be collected and preserved, whenever possible, with inhalant testing prioritized over other drugs and/or alcohol. Laboratories should also consider qualitative reporting and/or testing as a one-time analysis. By employing these best practices, an inhalant gas may be better collected and preserved, increasing the chances of detection.

Assessment of drugs of abuse in biological fluids requires thorough knowledge of stability of the drugs under various conditions, including sample collection, handling, transportation, and analysis, to ensure accurate interpretation of results. This systematic review provides an overview of the literature on the pre-analytical stability of selected clinically relevant drugs of abuse in urine. A systematic search of the PubMed and Embase databases was conducted in October 2020 and February 2024. The search strategy encompassed over 20 drugs and their relevant metabolites tested in urine, focusing on studies that examined the stability of opioids, amphetamine-like drugs (including ephedrine, cocaine and cathinone), and cannabis using mass spectrometry. A total of 2,688 records were identified, and 71 studies met the inclusion criteria. These studies evaluated storage conditions including room temperature, refrigeration, freezing, and deep freezing, as well as the effects of freeze-thaw cycles. Most drugs demonstrated stability for months when refrigerated or frozen, and deep freezing and freeze-thaw cycles generally had minimal impact on stability. However, storage at room temperature showed limited stability, with cathinone, cannabis, morphine, codeine, and cocaine being particularly prone to degradation under different conditions. This review offers valuable insights into the storage stability of a wide range of drugs of abuse in urine, serving as a practical resource for healthcare professionals and others working with these substances in laboratory settings.

Purpose: This study aimed to develop a highly sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of propranolol and its metabolites in human biological samples. By analyzing their presence in urine, postmortem biological fluids, and various solid tissues, the study could be of reliable forensic toxicological use for investigations in propranolol poisoning cases. In this study, the Standard Addition Method (SAM) was used for quantification, and its validation was mixed with one of the analyte's concentrations.

Methods: A 0.1 mL aliquot of each body fluid sample or 0.1 g each of homogenized solid tissue was mixed with one of the analyte concentration standards, extracted with methanol, spiked with an internal standard (IS) using the SAM, and purified using magnesium sulfate and sodium sulfate. Following centrifugation and filtration, samples were analyzed via LC-MS/MS. Urine samples underwent enzymatic hydrolysis with sulfatase and β-glucuronidase to measure conjugated metabolite forms prior to analysis.

Results: Phase I metabolites (propranolol, 4-hydroxypropranolol, propranolol glycol, N-desisopropylpropranolol, 1-naphthylenyloxyacetic acid, and 1-naphthol) and phase II metabolites (sulfate and glucuronide conjugates) were identified in urine. Among postmortem samples, propranolol was highest in the bile, followed by the lung tissue. Naphthoxylactic acid could be consistently detected in all samples except for the brain, suggesting its potential as a good biomarker for propranolol exposure.

Conclusion: A validated LC-MS/MS method for determining propranolol and its metabolites in forensic samples was established, and it could also be applied to the authentic human samples obtained from a propranolol poisoning case. The findings could offer substantial and reliable support for investigating propranolol-related fatalities and contribute to the comprehensive understanding of the metabolism of propranolol in the human body.