Qualitative urine drug screens (UDSs) are essential in clinical toxicology for assessments of drug exposure and are commonly employed by emergency departments to provide rapid results that inform care. Fentanyl, a potent synthetic opioid, is of significant public health concern due to rising overdose rates. However, fentanyl is also administered clinically as an analgesic, which may result in positive UDS assessments for patients with iatrogenic exposure. Positive UDS findings may cause patient anxiety, provider confusion, and lead to medicolegal concerns in clinical settings such as labor and delivery and neonatology. As such, it is essential that UDSs are utilized appropriately and that the positivity threshold for the assay is rigorously evaluated to serve the clinical need. This study examined UDS utilization within a quaternary care system over a one-year period. A subset of remnant urine specimens from UDSs were further analyzed using HRMS (high resolution mass spectrometry) and LC-MS/MS (liquid chromatography-tandem mass spectrometry) to focus on the impact of fentanyl immunoassay cutoff concentrations in high-volume populations. At our institution, the adult emergency department (ED) had the highest number of specimens ordered (5,901), while neonatal units had the least (727). Notably, fentanyl positivity rates were the highest in neonatal units at 34%. Quantitative analysis of remnant urine samples from the ED, labor and delivery (L&D), and neonatal units showed higher fentanyl concentrations in the ED, indicative of illicit use, and lower concentrations in L&D and neonates, consistent with analgesic administration. Raising the immunoassay cutoff from 1.0 ng/mL to 5.0 ng/mL would convert iatrogenic positives to negatives while retaining illicit positives. These findings underscore the need to evaluate UDS cutoff values for improved clinical performance in different populations.

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.

Analytical toxicology is a discipline of forensic toxicology which applies analytical techniques for the determination of drugs of abuse in biological and nonbiological matrices. To this concern, artificial intelligence (AI), particularly machine learning (ML), is innovating analytical toxicology by improving data processing and facilitating the identification of New Psychoactive Substances (NPS). The aim of this review was to explore the current application of AI in this field and to highlight the future perspectives. A literature search was performed in several scientific databases to review articles reporting the implementation of AI models for analytical toxicological purposes. The most frequent applications of these technologies were for compound identification, molecular structure prediction and retention time prediction. AI proved to be a valuable tool for analytical toxicologists for the capability to process large amount of data which are typically obtained by untargeted approaches.

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.

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.

A streamlined liquid chromatography quadrupole time-of-flight mass spectrometry method utilizing protein precipitation and filtration extraction was developed to achieve rapid and reliable screening and confirmation for blood and urine matrices. This method targets 946 drugs and metabolites across 35 drug classes via sequential window acquisition of all theoretical mass spectra with variable customized windows to enhance spectral clarity, and was validated per established guidelines to ensure high accuracy and reproducibility. Combined with complementary in-house methods, this approach meets and exceeds the testing requirements outlined in ANSI/ASB standards and recommendations for postmortem, drug-facilitated crime, and Tier I and II driving under the influence of drug analyses. The method demonstrated efficient and sensitive performance, achieving limits of detection as low as 0.1 ng/mL. It accurately identified expected detections across 67 proficiency test samples and 224 authentic case samples, with high accuracy and reliability in the detection of both traditional drugs and novel psychoactive substances. The method employs an in-house built library and incorporates in-batch standards analyzed alongside case samples to ensure contemporaneous identification criteria, making it suitable for confirmation and reporting purposes. By expanding the analytical capabilities to include a vast range of analytes, this method improves the likelihood of identifying substances that may otherwise go undetected and reduces the need for multiple separate tests, thereby enhancing the overall effectiveness of toxicological investigations.

Medetomidine is an alpha-2 agonist and non-opioid sedative. Its presence in illicit fentanyl and heroin drug supplies poses significant risks to user health caused by cardiac effects and sedation. Medetomidine exists in two enantiomeric forms: dexmedetomidine and levomedetomidine. Dexmedetomidine is used in humans in medical settings, while dexmedetomidine alone and a racemic mixture of dexmedetomidine and levomedetomidine are used in animals in veterinary settings. Little information is known about circulating blood concentrations of medetomidine or its enantiomers in humans in situations involving synthetic opioids (e.g. fentanyl) and other sedatives (e.g. xylazine, benzodiazepines). A new toxicological workflow using liquid chromatography-tandem quadrupole mass spectrometry (LC-QQQ-MS) was developed and validated for the quantitation of medetomidine and the qualitative enantiomeric separation of dexmedetomidine and levomedetomidine. The assays were applied to a case series of 100 authentic specimens from emergency department admissions and forensic postmortem investigations containing medetomidine, fentanyl, xylazine, and metabolites, among other substances. Medetomidine blood concentrations in non-fatal overdoses ranged 0.1-16 ng/mL (median: 1.5 ng/mL) and in fatal overdoses ranged 0.1-32 ng/mL (median: 0.31 ng/mL). Xylazine was co-detected in 76% of cases with higher median concentrations: 8.3 ng/mL (non-fatal) and 15 ng/mL (fatal). Fentanyl was co-detected in 93% of cases with median concentrations of 5.2 ng/mL (non-fatal) and 21 ng/mL (fatal). Dexmedetomidine and levomedetomidine were identified in 90% of cases; the remaining cases were confirmed or suspected medical-setting administration of dexmedetomidine. These findings demonstrate that medetomidine is arising from veterinary or clandestine sources, and we hypothesize the latter. Recreational medetomidine use causes adverse effects such as profound bradycardia and heightened sedation, especially when combined with fentanyl and xylazine. Forensic laboratories must remain aware of adulterants, like medetomidine, appearing in traditional opioid products (e.g. fentanyl, heroin), updating testing methods to capture these emerging adulterants in real-time.

Hydroxyzine (Vistaril©) is an unscheduled, first-generation antihistamine prescribed for nausea/vomiting, atopic dermatitis or eczema, and anxiety. It can produce adverse central nervous system (CNS) depressant effects such as fatigue, sedation, and impaired memory and concentration. Several clinical studies have shown hydroxyzine is able to impair driving and psychomotor function; however, no case series have been published highlighting driving performance and roadside observations in driving under the influence of drugs investigations (DUID). Between January 2017 and October 2024, 319 blood specimens submitted for DUID testing confirmed positive for hydroxyzine. Mean and median blood concentrations were 70 ± 79 and 48 ng/mL, respectively, with a range of 8.0-600 ng/mL. More than half of hydroxyzine positive drivers were female (57%) with a mean age of 42 years. Common drug combinations in this study involved antidepressants (74%), opioids (44%), and anticonvulsants (38%). Only seven cases have been reported involving a single substance, and four of these investigations have been presented. Behavioral observations included incoordination, slow and slurred speech, and difficulty following instructions on standardized field sobriety tests. Driving observations included erratic driving, crashes, driving in opposite lanes of travel, and running stop signs. This is concerning since hydroxyzine became the most identified antihistamine in this population in 2023 outpacing diphenhydramine.

This study aimed to develop and validate a green method for the detection of ketamine and its metabolites in human urine samples and subsequently determine which metabolite is more suitable for judgment of ketamine abuse. Ketamine and its metabolites were extracted from urine samples with 1-undecanol using liquid-liquid microextraction based on the solidification of floating organic droplet extraction. The extracts were directly analyzed using gas chromatography-mass spectrometry (GC-MS) without derivatization. The sample pretreatment procedure prior to GC-MS analysis was simple, fast, and required little solvent consumption. Parameters that affect the extraction efficiency, including pH of the urine sample and centrifugation speed, were optimized. Under the optimized conditions, the limits of detection for norketamine, dehydronorketamine, and ketamine were 1.5, 1.7, and 1.0 ng/mL, respectively. The method was used to analyze eight real urine samples from drug abusers and exhibited acceptable accuracy and precision. By analyzing real samples, this study revealed that detection of dehydronorketamine in urine samples for the judgment of ketamine abuse may be more suitable than detection of norketamine or ketamine. The method used in this study addresses the need for green analytical techniques in toxicology.

Concentrations of amitriptyline and nortriptyline in postmortem blood samples may not accurately reflect the concentrations at the time of death due to postmortem redistribution or degradation. The brain is suggested as an alternative matrix since it is less subjected to postmortem redistribution and more protected against trauma and putrefaction, but reference concentrations in brain tissue are scarce. In this study, we aimed to provide concentrations in brain tissue and brain–blood ratios in 53 postmortem cases, where amitriptyline and/or nortriptyline were detected. To establish reference levels, each case was assigned to one of three classes according to the cause of death: (i) lethal intoxication by the sum of amitriptyline and nortriptyline or nortriptyline alone, (ii) lethal intoxication by the drugs in combination with other drugs, and (iii) the cause of death was not influenced by amitriptyline and/or nortriptyline. A positive correlation between blood and brain concentrations was found with a Spearman coefficient of 0.98. In 42 cases, where both drugs were detected, the 10–90 percentiles in brain tissue ranged from 0.17–9.1 mg/kg (median: 0.78 mg/kg) for amitriptyline and 0.22–5.0 mg/kg (median: 1.43 mg/kg) for nortriptyline across all classes. In 11 cases where only nortriptyline was detected, the percentiles ranged from 0.32–7.2 mg/kg (median: 0.28 mg/kg) in brain tissue. A median brain–blood ratio of 3.4 was found for amitriptyline, 8.5 for nortriptyline as a metabolite of amitriptyline and 9.7 for nortriptyline as an individual ingested drug. No significant difference was found between the different classes. The obtained brain concentrations and brain–blood ratio can contribute to the alternative or complementary use of brain tissue for future toxicological investigations.