Cannabinoids is the most frequently reported illicit drug class in Driving Under the Influence of Drugs (DUID) investigation casework. In recent years, our laboratory observed an increasing rate of overlapping peaks for the cannabinoids confirmation performed using two-dimensional (2D)-High Performance Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). Starting in early 2018, the incidence of unresolved interfering substances increased, contributing to a higher rate of canceled testing that peaked at 3.7% in February 2019. The observed interference demonstrates a distinctive pattern affecting identifications and quantification of both Delta-9 THC and Delta-9 carboxy THC. An improved quantitative method was developed and validated to separate Delta-8 and -9 isomers and their metabolites in blood. All acceptance criteria were met with identical measurement ranges from the original method (lower limit of quantitation: 0.5 ng/mL for Delta-9 THC, 1.0 ng/mL for 11-Hydroxy Delta-9 THC, and 5.0 ng/mL for Delta-9 carboxy THC). Cannabinoids were extracted from whole blood using liquid-liquid extraction, separated in a 2D liquid chromatography system over a run-time of 10 min and detected by a tandem mass spectrometry system equipped with ESI source operating in positive ionization mode with scheduled multiple reaction mass spectrometric monitoring (MRM). The LC system consisted of a pair of Phenomenex® SecurityGuard™ C6 Phenyl (4x2 mm) cartridges for extracting the compounds with 5 mM ammonium formate buffer in deionized (DI) water and 0.1% formic acid in methanol as mobile phase, and a Phenomenex® Kinetex C18 column (100x3 mm) with 0.1% formic acid in DI water and 0.1% formic acid in methanol for LC separation at 45°C. Each set of isomers was fully resolved by the longer run-time method. To the authors’ knowledge, this is the first report of a method that successfully quantitates these primary cannabinoids in blood specimens where significant concentrations of both Delta-9 and Delta-8 isomers are present.

Mitragynine is the primary active alkaloid in the leaves of the tropical tree Mitragyna speciosa, and goes by the popular names “Kratom”, biak-biak and maeng da. Mitragynine is increasingly seen in forensic toxicology casework including driving under the influence of drugs and medicolegal death investigation cases. The toxicity of mitragynine continues to be debated in the scientific community as advocates highlight its long history of use in Southeast Asia and testimonials to its benefits by present-day users, while opponents point to an increasing number of adverse events tied to mitragynine use in Western societies. Quantitative reports of mitragynine in biological specimens from forensic investigations in the literature are sparse and may be influenced by poor analyte stability and inadequate resolution of mitragynine from its diastereomers, which could lead to falsely elevated concentrations and subsequently render those reported concentrations inappropriate for comparison to a reference range. Over the course of 27 months, 1,001 blood specimens submitted to our laboratory tested positive for mitragynine using a sensitive and specific quantitative LC-MS/MS method; concentrations ranged from 5.6–29,000 ng/mL, with mean and median concentrations of 410 ± 1,124 and 130 ng/mL, respectively. Mitragynine presents an analytical challenge that requires a method that appropriately separates and identifies mitragynine itself from its isomers and other related natural products. We describe a validated analytical method and present a short series of case reports that provide examples of apparent adverse events, and the associated range of mitragynine concentrations. This type of analytical specificity is required to appropriately interpret mitragynine concentrations detected in biological specimens from forensic casework and assess its potential toxicity.

This report describes updates to the National Safety Council’s (NSC) Alcohol, Drugs, and Impairment Division’s (ADID) recommendations for drug testing in Driving Under the Influence of Drugs (DUID) cases and motor vehicle fatalities. The updates are based on a survey of drug testing practices in laboratories in the United States and Canada, a comprehensive review of the prior recommendations, and data and research on drugs most frequently detected in DUID cases. A consensus meeting was held with representative forensic science practitioners and the authors of this report to update recommendations. No changes were made to the Tier I scope; however, there were changes to cutoffs of some analytes for blood, urine and oral fluid. Due to increased prevalence in DUID cases, trazodone and difluoroethane were added to the Tier II scope. For clarification, Tier I cutoffs reflect free concentrations, and hydrolysis is recommended but not required. The consensus panel concluded that urine is an inferior matrix to blood and oral fluid as it may represent historical use or exposure unrelated to observed impairment; therefore, future iterations of these recommendations will not include urine as a recommended matrix. Laboratories currently testing urine should work with traffic safety partners to encourage the use of blood and oral fluid as more appropriate specimens and adjust their capabilities to provide that testing.

Prior to 2017, heroin and other prescription opioids were the most prevalent opioids implicated in driving under the influence of drugs (DUID) investigation cases and fentanyl was rarely included in the scope of toxicological analysis. Fentanyl has become the most frequently identified opioid in DUID cases with many suspected heroin cases turning out to be only fentanyl. A review of fentanyl positive DUID cases at NMS Labs was performed to provide prevalence information, change in concentration, patterns of combined drug use, indicators of impairment, and driving behavior in order to assist with toxicological interpretation of DUID scenarios involving fentanyl. Fentanyl positive DUID cases received between January 2010 and December 2020 were examined. Blood results were confirmed and quantitated for fentanyl, norfentanyl and acetylfentanyl using a liquid chromatography–tandem mass spectrometry (LC–MS-MS) analysis with a limit of quantitation (LOQ) of 0.10, 0.20 and 0.10 ng/mL, respectively. Of 153,234 blood cases examined for DUID over 11 years, fentanyl confirmed positive in 6,779 (4.4%) cases. However, there were significant changes in positivity over time. Fentanyl percent positivity increased from 0.60% in 2010 to 12% in 2020. Of 5,976 confirmed fentanyl positive cases in 2018 through 2020, blood concentrations greater than 4.0 ng/mL were observed in 44% (2018), 55% (2019), and 59% (2020) of cases. Polypharmacy was common with 87% of blood samples confirming positive for fentanyl and at least one other compound. Stimulants was the most commonly identified drug class in cases where at least one additional drug class was present. This study illustrates the importance of including fentanyl in a routine blood DUID panel.

Designer benzodiazepine (DBZD) use has been increasing over the past decade and poses a threat to human health and safety, particularly when involved in driving under the influence of drugs (DUID) cases. Over a five-year period between 2017 and 2021, there were 1145 reported DBZDs in 805 blood samples submitted from law enforcement agencies for DUID testing. Eleven different DBZD were detected, including three metabolite pairs: etizolam/alpha-hydroxyetizolam, clonazolam/8-aminoclonazolam, diclazepam/delorazepam, flualprazolam, flubromazolam, flubromazepam, bromazolam, and bromazepam. Etizolam/alpha hydroxyetizolam (n=485) and flualprazolam (n=149) were the most frequently detected DBZD, at 60% and 18%, respectively. Driving behavior, standardized field sobriety tests (SFST) performance, and physical observations of individuals suspected of DUID, whose blood was toxicologically confirmed for one or more DBZD, were consistent with effects caused by central nervous system (CNS) depressants. Each DBZD has its own unique timeline, and toxicology testing had to be frequently updated to reflect the state of the novel psychoactive substance (NPS) market. DBZD play a role in impaired driving and can be the sole intoxicant in DUID cases.