The analysis of the first 1000 cases indicates that NPS in roadway crashes is a cause for concern in the United States. Eight NPS with a detection frequency of 26 were confirmed in 17 cases. Importantly, the NPS detected at the highest frequencies (bromazolam, para-fluorofentanyl, and mitragynine) are all categorized as CNS depressants. These drugs reduce the activity of the brain, resulting in sedation, drowsiness, and impaired cognitive and motor functions (Hetland and Carr, 2014). Previous studies have found an association between heightened crash risk and the type of drug used while driving with depressants resulting in the highest risk (Li et al., 2013). Benzodiazepine users have been found to be up to 80 percent more likely to be involved in a motor vehicle crash compared to non-users (Dassanayake et al., 2011). Bromazolam, the most frequently detected NPS in this study, is a designer benzodiazepine. This NPS is the brominated counterpart to the chlorinated drug alprazolam, most popularly prescribed as Xanax (Zawilska and Wojcieszak, 2019). In vitro studies predicted bromazolam to be a non-subtype selective agonist at the benzodiazepine site of GABA-A receptor with binding affinities of 2.81 nM at the α1 subtype, 0.69 nM at α2, and 0.62 nM at α5 (Clayton et al., 2015). Bromazolam was initially reported in a retrospective analysis of DUID cases submitted by law enforcement agents that tested positive for designer benzodiazepines between 2017 and 2021. Bromazolam was one of eight benzodiazepines included in 1145 designer benzodiazepine detections in 805 blood samples. However, it only accounted for five detections (0.4%) (Papsun et al., 2023). The relatively high detection of bromazolam in our cohort is consistent with recent research reporting 98 out of 52,585 (0.19%) impaired driving cases from 2021 to 2023 in which the driver tested positive for bromazolam via LC-TOF/MS and/or immunoassay screenings (Bierly et al., 2024). Our higher detection frequency of 0.7% is consistent with the rise of bromazolam as the most detected NPS in the US Drug Enforcement Administration Toxicology Testing Program (DEA TOX) that started in 2023. In almost all of these cases, bromazolam was detected along with fentanyl suggesting either their intentional co-ingestion or the co-presence of fentanyl and bromazolan in drug products, consistent with the increasing prevalence of “benzo dope”.

The second most detected NPS in our study, para-fluorofentanyl (pFF), was the most frequently detected NPS in the United States from 2021 until it was overtaken by bromazolam in 2023 (Trecki et al., 2022; U.S. Drug Enforcement Administration, Diversion Control Division, 2023b). This NPS was sold briefly on the US black market in the early 1980s, which served as the impetus for the introduction of the Federal Analog Act (Henderson, 1988). It made its comeback in 2020 when it was frequently detected as a fentanyl adulterant. pFF has been detected previously in a similar study conducted by the National Highway Traffic Safety Administration (NHTSA) between 2021 and 2022; pFF was detected 13 times over 2 years in 7,279 roadway users (0.18%) across seven different states (Thomas et al., 2022). One explanation for our higher detection frequency (0.4%) of pFF may be that some laboratories that performed the analyses in the NHTSA study do not have the ability to detect pFF.

Mitragynine, the primary psychoactive ingredient of Kratom, was the third most detected NPS in our study. Although Kratom was first brought to the United States in the 1980s, its recreational use only gained traction in 2010 (Post et al., 2019). Kratom has a stimulant effect in low doses, opioid-like effects in moderate to high doses, and sedative effects in very high doses (Prozialeck et al., 2012). Mitragynine was first reported in a DUI case in the United States in 2018 (Wright, 2018). The detection frequency in our cases (0.3%) is consistent with a previous retrospective study on the detection of mitragynine in DUID cases in Orange County, California between 2017 and 2019. In the 25,398 cases evaluated, mitragynine was detected in 60 cases (0.24%) (Kedzierski and Mata, 2023). The other NPS detected in our cases- N-methyl norfentanyl, acetyl fentanyl, protonitazene, etizolam, xylazine-have also been reported in the United States in the last 5 years (United States Drug Enforcement Administration, 2020; U.S. Drug Enforcement Administration, Diversion Control Division, 2023a).

Five other NPS (3 new synthetic opioids, 1 designer benzodiazepine, xylazine) were quantified in six cases. As a class, opioids were detected the most (16, 62%) in the entire cohort (Figure 1); notably, new synthetic opioids were detected almost twice as much as plant-derived opioids. When the position in the crash was considered, opioids overwhelmingly predominated (11 of 15, 73%) among drivers and bicyclists. Interestingly, designer benzodiazepines (6, 55%) and opioids (5, 45%) were almost equally detected in passengers and pedestrians. Of note, designer benzodiazepines were detected twice as much in passengers compared to drivers. However, with the limited number of NPS-positive cases detected so far in our study, it is hard to ascribe any meaning yet to these observations. The study is ongoing and with more NPS cases accumulating in our second year, a more representative sampling would soon be available.

While alcohol-impaired driving continues to have the most detrimental impact on traffic safety (Thomas et al., 2022), polysubstance abuse-impaired driving is a significant contributor. Under the DRUID project, Hels and colleagues found that the odds of being seriously injured in a motor vehicle collision are 28 times higher for drivers who test positive for alcohol with other drugs and 8 times higher for drivers who tested positive for more than one nonalcohol drug compared to those who tested negative. These numbers are even higher (31x and18x, respectively) for fatally injured drivers (Hels et al., 2011).

The percentage of drivers using multiple drugs has increased from 32.6 percent in 1993 to 45.85 percent in 2010 (Wilson et al., 2014). There are several factors to account for this, including but not limited to an increase in prescription drug use (Kantor et al., 2015) and an increase in NPS supply and use in the United States. This is consistent with our data, as all but one case of NPS detection involved polypharmacy. Fifteen of the 17 cases have at least one traditional recreational drug with fentanyl detected in 9 cases. NPS are commonly co-detected with TRDs due to co-existence in drug preparations and/or because users combine different pharmacological classes of drugs they abuse.

The current fentanyl epidemic in the United States started in 2015, and since then, fentanyl has been detected as an adulterant in common drugs of abuse, initially in heroin and later in stimulants like methamphetamine and cocaine. Seven of the fentanyl detections have either methamphetamine or cocaine. Additionally, NPS are also added to fentanyl drug preparations. The majority of the bromazolam and pFF detections in NPS surveillance in the United States are co-detected with fentanyl (U.S. Drug Enforcement Administration, Diversion Control Division, 2023a; U.S. Drug Enforcement Administration, Diversion Control Division, 2023b; U.S. Drug Enforcement Administration, Diversion Control Division, 2023c; U.S. Drug Enforcement Administration, Diversion Control Division, 2023d; U.S. Drug Enforcement Administration, Diversion Control Division, 2023e). This is reflected in our cases; fentanyl was detected in five of seven and four of five cases of bromazolam and pFF cases, respectively. The combination of these depressants undoubtedly exacerbates the impact of these drugs on driving. In the two cases in which bromazolam was found without fentanyl, methamphetamine was detected. Although the combination of drugs with opposing pharmacological effects may be antagonistic in some cases, both stimulants and depressants, on their own, impact driving. More importantly, it is difficult to determine whether the effects each class has on driving are mitigated by their combination. And, even if this is the case, obtaining the specific proportion required for one drug to antagonize another may be challenging to precisely achieve and may vary from one person to another.

Furthermore, our research provides a unique insight into not just the identification of NPS detected in roadway crashes, but their quantitative levels too. Previous studies primarily report qualitative data on NPS; in a few studies, quantitative data for a specific NPS like bromazolam was presented (Bierly et al., 2024; Papsun et al., 2023). Table 1 shows the quantitative levels of all eight NPS found in roadway crash victims in our study. While there is lack of consensus regarding the correlation between drug concentration and degree of impairment due to factors such as tolerance or drug-drug interactions (Reisfield et al., 2012), there are proposed cut-offs for psychoactive substances that have clear concentration dependent cognitive impairment (Blandino et al., 2022). Quantitative drug levels may be able to provide a window into the potential degree of intoxication for those involved in the crashes. This can, in turn, provide a clue to the degree of impairment that can be ascribed to drug levels detected. A cutoff blood alcohol level has been established to objectively determine whether a motorist may be experiencing alcohol-induced driving impairment. The same has not yet been established for any other drugs including common drugs of abuse, e.g., cannabis. Hence, the NPS levels reported in our study can provide initial data that may be useful in establishing guidelines for determining NPS-induced drug impairment. The geometric mean of serum levels that we measured for bromazolam was 5.41 ng/mL (range: 0.22–26.59 ng/mL). These levels are lower than those reported in overdose cases (12.34 ng/mL) and death cases (25.39 ng/mL) by the DEA TOX program in 2023. This is expected as overdose patients are incapacitated, making driving highly unlikely. However, the wide range of values observed in roadway crash victims likely reflects differences in drug tolerance. For pFF, the geometric mean observed in roadway crash victims was 0.45 ng/mL (range: 0.28–2.02), whereas in DEA TOX it was 5.26 ng/mL (U.S. Drug Enforcement Administration, Diversion Control Division, 2023b; U.S. Drug Enforcement Administration, Diversion Control Division, 2023c; U.S. Drug Enforcement Administration, Diversion Control Division, 2023d; U.S. Drug Enforcement Administration, Diversion Control Division, 2023e). No comparison to overdose cases can be made as almost all pFF cases in DEA TOX were deaths. This and the lower concentrations observed for pFF associated with roadway crashes and deaths may suggest its stronger potency in these cases. Alternatively, because almost all pFF cases also involve fentanyl, this may be suggestive of the synergistic effects of the two drugs.

Our work is the first to conduct a comprehensive targeted analysis of more than 1000 NPS, in combination with suspect and non-targeted screening in roadway collisions across a large population. While there have been reviews across several countries in Europe such as Spain, Germany, France, and Belgium (Fels et al., 2020; Richeval et al., 2019; Soria, 2018; Wille et al., 2018), in the United States there only exist case reports for NPS in individual drivers (Fassette and Martinez, 2016; Lemos, 2014; Rajotte et al., 2017), larger studies (n = 9,569) with limited NPS testing (only synthetic cannabinoids and cathinones) and detection frequency (n = 2, <0.1% detection frequency) (NTSB, 2022), and retrospective analyses for specific NPS like bromazolam and mitragynine in large cohorts (Bierly et al., 2024; Kedzierski and Mata, 2023; Papsun et al., 2023). Counterintuitively, the United States also experiences some of the highest rates of NPS use. The reasons for why this may be the case center around regulation and policy frameworks. The United States lacks an organized federal body akin to the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), which is focused on tracking the prevalence of NPS in public health that also looks specifically at road safety. This allows for a more coordinated and unified effort in which laws and testing protocols around NPS are harmonized, whereas in the United States federal and state jurisdiction disputes can complicate the implementation of consistent policies. Furthermore, there are specific policies, such as the EU Drug Driving Directive (2015/413) which targets drug-impaired driving, including NPS. There is also a larger extent and wider range of testing infrastructure in Europe including monitoring tools such as syringe residue and wastewater analysis, drug product and biological sample testing through the European Drug Emergencies Network, drug checking services, and expanded surveys (Wood and Dargan, 2020). In the United States, attempts at improving regulation through the Controlled Substances Analogue Enforcement Act (1986) as well as the Synthetic Drug Abuse Prevention Act (2012) largely failed as they were devised in a manner that leaves the classification of NPS open to various justifications in structural and functional arguments, complicating legal and testing systems. A lack of consistent legislation that accurately and quickly determines which substances are deemed NPS contributes to this problem in the United States.

Although our analysis is comprehensive, it is not without limitations. First, samples were collected from cases limited to two geographic locations- Los Angeles and Sacramento. Although these two cities may represent a large and medium-sized city in California, they do not represent the rest of the cities and towns in the state. To a greater extent, they do not represent the rest of the United States. There are known geographic differences in drug use patterns so our findings may not accurately be used to extrapolate what may be true for the rest of the country. Furthermore, our cohort does not include subjects from other at-risk populations, such as drivers or pedestrians who may not have been involved in roadway crashes that required an Emergency Department visit. Certain exclusionary criteria, such as the patient having a warrant for their arrest also limit data extrapolation. Additionally, the study was conducted over 7 months, from January to July 2024. The general findings of our larger ongoing study demonstrated differential detection frequency among drugs between months. The detection frequency of most drugs, for example, was trending up towards the summer months (unpublished data). It is difficult to predict the impact that collection from August to December would have had on our data. This time constraint also makes it difficult to establish trends in NPS use over longer periods of time. Longitudinal studies would provide a more comprehensive understanding of NPS use and its evolving impact on impaired driving. Finally, like any other comprehensive LC-QTOF/MS method, our method may not have the best sensitivity for a few NPS. Comprehensive methods are designed to optimize detection and confirmation of as many drugs (NPS) as possible. However, the wide range of structural differences and polarities among various classes of NPS make it impossible to have optimum sensitivity for all NPS classes. Although our method is good at detecting more than 98% of our target analytes at sensitivities appropriate for serum, it does not have good sensitivity to very small, strongly polar (e.g., GHB analogs) and large highly hydrophobic (e.g., delta-9-THC derivatives) compounds, so if the levels of these NPS are low in biological samples, we likely missed their detection. Moreover, substances of which there is no known reference standard or are very uncommon might be excluded which may further limit the scope of the study.

In summary, although the number of cases where we found NPS in our cohort is limited, our findings provide more evidence of the ongoing danger that NPS may pose to road safety and highlight the need for policy change aimed at expanding NPS testing infrastructure, specifically in cases of impaired driving. Future research should continue to look at NPS in roadway collisions, specifically in drivers, as this is the cohort most responsible for injury and death in crashes. If possible, it would also be beneficial to develop a dynamic roadside testing strategy that adapts to the highly detected and impairing NPS reported in nationwide drug surveillance close to, if not in, real-time.