Abstract
Background and aims:
Nitazenes are a novel subclass of synthetic opioids that have been increasingly implicated in the United States (US) overdose crisis. Despite their growing presence in the illicit drug supply, national trends have not been systematically evaluated. This study aimed to describe temporal and geographic patterns in nitazene detections and assess substances co-involved in nitazene-positive biospecimens.
Design:
Cross-sectional study using forensic data from two national sources: the US Drug Enforcement Administration’s National Forensic Laboratory Information System (NFLIS) and the Center for Forensic Science Research & Education’s (CFSRE) NPS Discovery Program.
Setting and cases:
Nitazene detections in all 50 US states and the District of Columbia between 2019 and 2024.
Measurements:
We quantified annual nitazene detections overall and by individual nitazene analog, US Census region and state. Temporal trends were modeled using piecewise linear regression with a Poisson distribution and log link, nationally and by region. NPS Discovery data were used to characterize substances co-involved with nitazene-positive biospecimens.
Findings:
Between 2019 and 2024, 7117 nitazene analog reports were submitted to NFLIS, increasing from 43 in 2019 to 1905 in 2024. Counts rose sharply from 2019 to 2021 [count ratio = 7.32; 95% confidence interval (CI) = 2.22–24.20] but did not increase statistically significantly from 2021 to 2024 (count ratio = 1.08; 95% CI = 1.00–1.17). Early detections were predominated by isotonitazene (97.7% of NFLIS nitazene reports in 2019) but later shifted toward metonitazene and protonitazene (29.5% and 30.1%, respectively, in 2024). NPS Discovery identified 361 nitazene-positive biospecimens, increasing from 11 in 2019 to 113 in 2024, with counts increasing by approximately 45% per year (count ratio = 1.45; 95% CI = 1.23–1.71). Nearly all nitazene-positive biospecimens (98.3%) had at least one co-detected substance, most commonly fentanyl (54.6%).
Conclusions:
Nitazene detections increased sharply across the United States between 2019 and 2024, with shifting patterns in the prevalence of individual nitazenes and extensive polysubstance involvement. These findings highlight the need to strengthen drug testing capacity, expand epidemiological surveillance and implement targeted public health interventions to mitigate harms associated with this emerging class of synthetic opioids.
Keywords: drug seizures, drug surveillance, fentanyl, nitazenes, polysubstance, synthetic opioids, toxicology testing
INTRODUCTION
The United States (US) remains mired in an overdose crisis increasingly driven by the spread of highly potent synthetic opioids [1]. Since the mid-2010s, illicitly manufactured fentanyl (IMF) has been the dominant synthetic opioid implicated in overdoses [2]. More recently, however, newer subclasses of synthetic opioids have emerged amid shifts in supply chains, regulatory oversight and drug scheduling [3]. In 2018, the US Drug Enforcement Administration (DEA) issued a temporary class-wide scheduling order placing all fentanyl-related substances under Schedule I of the Controlled Substances Act [4]. Although this action aimed to reduce IMF availability, it may have inadvertently created an opening for the introduction of a new subclass of synthetic opioids: nitazene analogs [3, 5, 6].
Nitazene analogs, also referred to as 2-benzylbenzimidazoles or simply ‘nitazenes’, are a class of novel synthetic opioids structurally derived from the prototype etonitazene. First synthesized in the 1950s by pharmaceutical companies exploring alternatives to morphine, these drugs were never approved for clinical use because of their high potency and poor safety profiles [4,7]. Their binding affinity and potency at the μ-opioid receptor vary considerably. Some analogs, such as isotonitazene, exhibit in vitro potencies up to 500 times greater than morphine [8]. These substances are typically synthesized in industrial laboratories overseas, often by Chinese chemical suppliers using online marketplaces [9], then pressed into counterfeit tablets or adulterated into heroin or fentanyl powders [10, 11].
Because these substances have no approved medical use, all known nitazene reports in the US are believed to reflect nonpharmaceutical, illicit availability [12, 13]. As of October 2025, the US DEA has scheduled 21 individual nitazene compounds, including 10 permanently and 11 temporarily [13]. However, to our knowledge, the agency has not articulated why the core nitazene scaffold, 2-benzylbenzimidazole, remains unscheduled. Broad class-wide scheduling of this structure may be impractical, given that the benzimidazole ring is a common motif in widely used therapeutic agents, including anti-parasitics such as albendazole and mebendazole, proton pump inhibitors such as omeprazole and pantoprazole, and antihypertensive medications such as telmisartan and candesartan [12].
The US Centers for Disease Control and Prevention (CDC) now includes nitazene analogs in its State Unintentional Drug Overdose Reporting System (SUDORS), an epidemiological surveillance system that monitors drug overdose deaths with available toxicology data across nearly all states [13]. Ohio and Tennessee were among the first to identify clusters of nitazene-related fatalities in SUDORS [14, 15]. In parallel, in 2020, the United Nations Office on Drugs and Crime issued early warning alerts documenting the emergence of nitazene analogs, and in 2022, the World Health Organization released critical review reports on several analogs such as protonitazene and N-pyrrolidino etonitazene [15-17]. Despite these efforts, no national study to date has systematically examined forensic trends in nitazene analogs across the US.
To address this gap, we conducted a cross-sectional study to characterize temporal and geographic trends in US nitazene detections using two primary sources: (1) the US DEA’s National Forensic Laboratory Information System (NFLIS) and (2) the Center for Forensic Science Research and Education’s (CFSRE’s) NPS Discovery program. We quantified nitazene detections overall and by individual analog, US Census region and state. Using CFSRE data, we also examined substances co-detected in biospecimens to situate nitazenes within the growing polysubstance overdose crisis.
METHODS
Two sources of data were used in this investigation. First, we obtained data on annual nitazene reports from 2019 to 2024 using the NFLIS Public Data Query System (DQS) [18], most recently accessed on 19 June 2025. Although sporadic nitazene reports appeared in earlier years [2 reports in Michigan in 1999; 1 report in Michigan in 2000; 21 reports in 2003 (18 in Utah, 2 in Michigan and 1 in New York); and 2 reports in Michigan in 2004], we restricted our analysis to 2019 to 2024, when reports began to appear more consistently. NFLIS systematically collects drug identification data from 284 federal, state and local forensic laboratories in all 50 states and the District of Columbia (DC) that participate voluntarily [19]. Although specific forensic procedures vary by laboratory, 99% reported using gas chromatography–mass spectrometry (GC/MS) and 96% used Fourier transform infrared (FTIR) spectrophotometry for drug confirmation [20]. These data represent raw, unweighted counts of seized nitazene-positive drug samples and are not adjusted for variations in laboratory participation, reporting volume or missing data [20]. Consistent with our objective of characterizing absolute temporal and geographic changes, we did not calculate rates. Absolute counts have been used in prior studies, are correlated with overdose rates and are widely used proxies for street-level drug availability [21-24].
Second, we obtained data on nitazene-positive biospecimens from the CFSRE’s NPS Discovery program, which conducts drug market surveillance using case samples submitted from across the US. This program conducts comprehensive testing for novel psychoactive substances identified in both seized drug samples and biospecimens submitted by collaborating crime laboratories, medical examiners and clinical partners. Drug identifications are made using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), with supplemental mass spectrometry platforms used as needed [25]. Because the NFLIS dataset includes only seized drug samples, we filtered the NPS Discovery dataset to only biospecimens to provide complementary information across the two sources. NFLIS data are structured at the report level, with separate entries for each identified analog, whereas NPS Discovery data are structured at the unique biospecimen level, ensuring that multiple nitazene analogs detected within the same biospecimen are not double counted. NPS Discovery data are neither nationally representative nor weighted by submission volume. However, they provide timely signals of emerging drug trends as the data are consistently acquired and analyzed using standardized laboratory protocols. In both databases, nitazene analogs were identified as any drug containing the string ‘–nitaze–’. We additionally conducted manual review of drug names in both datasets to ensure no nitazenes were excluded.
Data analysis
The primary research questions and analysis plan were not pre-registered on a publicly available platform; therefore, all analyses should be considered exploratory. We descriptively analyzed the total number of nitazene reports submitted to NFLIS by year, individual nitazene analog, US Census region and state. Similarly, we tabulated NPS Discovery data by year and analog. We also calculated the percentages of biospecimens with co-detected substances. Polysubstance patterns were examined exclusively using NPS Discovery data because its analytical pipeline enables identification of multiple drugs within the same physical admixture. In contrast, the ‘co-reported drugs’ field in NFLIS reflects substances identified within the same submission, but not necessarily within the same mixture and, therefore, does not reliably capture true polysubstance involvement [26].
We then conducted temporal trend analyses examining the total number of nitazene reports submitted to NFLIS nationally and by US Census region from 2019 to 2024, as well as the total number of nitazene-positive biospecimens detected by NPS Discovery over the same period. Annual trends were modeled using piecewise linear regression with a Poisson distribution and log link. Given evidence of overdispersion, all final models used quasi-Poisson specifications. For each time series, the number and location of any breakpoints in the trend were selected according to the Davies test and model fit, as we have done in previous work [22, 27]. If the Davies test provided evidence of a breakpoint, segmented and non-segmented models were compared using F-tests to determine whether the segmented specification provided a significantly improved fit with a two-sided α = 0.05. All analyses and visualizations were conducted using R version 4.2.1 (R Core Team).
This analysis was deemed exempt from institutional review board approval by the National Institute of Justice (US Department of Justice) because it focused on deidentified, non-human data.
RESULTS
Annual nitazene reports submitted to NFLIS
As shown in Table 1, we identified 7117 total nitazene reports submitted to NFLIS between 2019 and 2024, encompassing 21 distinct nitazene analogs. The number of distinct nitazene analogs increased from two in 2019 to 18 in 2024. The majority of nitazene reports were metonitazene (n = 3297; 46.3%), followed by protonitazene (n = 1334, 18.7%) and isotonitazene (n = 912, 12.8%). Figure 1 illustrates the relative share of specific nitazene analogs over time. Early reports were predominated by isotonitazene, which accounted for 97.7% of nitazene reports in 2019, but was subsequently overtaken by metonitazene in 2021 (first detected in 2020) and by protonitazene in 2023 (first detected in 2021). Three nitazene analogs identified in NFLIS (ethyleneoxynitazene, N,N-dimethylamino etonitazene and ethylene etonitazene) were not detected in NPS Discovery biospecimens. In addition, five nitazene analogs captured by NPS Discovery (flunitazene, butonitazene, etonitazene, N-piperidinyl etonitazene and N-pyrrolidino isotonitazene) fell outside the analytic dataset and were, therefore, absent from the present analysis.
TABLE 1.
Annual Nitazene Reports to the US Drug Enforcement Administration’s (DEA) National Forensic Laboratory Information System (NFLIS), 2019–2024.
| No. of reports by year | |||||||
|---|---|---|---|---|---|---|---|
| Drug | Total, n (%) | 2019, n (%) | 2020, n (%) | 2021, n (%) | 2022, n (%) | 2023, n (%) | 2024, n (%) |
| Any nitazene | 7117 (100.0) | 43 (100.0) | 315 (100.0) | 1603 (100.0) | 1444 (100.0) | 1807 (100.0) | 1905 (100.0) |
| Specific nitazene analog | |||||||
| Metonitazene | 3297 (46.3) | — | 143 (45.4) | 960 (59.9) | 714 (49.4) | 918 (50.8) | 562 (29.5) |
| Protonitazene | 1334 (18.7) | — | — | 28 (1.7) | 187 (13.0) | 546 (30.2) | 573 (30.1) |
| Isotonitazene | 912 (12.8) | 42 (97.7) | 159 (50.5) | 357 (22.3) | 213 (14.8) | 98 (5.4) | 43 (2.3) |
| N-pyrrolidino etonitazene | 704 (9.9) | — | — | 165 (10.3) | 159 (11.0) | 75 (4.2) | 305 (16.0) |
| N-pyrrolidino protonitazene | 170 (2.4) | — | — | — | — | 37 (2.0) | 133 (7.0) |
| Etodesnitazene | 165 (2.3) | — | 1 (0.3) | 53 (3.3) | 56 (3.9) | 37 (2.0) | 18 (0.9) |
| N-desethyl isotonitazene | 126 (1.8) | — | — | — | 4 (0.3) | 26 (1.4) | 96 (5.0) |
| N-desethyl etonitazene | 115 (1.6) | — | — | — | — | 19 (1.1) | 96 (5.0) |
| Flunitazene | 81 (1.1) | — | 1 (0.3) | 18 (1.1) | 45 (3.1) | 3 (0.2) | 14 (0.7) |
| Butonitazene | 76 (1.1) | — | — | 13 (0.8) | 50 (3.5) | 8 (0.4) | 5 (0.3) |
| Etonitazene | 39 (0.5) | 1 (2.3) | 10 (3.2) | 9 (0.6) | 4 (0.3) | 9 (0.5) | 6 (0.3) |
| Ethyleneoxynitazene | 14 (0.2) | — | — | — | — | 14 (0.8) | — |
| Methylenedioxynitazene | 18 (0.3) | — | — | — | — | — | 18 (0.9) |
| N-pyrrolidino metonitazene | 14 (0.2) | — | — | — | — | 10 (0.6) | 4 (0.2) |
| N-piperidinyl etonitazene | 11 (0.2) | — | — | — | 10 (0.7) | 1 (0.1) | — |
| N-pyrrolidino isotonitazene | 10 (0.1) | — | — | — | — | 2 (0.1) | 8 (0.4) |
| N-desethyl protonitazene | 9 (0.1) | — | — | — | — | — | 9 (0.5) |
| 5-methyl etodesnitazene | 6 (0.1) | — | — | — | — | 2 (0.1) | 4 (0.2) |
| Metodesnitazene | 4 (0.1) | — | 1 (0.3) | — | 2 (0.1) | 1 (0.1) | — |
| N,N-dimethylamino etonitazene | 8 (0.1) | — | — | — | — | — | 8 (0.4) |
| Ethylene etonitazene | 4 (0.1) | — | — | — | — | 1 (0.1) | 3 (0.2) |
Note: ‘—’ indicates no reports. Percentages were calculated using the total number of nitazenes for that year as the denominator. ‘Total’ refers to the period of 2019–2024.
Abbreviation: US, United States.
FIGURE 1. Annual Nitazene Reports to the US Drug Enforcement Administration’s (DEA) National Forensic Laboratory Information System (NFLIS) by Nitazene Analog and Census Region, 2019–2024.
Caption: Area plots showing the annual number of nitazene detections reported to the DEA’s NFLIS, stratified by individual nitazene analog and US Census region, from 2019 to 2024.
Overall, nitazene reports submitted to NFLIS increased from 43 in 2019 to 1905 in 2024 (Table 2, Figure 2). The Davies test provided significant evidence of a breakpoint in 2021 (P < 0.001), and the F-test indicated that the segmented model provided a significantly better fit than the non-segmented model (P = 0.02). Counts increased sharply from 2019 to 2021, with an estimated seven-fold increase per year (count ratio = 7.32; 95% CI = 2.22–24.20), and did not increase significantly from 2021 to 2024 (count ratio = 1.08; 95% CI = 1.00–1.17).
TABLE 2.
Annual Trends in the Number of Nitazenes Detected in the United States, 2019–2024.
| Count |
Overall trend | Trend 1 |
Trend 2 |
||||
|---|---|---|---|---|---|---|---|
| 2019 | 2024 | Count ratio (95% CI) | Years | Count ratio (95% CI) | Years | Count ratio (95% CI) | |
| DEA’s NFLIS | |||||||
| United Statesa | 43 | 1905 | 1.42 (1.08–1.88) | 2019–2021 | 7.32 (2.22–24.20) | 2021–2024 | 1.08 (1.00–1.17) |
| Northeasta | 0 | 362 | 2.24 (1.68–2.98) | 2019–2023 | 4.55 (3.55–5.84) | 2023–2024 | 1.27 (1.12–1.45) |
| Midwestb | 28 | 887 | 1.33 (0.97–1.83) | 2019–2021 | 7.85 (0.66–93.97) | 2021–2024 | 0.98 (0.81–1.18) |
| Southa | 14 | 584 | 1.40 (1.04–1.90) | 2019–2021 | 5.78 (4.72–7.09) | 2021–2024 | 0.95 (0.89–1.01) |
| Westb | 1 | 72 | 1.75 (1.15–2.66) | – | – | – | – |
| CFSRE’s NPS Discovery | |||||||
| United Statesb | 11 | 113 | 1.45 (1.23–1.71) | – | – | – | – |
Abbreviations: DEA NFLIS, United States Drug Enforcement Administration’s National Forensic Laboratory Information System; CFSRE’s NPS Discovery, Center for Forensic Science Research and Education’s NPS Discovery Program.
Segmented quasi-Poisson models preferred because the segmented model provided a significantly better fit than the non-segmented model based on F-tests comparing nested models.
Non-segmented quasi-Poisson models preferred because F-tests indicated that segmentation did not significantly improve model fit.
FIGURE 2. Observed and Fitted Trends in Annual Nitazene Reports to the US Drug Enforcement Administration’s (DEA) National Forensic Laboratory Information System (NFLIS) and the Center for Forensic Science Research & Education’s (CSFRE) NPS Discovery Program, 2019–2024.
Caption: Line graphs showing the annual total of nitazene detections reported to the DEA’s NFLIS and to the CFSRE’s NPS Discovery Program from 2019 to 2024. Reports to the DEA were also stratified by US Census region. Fitted trends were estimated using piecewise linear regression with a Poisson distribution and log link, allowing up to one breakpoint.
By US Census region (Table S1), we found that more than half of all nitazene reports from 2019 to 2024 were from the Midwest (n = 3777; 53.1%), followed by the South (n = 2407; 33.8%), Northeast (n = 783; 11.0%) and West (n = 150; 2.1%). As shown in Figure 1, temporal patterns varied by individual nitazene analog and region. In the Midwest, nitazene reports were predominated by isotonitazene from 2019 to 2020 (n = 154), followed by metonitazene from 2021 to 2023 (n = 1431) with increasing contributions by protonitazene (n = 302) and N-pyrrolidino etonitazene (n = 220) in 2024. In the South, isotonitazene accounted for all nitazene reports in 2019, after which metonitazene predominated from 2020 to 2024 (n = 1193). In the Northeast, relatively few nitazene reports were identified until 2022, and from 2022 to 2024, metonitazene comprised most of the Northeastern reports (n = 348), followed by protonitazene (n = 214). Similarly, in the West, nearly all nitazene reports occurred from 2021 to 2024, initially involving metonitazene (n = 14) and etonitazene (n = 7) in 2021, followed by etodesnitazene in 2022 (n = 8) and by protonitazene from 2023 to 2024 (n = 44).
Regional trend analyses are shown in Table 2 and Figure 2. Between 2019 and 2024, reports increased from zero to 362 in the Northeast, from 14 to 584 in the South, from 28 to 887 in the Midwest and from one to 72 in the West. Significant breakpoints were identified in the Northeast in 2023 and South in 2021 (Davies test P < 0.001 for both), with segmented models providing significantly improved fit (F-test P = 0.02 and P = 0.01, respectively). In the Northeast, counts increased rapidly through 2023 (count ratio = 4.55; 95% CI = 3.55–5.84), followed by slower but continued growth from 2023 to 2024 (count ratio = 1.27; 95% CI = 1.12–1.45). In the South, counts rose nearly six-fold per year through 2021 (count ratio = 5.78; 95% CI = 4.72–7.09) and stabilized thereafter (count ratio = 0.95; 95% CI = 0.89–1.01).
In contrast, although a breakpoint was suggested in the Midwest (Davies test P < 0.001), the segmented model did not significantly improve fit (F-test P = 0.08); therefore, the non-segmented model was retained (Table 2, Figure 2). No breakpoint was identified in the West (Davies test P = 0.50). Over the full study period, counts in the Midwest increased by an estimated 33% per year, although this increase was not significant (count ratio = 1.33; 95% CI = 0.97–1.83), whereas submissions in the West increased significantly by approximately 75% per year (count ratio = 1.75; 95% CI = 1.15–2.66).
At the state level, nitazene reports were observed in nearly all states and DC, with the exception of New Mexico, Hawaii, Montana, Oklahoma and South Dakota (Figure S1). More than half of all reports occurred in just three states: Ohio (n = 2678; 37.6%), Florida (n = 754; 10.6%) and Tennessee (n = 479; 6.7%). In Ohio, the most common nitazene analogs were metonitazene (n = 1046), protonitazene (n = 525) and isotonitazene (n = 504). In Florida, metonitazene (n = 293), protonitazene (n = 173) and N-pyrrolidino etonitazene (n = 169) predominated, while isotonitazene was rare (n = 19). In Tennessee, metonitazene accounted for most reports (n = 403), with protonitazene (n = 26) and isotonitazene (n = 29) less frequent. Metonitazene first appeared in 2020 and peaked in 2021 with 368 reports in Ohio and 192 in Tennessee. Protonitazene emerged in 2021 and peaked in 2024 with 263 reports in Ohio. Isotonitazene was first detected in 2019 and peaked in 2021, also in Ohio, with 264 reports.
Annual nitazene reports submitted to the NPS Discovery program
As shown in Table 3, NPS Discovery identified 361 unique nitazene-positive biospecimens between 2019 and 2024, corresponding to 519 nitazene analog detections, as individual biospecimens may contain more than one analog. In total, 19 distinct nitazene analogs were identified, and of these, six compounds (4′-hydroxy nitazene, 5-amino isotonitazene, 5-amino protonitazene, 5-amino metonitazene, N-pyrrolidino 4′-hydroxy nitazene and N-desethyl metonitazene) were not reported in NFLIS drug sample submissions. Most NPS Discovery biospecimens involved metonitazene (n = 145; 40.2%), followed by protonitazene (n = 82, 22.7%) and isotonitazene (n = 66, 18.3%). Biospecimens consisted primarily of isotonitazene from 2019 to 2020, followed by metonitazene from 2021 to 2024 and protonitazene from 2023 to 2024.
TABLE 3.
Annual Unique Nitazene-Positive Biospecimens from the Center for Forensic Science Research and Education’s (CSFRE) NPS Discovery Program, 2019–2024.
| No. of reports by year | |||||||
|---|---|---|---|---|---|---|---|
| Drug | Total, n (%) | 2019, n (%) | 2020, n (%) | 2021, n (%) | 2022, n (%) | 2023, n (%) | 2024, n (%) |
| Nitazenes | |||||||
| Any nitazene | 361 (100.0) | 11 (100.0) | 30 (100.0) | 62 (100.0) | 52 (100.0) | 93 (100.0) | 113 (100.0) |
| Specific nitazene analoga | |||||||
| Metonitazene | 145 (40.2) | — | 7 (23.3) | 25 (40.3) | 22 (42.3) | 51 (54.8) | 40 (35.4) |
| Protonitazene | 82 (22.7) | — | — | 10 (16.1) | 6 (11.5) | 34 (36.6) | 32 (28.3) |
| Isotonitazene | 66 (18.3) | 10 (90.9) | 22 (73.3) | 8 (12.9) | 18 (34.6) | 6 (6.5) | 2 (1.8) |
| N-pyrrolidino etonitazene | 46 (12.7) | — | — | 20 (32.3) | 7 (13.5) | 1 (1.1) | 18 (15.9) |
| N-pyrrolidino protonitazene | 33 (9.1) | — | — | — | — | 15 (16.1) | 18 (15.9) |
| N-desethyl protonitazene | 30 (8.3) | — | — | 4 (6.5) | 2 (3.8) | 8 (8.6) | 16 (14.2) |
| N-desethyl isotonitazene | 25 (6.9) | 3 (27.3) | 5 (16.7) | 3 (4.8) | 5 (9.6) | 4 (4.3) | 5 (4.4) |
| Etodesnitazene | 18 (5.0) | — | 1 (3.3) | 7 (11.3) | 4 (7.7) | 2 (2.2) | 4 (3.5) |
| 4′-hydroxy nitazene | 9 (2.5) | 1 (9.1) | 1 (3.3) | 5 (8.1) | 1 (1.9) | — | 1 (0.9) |
| N-desethyl metonitazene | 18 (5.0) | — | — | 5 (8.1) | — | 6 (6.5) | 7 (6.2) |
| N-pyrrolidino metonitazene | 8 (2.2) | — | — | — | — | 4 (4.3) | 4 (3.5) |
| 5-amino isotonitazene | 8 (2.2) | — | 2 (6.7) | 3 (4.8) | 1 (1.9) | — | 2 (1.8) |
| 5-amino protonitazene | 9 (2.5) | — | — | 1 (1.6) | 2 (3.8) | 5 (5.4) | 1 (0.9) |
| 5-methyl etodesnitazene | 5 (1.4) | — | — | — | — | — | 5 (4.4) |
| N-desethyl etonitazene | 5 (1.4) | — | — | — | — | — | 5 (4.4) |
| 5-amino metonitazene | 4 (1.1) | — | — | 4 (6.5) | — | — | — |
| N-pyrrolidino 4′-hydroxy nitazene | 4 (1.1) | — | — | — | — | — | 4 (3.5) |
| Methylenedioxynitazene | 2 (0.6) | — | — | — | — | — | 2 (1.8) |
| Metodesnitazene | 2 (0.6) | — | — | — | — | — | 2 (1.8) |
Note: ‘—’ indicates no reports. Percentages were calculated using the total number of nitazenes for that year as the denominator. ‘Total’ refers to the period of 2019–2024.
Percentages may exceed 100% because multiple nitazene analogs can be detected within a single biospecimen.
Overall, the total number of NPS Discovery nitazene-positive biospecimens increased from 11 in 2019 to 113 in 2024 (Table 2, Figure 2). There was no significant evidence of a breakpoint (Davies test P = 0.20); therefore, the non-segmented model was retained. From 2019 to 2024, counts increased significantly by approximately 45% per year (count ratio = 1.45; 95% CI = 1.23–1.71).
Tables S2 and S3 describe the number of co-involved substances and the most commonly co-involved substances within nitazene-positive biospecimens, respectively. Polysubstance involvement was common, with a mean of 7.6 co-detected substances per biospecimen (SD = 5.0) and a median of 7 [interquartile range (IQR) = 3–10]. Most nitazene-positive biospecimens contained at least one additional substance (n = 355; 98.3%). Nearly one-third included 10 or more additional substances (n = 111; 30.7%). Fentanyl was the most frequently co-detected substance (n = 197; 54.6%), with fentanyl-related substances such as 4-ANPP (n = 131; 36.3%) and norfentanyl (n = 82; 22.7%) also commonly co-detected. Methamphetamine (n = 114; 31.6%) and cocaine (n = 108; 29.9%) were the most common co-detected stimulants. Bromazolam and xylazine were the most frequently co-detected sedatives, each present in 86 nitazene-positive biospecimens (23.8%).
Polysubstance patterns varied by nitazene analog (Figure 3). Fentanyl was co-detected in 59.3% of metonitazene-positive biospecimens (n = 86) compared with 40.2% of protonitazene-positive biospecimens (n = 33). Xylazine was detected in 29.0% of metonitazene-positive biospecimens (n = 42), likely because of high fentanyl co-involvement but it did not appear among the 10 most frequent co-detections for either protonitazene- or isotonitazene-positive biospecimens. Flualprazolam was present in 33.3% of isotonitazene-positive biospecimens (n = 22), but not among the most common co-detections for either metonitazene- or protonitazene-positive biospecimens.
FIGURE 3. Annual Nitazene-Positive Biospecimen Reports from the Center for Forensic Science Research & Education’s NPS Discovery Program by Co-involved Substances, 2019–2024.
Caption: Bar charts depicting the top 10 substances co-detected with nitazenes in biospecimens submitted to the Center for Forensic Science Research & Education’s (CFSRE’s) NPS Discovery Program from 2019 to 2024. Data are shown for any nitazene, isotonitazene, metonitazene, and protonitazene.
DISCUSSION
To our knowledge, this is the first national study to characterize temporal and geographic trends in nitazene analog detections in the US. From 2019 to 2024, annual nitazene detections increased substantially, rising from 43 to 1905 reports in NFLIS and from 11 to 113 nitazene-positive biospecimens in NPS Discovery. Over the same period, the diversity of nitazene analogs expanded greatly, increasing from just 2 to 3 analogs in 2019 to more than 20 by 2024. Across both datasets, isotonitazene was the first nitazene analog to emerge and initially predominate, accounting for nearly all detections in 2019. However, isotonitazene declined sharply by 2024 as metonitazene and protonitazene became more prominent. Temporal analyses showed that NFLIS reports increased more than seven-fold from 2019 to 2021, followed by a period of relative stabilization. This apparent plateau suggests that nitazenes may now represent a more stable component of the US illicit drug supply. In comparison, nitazene-positive biospecimens increased steadily over time, with an estimated annual growth of 45%. Overall, these findings emphasize the need for sustained investment in drug testing, epidemiological surveillance, clinical education and public health messaging to monitor and respond to the spread of nitazenes across the US.
Our findings indicate that nitazene detections have increased rapidly in the US in recent years, mirroring broader global trends. A 2025 report by the European Union Drugs Agency (EUDA) identified 22 distinct nitazene analogs across 21 European countries between 2019 and 2024 (reporting only parent compounds) [28]. In comparison, our analysis identified 27 distinct nitazene analogs across the NFLIS and NPS Discovery datasets (reporting both parent compounds and metabolites). We detected 11 compounds that were not included on the EUDA list, including metabolites of protonitazene (e.g. 5-amino protonitazene, N-desethyl metonitazene), isotonitazene (e.g. 5-amino isotonitazene, N-pyrrolidino isotonitazene) and metonitazene (e.g. 5-amino metonitazene), as well as etonitazene-related compounds (e.g. etonitazene, ethylene etonitazene), desnitro nitazenes (e.g. 5-methyl etodesnitazene), hydroxylated nitazenes (e.g. 4′-hydroxy nitazene, N-pyrrolidino 4′-hydroxy nitazene) and ring-substituted analogs (e.g. methylenedioxynitazene). Conversely, six nitazene analogs reported by the EUDA were not observed in our data, including other desnitro nitazenes (e.g. 6-methyl desnitroetonitazene, desnitroclonitazene), non-nitro analogs (e.g. etomethazene), fluoro-substituted nitazenes (e.g. fluetonitazene, N-pyrrolidino fluetonitazene) and branched-chain analogs (e.g. isobutonitazene) [28]. Although some discrepancies reflect differences in metabolite reporting, others suggest meaningful geographic variation in parent compounds circulating in Europe versus the US.
These differences emphasize the importance of harmonized international drug surveillance, strengthened cross-border collaboration and timely data sharing to support early warning systems and coordinated public health responses. For example, closer collaboration between US public health surveillance agencies and the European Union (EU) Early Warning System could improve the early detection of new compounds. This is especially relevant for heavily affected regions in Northern Europe, particularly the Baltic states (Estonia, Latvia and Lithuania), which collectively accounted for approximately 75% of novel synthetic opioid adulterant seizures reported to the EUDA, including nitazenes and carfentanil [29-32]. Prior evidence indicates that nitazenes were involved in 66% and 42% of overdose deaths in Latvia and Estonia in 2023, respectively, with only modest declines to 43% and 42% in 2024. Strengthening global collaboration may, therefore, enhance surveillance of emerging nitazene analogs and enable more timely clinical and public health responses.
We observed temporal patterns in the relative share of specific nitazene analogs, particularly isotonitazene, metonitazene and protonitazene. These shifts likely reflect a combination of illicit drug market dynamics, regulatory actions and public health interventions. Notably, we found that isotonitazene detections declined sharply across both datasets from 2020 to 2024, which appears to coincide with the compound’s temporary scheduling in 2020 and permanent scheduling in 2021 [33]. In NFLIS, isotonitazene accounted for 51% of all nitazene submissions in 2020, but fell to just 2% by 2024. Similarly, in NPS Discovery, isotonitazene-positive biospecimens decreased from 73.3% of all nitazene-positive biospecimens in 2020 to 2% in 2024.
Despite this decline, overall nitazene detections in both NFLIS and NPS Discovery continued to rise over the study period, driven largely by increases in metonitazene and protonitazene. Corroborating these findings, a study from Tennessee found that isotonitazene was involved in 90% of nitazene-related overdose deaths in 2020, but was subsequently displaced by metonitazene, which accounted for 86% of such deaths in 2021 [15]. National SUDORS data show a similar pattern, which is that isotonitazene-involved overdose deaths rose from 25 in 2020 (across seven states) to 71 in 2021, before declining to 47 in 2022 and 27 in 2023 [14]. In contrast, metonitazene was rarely reported in 2020 (1 death in Ohio), but increased rapidly to 109 deaths in 2021, 86 in 2022 and 190 in 2023 [14].
Metonitazene entered temporary scheduling in 2022 and was permanently scheduled in 2023 [34]. In NFLIS, metonitazene accounted for approximately 45% to 60% of all annual nitazene reports from 2020 through 2023, before declining to 30% in 2024. Similarly, in NPS Discovery, metonitazene-positive biospecimens increased from 23% of all nitazene-positive biospecimens in 2020 to 55% in 2023, followed by a decline to 35% in 2024. Although metonitazene detections remained prevalent throughout the study period, the declines observed after scheduling suggest a possible moderating effect of regulatory action; however, formal evaluation of this effect warrants further investigation.
In contrast, protonitazene appeared less responsive to scheduling. Although it entered temporary scheduling in 2022 and was permanently scheduled in 2024 [35], its detections continued to rise. Protonitazene increased from 13% to 30% of all nitazene-positive NFLIS reports between 2022 and 2024, while in NPS Discovery, protonitazene-positive biospecimens increased from 12% to 28% of all nitazene-positive biospecimens over the same period. These early postscheduling patterns suggest that protonitazene has not yet been substantially affected by regulatory controls, although longer-term research and monitoring are needed.
We further note that 11 nitazene analogs underwent temporary scheduling (N-desethyl isotonitazene, N-piperidinyl etonitazene, N-pyrrolidino metonitazene, N-pyrrolidino protonitazene, ethyleneoxynitazene, methylenedioxynitazene, 5-methyl etodesnitazene, N-desethyl etonitazene, N-desethyl protonitazene, N,N-dimethylamino etonitazene and N-pyrrolidino isotonitazene), while seven analogs were permanently scheduled (butonitazene, clonitazene, etodesnitazene, etonitazene, flunitazene, metodesnitazene and N-pyrrolidino etonitazene) during the study period [36]. Across these compounds, we did not observe consistent or sustained changes in detection trends in either NFLIS or NPS Discovery that aligned temporally with their respective scheduling actions. This likely reflects the stronger and more timely response of international scheduling efforts under the United Nations Conventions, but may also be due to infrequent detections, limited sample sizes or more complex scheduling, emphasizing the need for longer-term follow-up to more fully assess the impact of scheduling on nitazene market dynamics.
We also observed differences in the specific nitazene analogs identified between the two datasets in this study. Three compounds (ethyleneoxynitazene, N,N-dimethylamino etonitazene and ethylene etonitazene) were detected in NFLIS drug submissions, but not in NPS Discovery biospecimens, and five compounds (flunitazene, butonitazene, etonitazene, N-piperidinyl etonitazene and N-pyrrolidino isotonitazene) were absent from the NPS Discovery forensic toxicology dataset analyzed but detected at the laboratory. One plausible explanation is variation in pharmacologic potency as nitazene analogs differ substantially in μ-opioid receptor potency; lower-potency compounds may be less likely to result in severe or fatal clinical presentations even when present in the illicit drug supply. This may partially explain the absence of compounds such as flunitazene and ethyleneoxynitazene in a substantial number of NPS Discovery biospecimens [37]. However, because our analysis did not distinguish between post-mortem and ante-mortem biospecimens, further work is needed to triangulate these findings with analog-specific overdose mortality data. Another possible explanation relates to differences in geographic coverage and submission practices. Unlike NFLIS, which captures drug submissions from law enforcement laboratories nationwide, the NPS Discovery program is not fully nationally representative and reflects submissions from a subset of jurisdictions. As a result, some nitazene analogs may be circulating in regions outside the NPS Discovery testing network. Differences in participating laboratories and submission practices between NFLIS and CFSRE may contribute to this pattern as well.
Conversely, six compounds (4′-hydroxy nitazene, 5-amino isotonitazene, 5-amino protonitazene, 5-amino metonitazene, N-pyrrolidino-4′-hydroxy nitazene and N-desethyl metonitazene) were detected exclusively in the NPS Discovery dataset and not in NFLIS drug submissions. This pattern is expected, as these compounds primarily represent metabolites identified in biospecimens rather than parent compounds typically detected in seized drug samples. Differences in analyte reporting may also play a role; for example, NFLIS captures several N-desethyl metabolites, including N-desethyl protonitazene, N-desethyl isotonitazene and N-desethyl etonitazene, but does not capture N-desethyl metonitazene, which was observed in NPS Discovery biospecimens. These findings highlight the importance of triangulating complementary data sources to systematically monitor the emergence, spread and public health impact of nitazene analogs.
Further research is also needed to evaluate differences between nitazene analogs detected in drug samples versus biospecimens and to assess temporal sequencing, whereby new analogs may be detected first in law enforcement seizures before appearing in clinical or post-mortem biospecimens. Although this could not be evaluated in the present study because NFLIS lacks more granular temporal data (e.g. monthly or quarterly), understanding this sequencing is critical for future monitoring efforts, as consistent seizure-first detection would strengthen the use of forensic data as an early warning system for nitazene-related overdose risks.
Our subgroup analyses by US Census region and state emphasize the need for regionally tailored strategies. Ohio, Florida and Tennessee collectively accounted for more than half (54.9%) of the 7117 nitazene analogs submitted to NFLIS. Florida had a particularly high share of protonitazene, comprising nearly one-quarter of all nitazene reports, consistent with state-issued alerts of highly potent ‘Frankenstein opioids’ (a media term sometimes used to describe nitazene analogs) [38]. In the Northeast, reports rose from six in 2020 to 362 in 2024, largely driven by growth in New York and Pennsylvania. In contrast, reports in the Western US remained low, accounting for 2.1% of all nitazene reports to NFLIS. These geographical patterns may reflect different trafficking and distribution networks, including maritime entry points in Florida, distribution hubs centered in or near Ohio and major highway corridors along the Mid-Atlantic. Consistent with this interpretation, prior evidence indicates that Ohio and Florida have experienced concentrated clusters of other high-potency synthetic opioids, including carfentanil [39]. Further research is needed to map distribution pathways, assess their influence on regional drug availability and inform regionally tailored harm reduction strategies. Ongoing monitoring is also critical to understand differences in market structure and evolving trends in dominant nitazene analogs within these geographic hotspots. For example, early trends in Ohio were predominated by metonitazene and isotonitazene, whereas early trends in Florida were driven by metonitazene and N-pyrrolidino etonitazene. In both states, protonitazene emerged later and became one of the three most frequently reported nitazene analogs by the end of the study period.
Another key finding was the near-universal polysubstance involvement among nitazene detections. Among nitazene-positive biospecimens identified by NPS Discovery, which can assess polysubstance patterns because its analytical pipeline identifies multiple drugs within the same physical admixture, 98.3% contained at least one additional drug, with a median of seven co-detected substances. Fentanyl appeared in approximately 55% of all nitazene-positive biospecimens, with higher co-occurrence for metonitazene- and isotonitazene-positive biospecimens (59% and 55%, respectively) and lower co-occurrence for protonitazene-positive biospecimens (40%). These patterns are consistent with a Tennessee study from 2019 to 2021, which reported that all nitazene-involved biological samples contained at least one additional substance, with fentanyl being the most commonly co-detected substance (60%) [15]. Similarly, a study of fatal overdoses in Knox County, Tennessee, from 2020 to 2021 found that metonitazene was implicated in 26 of 770 unintentional overdose deaths (4.6%), almost universally co-present with fentanyl or stimulants, highlighting the growing role of nitazenes in polysubstance overdose deaths [40].
Protonitazene showed particularly high co-positivity with stimulants, with methamphetamine and cocaine co-detected in 38% and 32% of protonitazene-positive biospecimens, respectively. This pattern is consistent with findings from Tennessee, where methamphetamine was co-involved in 46% of nitazene overdose deaths [15]. Our analysis also identified other substances, including xylazine, which was detected in 24% of all nitazene-positive biospecimens and was especially prevalent in metonitazene-positive biospecimens (29%). These findings align with emerging evidence indicating that xylazine is frequently co-involved with nitazenes; for example, a forensic case series of 85 nitazene reports from North America and the United Kingdom identified xylazine in 26 cases (31%) [41]. Altogether, these findings highlight the need to monitor emerging nitazene-related polysubstance combinations within the evolving drug supply. Further research is also needed to triangulate forensic and toxicological data with overdose mortality (e.g. SUDORS) and morbidity data to better understand the population-level impact of the emergence and spread of nitazenes in the ongoing overdose crisis.
Strengthening epidemiological surveillance is essential to monitor the proliferation of nitazene analogs. Nitazene test strips represent a promising point-of-care drug checking tool. Recent laboratory evaluations found that test strips were able to detect 24 of 33 nitazene analogs at concentrations ≤9000 ng/mL, indicating broad but incomplete cross-reactivity with currently circulating analogs [42]. Similarly, another study demonstrated reliable detection of 28 of 36 nitazene analogs, with limits of detection ranging from 250 ng/mL to 100 μg/mL [43]. However, important limitations remain. Certain structurally distinct nitazene analogs, such as desnitazenes lacking the 5-nitro group, were not detected. Further, testing was conducted using high-purity samples, which may not reflect the complexity of real-world polysubstance mixtures. For example, one study reported falsepositive results in the presence of caffeine (a common adulterant) at concentrations exceeding 300 μg/mL, raising concerns about the accuracy of nitazene detection in polysubstance mixtures [43]. Finally, test strips are only able to detect the presence of a nitazene, but cannot provide information on specific analog identity, dosage or the presence of adulterants.
To address these gaps, national public health surveillance efforts should prioritize validation of next-generation assays, standardization of toxicology testing across jurisdictions and expansion of drug checking programs capable of detecting a broad range of synthetic compounds, including nitazenes. Additional efforts could include scaling up wastewater surveillance for nitazene analogs, building on approaches used by public health systems in the EU to monitor population-level nitazene trends [7, 44]. At the same time, continued monitoring and research are needed to better characterize nitazene trafficking patterns and supply chain dynamics. Although studies from the US, Australia and Europe (including the United Kingdom, Norway and Estonia) have documented a high prevalence of polysubstance involvement with nitazenes [6, 32, 45-47], there remains a very limited understanding of where along the drug supply chain nitazenes are introduced. A recent report by the Inter-American Drug Abuse Control Commission of the Organization of American States noted that while many seizures involve international trafficking networks (e.g. from China to the US and Mexico), empirical evidence on nitazene production, trafficking pathways and distribution pathways remains limited [11]. Further research is needed to determine whether nitazenes are mixed into heroin or fentanyl powders before entering US markets or trafficked as standalone high-potency products and subsequently mixed locally. Clarifying this distinction is crucial for informing effective public health surveillance, harm reduction strategies and regulatory responses.
Finally, enhanced surveillance of nitazene-involved overdoses is critical to inform prevention efforts. Notably, SUDORS data show that nitazene-involved overdose deaths in the US increased sharply from 27 in 2020 (primarily isotonitazene) to 320 in 2023 (primarily metonitazene) [14]. Although this increase may partially reflect the expanded toxicological testing for nitazenes, it also signals an alarming rise in nitazene-related mortality. Similar patterns have been observed internationally, particularly in Northern Europe [29, 30]. In England, 179 nitazene-involved overdose deaths were reported between 2023 and 2024, most commonly involving protonitazene [48]. In Scotland, nitazenes were implicated in 76 drug overdose deaths in 2024, more than a threefold increase from 2023 [49]. Estonia documented 136 nitazene-involved overdose deaths between 2019 and 2024 [32]. Consistent with our findings, early deaths were largely associated with isotonitazene, while later years were predominated by metonitazene and protonitazene. Norway reported 36 nitazene-involved overdose deaths between 2021 and 2024, with most occurring after 2023 [47]. Notably, more than 90% of Norwegian cases co-involved other novel psychoactive substances, mirroring the high degree of polysubstance involvement observed in our analysis. Overall, these findings highlight the international spread of nitazenes and reinforce the need to expand drug testing infrastructure to inform both clinical care and public health responses.
Limitations
This study has several limitations. First, although NFLIS and NPS Discovery data are widely used as proxies for the US illicit drug supply, they do not fully capture the true prevalence or distribution of nitazene analogs nationwide [26]. NFLIS detections are influenced by public safety priorities, regional testing capacity and laboratory reporting practices; therefore, higher counts may reflect increased testing capacity or enforcement activity rather than true differences in availability. Second, both NFLIS and NPS Discovery rely on voluntary submissions that vary across jurisdictions, resulting in uneven geographic coverage. NFLIS includes only drugs that are both seized and tested, excluding seized materials that are not analyzed. In addition, caution is warranted when comparing raw annual NFLIS counts, as year-to-year changes in the number of reporting laboratories may bias temporal comparisons. Third, polysubstance profiles were derived exclusively from NPS Discovery biospecimens. Although NFLIS ‘co-reports’ other substances in the same seizure, it does not specify whether they were physically mixed, a well-recognized limitation that precludes valid inference about true polysubstance patterns [26]. Within this context, complementing NFLIS with NPS Discovery data is a key strength of this study, because it enables direct assessment of polysubstance patterns. Nevertheless, reliance on NPS Discovery data may not capture the full range of substances co-involved with nitazene analogs. For example, not all biospecimens were tested for cannabinoid analytes, limiting assessment of cannabis co-positivity. Moreover, because the NPS Discovery data are derived from biological specimens, we cannot determine whether detected substances were used concurrently or sequentially. Finally, the absence of demographic or clinical information precludes more detailed analysis of individual-level health effects and public health implications.
CONCLUSION
Overall, nitazene analog detections increased sharply in both the NFLIS drug sample submissions and NPS Discovery biospecimens between 2019 and 2024. The distribution of nitazene analogs varied substantially over time and across regions, with distinct geographic hotspots, including Ohio, Florida and Tennessee, and notably polysubstance patterns, particularly with fentanyl. These trends emphasize the need to expand public health surveillance and harm reduction strategies tailored to the rapidly evolving nitazene landscape. Further research that triangulates forensic crime laboratory and toxicological data with clinical and overdose mortality data, alongside expanded overdose prevention efforts, will be critical to addressing the growing public health impact of nitazenes in the US.
Supplementary Material
Additional supporting information can be found online in the Supporting Information section at the end of this article.
ACKNOWLEDGEMENTS
The Center for Forensic Science Research and Education received funding from the National Institute of Justice, Office of Justice Programs, US Department of Justice (Award Numbers 15PNIJ-22-GG-04434-MUMU and 15PNIJ-24-GK-00981-COAP). This study was also supported by the National Institute on Drug Abuse of the National Institutes of Health under Award Numbers R01DA057289 (J.J.P.), U01DA051126 (J.J.P.) and T32DA031099 (Hasin and Martins). The opinions, findings, conclusions and/or recommendations expressed in this publication are those of the author(s) and do not necessarily represent the official position or policies of the US Department of Justice or National Institutes of Health.
Funding information
The Center for Forensic Science Research and Education received funding from the National Institute of Justice, Office of Justice Programs, US Department of Justice (award numbers 15PNIJ-22-GG-04434-MUMU and 15PNIJ-24-GK-00981-COAP). This study was also supported by the National Institute on Drug Abuse of the National Institutes of Health under award numbers R01DA057289 (J.J.P.), U01DA051126 (J.J.P.) and T32DA031099 (Hasin and Martins).
Footnotes
DECLARATION OF INTERESTS
J.J.P. has consulted for the Washington-Baltimore High Intensity Drug Trafficking Areas program. The authors have no other potential conflicts of interest to report.
DATA AVAILABILITY STATEMENT
Data from the US Drug Enforcement Administration’s National Forensic Laboratory Information System that support this study’s findings are openly available at https://www.nflis.deadiversion.usdoj.gov/. Data from the Center for Forensic Science Research and Education’s NPS Discovery program are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Data from the US Drug Enforcement Administration’s National Forensic Laboratory Information System that support this study’s findings are openly available at https://www.nflis.deadiversion.usdoj.gov/. Data from the Center for Forensic Science Research and Education’s NPS Discovery program are available from the corresponding author upon reasonable request.