LC–ESI–MS/MS Fragmentation Profiling for Identification of Known and Novel Nitazene Analogs

Nitazene analogs are potent synthetic opioids with fentanyl-like effects, posing a growing forensic challenge as new variants emerge despite 12 analogs being Schedule I controlled substances in the United States.

J. Tyler Davidson of the Department of Forensic Science at Sam Houston State University (Huntsville, Texas) used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI–MS/MS) to structurally characterize 38 nitazene analogs and propose fragmentation mechanisms that generate diagnostic product ions for analog differentiation. Common ions (m/z 100, 72, 44, 107) arise from substitutions on the amine or benzyl groups, while unique structures—such as piperidine or pyrrolidine rings—produce distinctive ions (m/z 112, 98). The findings establish characteristic fragmentation patterns that can aid in identifying both known and novel nitazene analogs in forensic analysis. LCGC International spoke to Davidson about his work, and the paper that resulted from it (1).

What factors have contributed to the rise of nitazene analogs in the illicit drug market since 2019, and how do they differ structurally from fentanyl analogs?

Changing legislation within the United States and China was likely the biggest factor that led to the rise of nitazene analogs in the illicit drug market. In February of 2018, the Drug Enforcement Administration (DEA) placed all fentanyl-related substances into Schedule I of the Controlled Substances Act (2), which is the highest level of regulation. Then, in May of 2019, China added all fentanyl-related substances to the supplementary list of controlled narcotic drugs and psychotropic substances with non-medical use (3). The combination of these two legislative actions increased the control and regulation of fentanyl-related substances, making it more difficult for these compounds to be manufactured in China and increasing the consequences for those caught in possession of fentanyl-related substances.

Another factor that contributed to the rise of nitazene analogs in the illicit drug market is their potency and the existence of scientific literature describing their synthesis and analgesic properties. The technical name for nitazene analogs is 2-benzylbenzimidazoles. These compounds were originally pursued as pharmaceutical targets back in the 1950s before the high risk of respiratory depression was discovered (4). However, clandestine manufacturers were able to reproduce scientific literature, and in 2019, isotonitazene began to be identified in postmortem blood specimens across the United States.

Nitazene analogs have a very different core structure compared to fentanyl analogs, which led to their rise as clandestine manufacturers attempted to avoid legislative restrictions. Not only are the core structures different, nitazene analogs and fentanyl analogs also contain different types of substitutions to their core structures, resulting in the formation of two distinct classes of compounds, each containing closely related analogs.

How has the scheduling of nitazene analogs by the DEA influenced their continued emergence in forensic toxicology casework?

As new nitazene analogs are scheduled by the DEA, we typically see a decrease in those analogs in forensic casework, followed by the emergence of novel analogs. This isn’t always the case, as metonitazene and protonitazene, which are currently Schedule I controlled substances, are still among the most prominent analogs observed in forensic casework. Unfortunately, because the DEA schedules most compounds on a compound-by-compound basis, this becomes a real cat-and-mouse game. For example, in June, the acting Administrator of the DEA issued an intent to publish a temporary order to schedule seven additional benzimidazole-opioid substances into Schedule I of the Controlled Substances Act, so we may see novel analogs emerge on the illicit drug market. However, the recent blanket scheduling of nitazene analogs in China may lead to the rise of another class of novel synthetic opioids (NSOs) altogether (5).

What makes the identification of nitazene analogs particularly challenging in forensic laboratories, especially in terms of analytical detection?

The identification of nitazene analogs is particularly challenging due to the high degree of structural similarity between analogs, which affects their corresponding analytical properties. For example, the data derived from traditional forensic science techniques, such as gas chromatography-mass spectrometry (GC-MS), is quite similar for many nitazene analogs. Nitazene analogs produce electron ionization (EI) mass spectra, which are dominated by the presence of low molecular weight iminium ions and typically devoid of molecular ions (6,7). In addition, these compounds are extremely potent, meaning that forensic practitioners are often trying to identify or quantify very low levels of nitazene analogs in seized drug mixtures or biological fluids.

Why is LC–ESI–MS/MS favored over gas chromatography-electron ionization-mass spectrometry (GC–EI–MS) for the structural characterization of nitazene analogs? What advantages does it offer?

I wouldn’t necessarily say that LC-ESI-MS/MS is favored over GC-EI-MS, but rather it is a complementary technique to gain additional information about nitazene analogs. Our previous work focused on characterizing nitazene analogs using GC-EI-MS, which helped identify limitations with this instrumentation, including long analysis times, fragment-poor EI mass spectra, and the lack of molecular ions for many nitazene analogs (6,7). In comparison, LC-ESI-MS/MS is better for determining the molecular weight based on the presence of [M+H]⁺ protonated molecules, although there are still challenges with relatively fragmentation-poor product ion spectra and similar retention times. Regardless, it is important to understand the fragmentation behavior for nitazene analogs using common instrumentation to establish a foundation for identifying novel nitazene analogs.

How do collision-induced dissociation (CID) and electron activated dissociation (EAD) compare in terms of utility for analyzing nitazene analogs in mass spectrometry?

CID is the most widely available activation technique to generate fragmentation information for molecules analyzed with soft ionization sources, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). Although soft ionization is advantageous for identifying the molecular weight of an unknown compound, an activation technique is required to gain structural information. With CID, collisions of isolated precursor ions lead to fragmentation that provides structural information about the analyte. In comparison, EAD is used to gain structural information through fragmentation induced by interaction with low-energy electrons. Interestingly, very similar fragmentation was observed with CID in our study compared to the EAD work of others (8), which is likely due to the fragmentation-poor nature of nitazene analogs.

Can you explain how diagnostic ions, such as m/z 121 for methoxy substitutions, assist in distinguishing nitazene analogs, and why these are crucial for multiple reaction monitoring (MRM) and precursor ion scan (PIS) method development?

The goal of identifying diagnostic ions is to establish a chemical signature for the structural components of nitazene analogs. Regardless of the mass spectrometry technique used for analysis, it is important to understand how to identify the structural components present based on the resulting mass spectra. To identify these chemical signatures, representative nitazene analogs must be characterized under various instrumental conditions and the resulting mass spectra studied to identify trends in the fragmentation behavior. These trends can then be applied to identifying novel nitazene analogs.

A good example of how diagnostic ions can be put into practice is the product ion at m/z 121 for methoxy substitutions to the phenyl ring. Based on the compounds analyzed in this study, the diagnostic product ion at m/z 121 was only ever identified in nitazene analogs with a methoxy substitution to the phenyl ring. This phenomenon is due to the methoxy substitution being too short to undergo secondary fragmentation to form the product ion at m/z 107 that is commonly observed for many nitazene analogs. By understanding the chemical signature of methoxy substitutions to the phenyl ring, we now have a mechanism to identify the presence of a methoxy substitution to the phenyl ring of novel nitazene analogs. This information can then be incorporated into MRM or PIS methods.

For example, the transition from m/z 383 to m/z 121 can be implemented in MRM methods to help identify metontizene. Likewise, monitoring for precursor ions that produce a product ion at m/z 121 can help identify the presence of a methoxy substitution to the phenyl ring. These structural trends can therefore be useful for either targeted or non-targeted methods for nitazene analog identification.

What role do doubly charged ions and retention time data play in differentiating desnitazene compounds and why are these compounds especially difficult to identify?

Desnitazene compounds are defined as nitazene analogs without a nitro group substitution to the benzimidazole core. These analogs are challenging to identify because they typically produce very few product ions, and the product ions they do produce tend to be relatively small (less than m/z 200), which are less informative than larger product ions. On one hand, these characteristics make it relatively easy to identify a desnitazene compound compared to its non-desnitazene (for example, nitro group-containing) counterparts; however, determining which desnitazene compound is present is very challenging. Of the 10 desnitazene analogs analyzed in the current study, none could be unequivocally identified based on their product ion spectra alone. Interestingly, the full scan mass spectra of desnitazene compounds contained the presence of a doubly charged precursor ion (i.e., [M+2H]⁺), which was not observed for any of the other non-desnitazene analogs analyzed in this study. Because the doubly charged precursor ions are dependent on the molecular weight of the desnitazene compound, the doubly charged ions can be informative for identifying desnitazene compounds. Ultimately, the combination of the doubly charged precursor ion and retention time information may suffice for desnitazene analog identification.

How did this study utilize isotopic labeling and high-resolution mass spectrometry to map fragmentation pathways of 38 nitazene analogs?

The combination of isotopic labeling and high-resolution mass spectrometry is a common approach to study fragmentation pathways and the corresponding mechanisms through which ions fragment. Isotopic labeling of specific atoms within a molecule enables the isotopically labeled atoms to be tracked in the resulting product ion spectra. The presence of an isotopically labeled atom will shift the observed m/z value (for example, 1 Da for deuterium instead of hydrogen, or ¹³C instead of ¹²C). Thus, there are shifts in the observed product ions due to the presence of isotopic labels, and product ions that are unaffected indicate the absence of isotopic labels. Because we know where the isotopic labels are located within the molecule, their location can be tracked throughout the fragmentation pathways of the molecule, which helps us understand the fragmentation behavior.

Likewise, accurate mass measurements with high-resolution mass spectrometry enable the determination of elemental formulae for product ions based on the certainty with which the ions can be measured. You can think about this certainty as using an expensive analytical balance to measure the weight of an unknown sample compared to the certainty you would find from an at-home protein scale. Because we can know the m/z value of each product ion with more certainty, and all elements have known accurate masses, it is possible to identify the most likely elemental formula for each product ion.

Given the lag between a compound's emergence and its identification in labs, how can forensic workflows be adapted to more quickly detect novel nitazene analogs?

The biggest challenge with identifying novel nitazene analogs is that they won’t exist in mass spectral libraries, and there aren’t certified analytical reference materials available for comparison. Instead, laboratories must interpret their analytical data and utilize alerts from organizations, such as the Novel Psychoactive Substances (NPS) Discovery program (9), that work to share the latest news about what compounds are being identified in the illicit drug market. To assist forensic laboratories with identifying novel nitazene analogs, we have structurally characterized nitazene analogs using electron ionization (EI) mass spectra and electrospray ionization (ESI) product ion spectra collected with CID to enhance our understanding of nitazene analog fragmentation and developed general workflows for the structural elucidation of nitazene analogs. Being able to recognize specific ions and subsequently identify the structures that these ions indicate will assist analysts with more rapid identification of novel nitazene analogs. Unfortunately, there will always be a delay between the emergence of novel analogs and their identification in forensic laboratories. My research group has worked to help provide the necessary resources and tools to help practitioners identify novel analogs as they appear in forensic laboratories.

In your view, what future steps are necessary to support forensic labs in keeping pace with the evolution of the NSO landscape, especially concerning unscheduled nitazene variants?

We will have to wait and see the impact of the recent blanket scheduling of nitazene analogs in China on the NSO landscape. Perhaps this legislation will help simplify the NSO landscape, but it may also lead to the emergence of a new subclass of NSOs. Regardless of future shifts in the NSO landscape, the two biggest resources that forensic laboratories need to keep pace with the evolution of the NSO landscape are information about current drug trends, both regionally and nationally, and resources to help with the interpretation of analytical data from novel compounds. As the NPS and NSO drug markets have continued to evolve, the forensic science community has learned the value of sharing regional and national drug trends. It is imperative to understand what new compounds are emerging locally, as well as what compounds are beginning to emerge elsewhere. Thankfully, resources such as NPS Discovery hosted through the Center for Forensic Science Research and Education (CFSRE) and the National Institute of Standards and Technology (NIST) Rapid Drug Analysis and Research (RaDAR) program are available to help address this need (9,10).

The second necessary resource is information and tools to assist with the interpretation of novel analogs based on available analytical data. This could come in the form of scientific literature regarding fragmentation behavior for specific compound classes, the development of software tools to assist with identifying novel analogs, or even statistical or chemometric treatment of data. This is the area where the research community can provide the most support for forensic practitioners through the development of the necessary resources and tools to support novel analog identification.

References:

1. Hardwick, E. K.; Davidson, J. T. Structural Characterization of Nitazene Analogs Using Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS). Drug Test Anal. 2025.DOI: 10.1002/dta.3921
2. Schedules of Controlled Substances: Temporary Placement of Fentanyl-Related Substances in Schedule I. Fed. Regist.: Drug Enforcement Administration, Department of Justice, 2018, 5188–5192
3. Bao, Y.; Meng, S.; Shi, J. et al. Control of Fentanyl-Related Substances in China. Lancet Psychiatry 2019, 6 (7), e15. DOI: 10.1016/S2215-0366(19)30218-4
4. Pergolizzi, J.; Raffa, R.; LeQuang, J. A. K. et al. Old Drugs and New Challenges: A Narrative Review of Nitazenes. Cureus 2023, 15 (6), e40736. DOI: 10.7759/cureus.40736
5. United Nations Office on Drugs and Crime Laboratory and Scientific Service Portals. July 2025 – China: Announcement of Class Scheduling of Nitazene Analogues. https://www.unodc.org/LSS/Announcement/Details/7e29daf9-1d49-45e6-95e7-8ce932bc94e1 (accessed 2025-07-15)
6. Hardwick, E. K.; Davidson, J. T. Structural Characterization of Nitazene Analogs Using Electron Ionization–Mass Spectrometry (EI–MS). Forensic Chem. 2024, 40, 1–11. DOI: 10.1016/j.forc.2024.100605
7. Phelps, C.; Hardwick, E. K.; Couch, A. N. et al. Development and Validation of a Combined Selected Ion Monitoring-Scan GC-EI-MS Method for Nitazene Analogs. J. Forensic Sci. 2025.DOI: 10.1111/1556-4029.70084
8. Liu, C. M.; Huang, B. Y.; Hua, Z. D. et al. Characterization of Mass Spectrometry Fragmentation Patterns Under Electron-Activated Dissociation (EAD) for Rapid Structure Identification of Nitazene Analogs. Rapid Commun. Mass Spectrom. 2025, 39 (12), e10030. DOI: 10.1002/rcm.10030
9. The Center for Forensic Science Research and Education: NPS Discovery. https://www.cfsre.org/nps-discovery/ (accessed 2025-07-15)
10. National Institute of Standards and Technology: Rapid Drug Analysis and Research (RaDAR). https://www.nist.gov/programs-projects/radar (accessed 2025-07-15)