Evaluation of enzyme-linked immunosorbent assay screening kits for the detection of nitazene analogs
Open accessAbstract
Nitazene analogs are a highly potent class of novel psychoactive substances (NPS) that were first identified in forensic casework in the United States in 2019. While enzyme-linked immunosorbent assay (ELISA) remains a prevalent screening tool in forensic toxicology laboratories, no nitazene-specific ELISA kits are commercially available, supporting the use of high-resolution mass spectrometry (HRMS) methods as a more adaptable alternative for screening. However, even with the growth in popularity of HRMS techniques, it is important to understand the cross-reactivity of novel substances, such as nitazenes, with routinely used ELISA kits. Cross-reactivity is particularly important for forensic toxicology casework since it can impact the reliability of screening results, potentially leading to non-detection of novel substances or false-positive identifications in the presence of non-target analytes. This study tested the cross-reactivity of seven nitazene analogs (4'-OH nitazene, 5-methyl etodesnitazene, isotonitazene, metodesnitazene, N-piperidinyl etonitazene, N-pyrrolidino etonitazene, and protonitazene) in whole blood using 13 commercial ELISA kits from three manufacturers. Various drug/class kits were selected based on reported or potential co-positivity with nitazenes (opiates, opioids, fentanyl) or by structural similarities (LSD, zolpidem). Across tested concentrations for the seven selected analytes, none of the tested kits produced a signal sufficient for a positive result, confirming that their presence at these levels does not compromise the screening specificity of the target analytes. However, these findings highlight the need for laboratories to adopt mass spectral-based screening methods like HRMS or advocate for the development of nitazene-specific ELISA kits for effective nitazene analog screening.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
5 of 12 cited sources checked · 3 supported · 0 flagged · accuracy not yet measured
- Premisecitation
The rise in HRMS use in screening from 2017 to 2021 demonstrates the shift toward broad, mass spectral-based screening methods.
“However, the rise in HRMS use in screening from 2017 to 2021 demonstrates the shift toward broad, mass spectral-based screening methods.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [19] 19. Logan BK, D'Orazio AL, Mohr ALA, Limoges JF, Miles AK, Scarneo CE, et al. Recommendations for toxicological investigation of drug- impaired driving and motor vehicle fatalities-2017 update. J Anal Toxicol. 2018;42(2):63-8. https:// doi. org/ 10. 1093/ jat/ bkx082 source
- cites [20] 20. D'Orazio AL, Mohr ALA, Chan-Hosokawa A, Harper C, Huestis MA, Limoges JF, et al. Recommendations for toxicological investigation of drug-impaired driving and motor vehicle fatalities-2021 update. J Anal Toxicol. 2021;45(6):529-36. https:// doi. org/ 10. 1093/ jat/ bkab064 source
The Introduction says laboratories gained greater access to sensitive screening techniques, including HRMS, and the Results > Toxicology survey reports LC–HRMS use for blood and urine screening. The Results also identify a shift from GC to LC technology between the 2016 and 2020 surveys. Our copy does not specifically establish a rise in HRMS screening use from 2017 to 2021 or characterize that shift as toward broad, mass spectral-based screening. (LLM, conf=0.79)
Experimental — accuracy not yet measured
- Premisecitation
Some nitazene analogs are more potent than fentanyl.
“Importantly, some analytes included in the nitazene class are reported to be more potent than fentanyl, posing a greater risk to drug users while potentially increasing analytical challenges in the laboratory, particularly for screening due to the need for low limits of detection [14][15][16][17][18].”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [14] 14. De Vrieze LM, Walton SE, Pottie E, Papsun D, Logan BK, Krotulski AJ, et al. In vitro structure -activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opi- oids. Arch Toxicol. 2024;98:2999-3018. https:// doi. org/ 10. 1007/ s0020 4-024-03774 -7 source
- cites [15] 15. Vandeputte MM, Verougstraete N, Walther D, Glatfelter GC, Malfliet J, Baumann MH, et al. First identification, chemical analysis and pharmacological characterization of N-piperidinyl etonitazene (etonitazepipne), a recent addition to the 2-benzylbenzimidazole opioid subclass. Arch Toxicol. 2022;96:1865-80. https:// doi. org/ 10. 1007/ s0020 4-022-03294 -2 source
- cites [16] 16. Vandeputte MM, Krotulski AJ, Walther D, Glatfelter GC, Papsun D, Walton SE, et al. Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerg- ing 2-benzylbenzimidazole 'nitazene' synthetic opioid. Arch Toxicol. 2022;96:1845-63. https:// doi. org/ 10. 1007/ s0020 4-022-03276 -4 source
- Findingown data
None of the 13 tested ELISA kits produced a signal sufficient for a positive result for any of the seven nitazene analogs at the tested concentrations (kit cutoff, five times cutoff, and ten times cutoff).
“Of the 13 ELISA kits tested, none produced a sufficient response to qualify as a positive result using the prepared nitazene mixture, even at 10 times the manufacturer cutoff concentration for the target analyte.”
- Also stated in: § 4 | CON CLUS IONS · plain
“None of the tested kits produced a response at tested concentrations (kit cutoff, five times kit cutoff, and ten times kit cutoff).”
- Also stated in: § Abstract · plain
“Across tested concentrations for the seven selected analytes, none of the tested kits produced a signal sufficient for a positive result,”
Quote in the paperSubstantial claim - Also stated in: § 4 | CON CLUS IONS · plain
- Findingown data
No dose-response curves or cutoff values could be determined from the ELISA data.
“Therefore, no dose-response curves or cutoff values were able to be determined.”
Quote in the paperSubstantial claim - Interpretationown data
Based on the data, nitazenes would not be at risk of causing a false-positive response in any of the tested ELISA kits.
“Based on these data, nitazenes would not be at risk of causing a false-positive response in any of these kits.”
Quote in the paperSubstantial claim
- Premisecitation
The specific site of protein binding during antibody production influences how the immunogen will bind to the antibodies, affecting the overall cross-reactivity profile of the ELISA kit.
“The specific site of protein binding during antibody production influences how the immunogen will bind to the antibodies, affecting the overall cross-reactivity profile of the ELISA kit [22].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [22] 22. Smith ML. Immunoassay. In: Levine BS, Kerrigan S, editors. Principles of forensic toxicology. Cham, Switzerland: Springer; 2020. p. 177-96. source
- Premisecitation
The 2-benzylbenzamidazole (nitazene) class is structurally distinct from other opioids.
“Specifically, the 2-benzylbenzamidazole (nitazene) class is structurally distinct from other opioids and is characterized by a benzene ring linked to a benzimidazole core with varying substitutions at R 1 , R 2 , and R 3 (Figure 1).”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [7] 7. Krotulski AJ, Newman R, Gilbert M, Walton SE, Fogarty MF, Logan BK. N-desethyl isotonitazene -NPS discovery new drug mono- graph. Horsham, PA: Center for Forensic Science Research and Education; 2022.
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [8] 8. Krotulski AJ, Manini A, Calello D, Brent J, Wax P, Aldy K, et al. N- piperidinyl etonitazene -NPS discovery new drug monograph. Horsham, PA: Center for Forensic Science Research and Education; 2021.
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [9] 9. Krotulski AJ, Walton SE, Papsun DM, Fogarty MF, Logan BK. N- pyrrolidino etonitazene -NPS discovery new drug monograph. Horsham, PA: Center for Forensic Science Research and Education; 2021.
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Interpretationown data
Even at concentrations 10 times above each kit's cutoff, none of the seven nitazenes would interfere with screening of traditional substances using the tested ELISA kits.
“Even at concentrations 10 times above each kit's respective cutoff, none of the seven included nitazenes would interfere with screening of traditional substances using the tested ELISA kits.”
Quote in the paperSubstantial claim - Interpretationown data
No tested kit target was structurally similar enough to nitazene analogs to produce a response.
“no tested kit target was structurally similar enough to nitazene analogs to produce a response”
Quote in the paperSubstantial claim - Premisereasoning
Cross-reactivity can impact the reliability of screening results, potentially leading to non-detection of novel substances or false-positive identifications.
“Cross-reactivity is particularly important for forensic toxicology casework since it can impact the reliability of screening results, potentially leading to non-detection of novel substances or false-positive identifications in the presence of non-target analytes.”
Quote in the paperSubstantial claim - Interpretationown data
The current study supports the shift toward mass spectral-based screening methods, especially for nitazene analogs.
“The current study supports this shift, especially for nitazene analogs, given that no commercial kits are currently available.”
Quote in the paperSubstantial claim- Rests on an assumption: No nitazene-specific ELISA kits are commercially available.
- Interpretationown data
None of the selected ELISA kits could be used off-label for nitazene screening.
“demonstrating that none of the selected kits could be used off-label for nitazene screening.”
Quote in the paperSubstantial claim - Interpretationreasoning
The lack of cross-reactivity observed in the opioid kits tested is not unexpected due to the structural dissimilarities between the core opioid/fentanyl structures and nitazene analogs.
“The lack of cross-reactivity observed in the opioid kits tested is not unexpected due to the structural dissimilarities between the core opioid/fentanyl structures and nitazene analogs.”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 8
- May combine two claims. 4
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationAs of 2021, GC/MS remained the most employed mass spectrometry-based screening method in forensic toxicology laboratories, with LC-MS/MS following closely behind (35% and 31%, respectively).
“As of 2021, gas chromatography mass spectrometry (GC/MS) remained the most employed mass spectrometry-based screening method in forensic toxicology laboratories, with liquid chromatography tandem mass spectrometry (LC-MS/MS) following closely behind (35% and 31%, respectively) [20].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [20] 20. D'Orazio AL, Mohr ALA, Chan-Hosokawa A, Harper C, Huestis MA, Limoges JF, et al. Recommendations for toxicological investigation of drug-impaired driving and motor vehicle fatalities-2021 update. J Anal Toxicol. 2021;45(6):529-36. https:// doi. org/ 10. 1093/ jat/ bkab064 source
The Results (Toxicology survey) report GC–MS at 35% and LC–MS-MS at 31% for screening drugs in blood in DUID cases, making them the two most reported mass-spectrometry methods in that category. The claim omits the blood and DUID-case limits and presents those percentages as applying to forensic toxicology screening generally; the Methods identify the underlying survey as conducted in 2020. (LLM, conf=0.87)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenEach nitazene sample and corresponding positive QC was prepared in duplicate for all ELISA plates tested.
“Each nitazene sample and corresponding positive QC was prepared in duplicate for all ELISA plates tested.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductnone givenAny sample that demonstrated an absorbance greater than that of the positive QC was deemed negative.
“Any sample that demonstrated an absorbance greater than that of the positive QC was deemed negative.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Premisenone givenThe targeted or semi-targeted nature of mass spectral methods increases sensitivity, allowing for the low detection limits required for potent analytes such as nitazenes.
“Additionally, the targeted or semi-targeted nature of mass spectral methods increases sensitivity, allowing for the low detection limits required for potent analytes such as nitazenes.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
Study design and conductnone givenAbsorbance readings were taken at the manufacturer recommended wavelength (450 nm for all kits), and values for duplicate wells were averaged for all samples and QCs.
“Absorbance readings were taken at the manufacturer recommended wavelength (450 nm for all kits), and values for duplicate wells were averaged for all samples and QCs.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningCross-reactivity not observed from the zolpidem and LSD kits indicates that either those kits do not bind at their respective amine groups, or that nitazene analogs do not react with the antibodies for those kits at the diethylamine moiety.
“Cross-reactivity not observed from the zolpidem and LSD kits indicates that either those kits do not bind at their respective amine groups, or that nitazene analogs do not react with the antibodies for those kits at the diethylamine moiety.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationFor kits without manufacturer-recommended cutoffs, guidance from ANSI/ASB 119 and ANSI/ASB 120 for blood screening was used to determine the cutoff concentration to be tested.
“For kits without manufacturer-recommended cutoffs, guidance from ANSI/ASB 119 and ANSI/ASB 120 for blood screening was used to determine the cutoff concentration to be tested [24,25].”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [24] 24. Academy Standards Board. ANSI/ASB 119: Standard for the analyt- ical scope and sensitivity of forensic toxicological testing of blood in medicolegal death investigations. Available from: https:// www. aafs. org/ sites/ defau lt/ files/ media/ docum ents/ 119_ Std_ e1. pdf (2021). Accessed 13 Dec 2024.
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [25] 25. Academy Standards Board. ANSI/ASB Standard 120: Standard for the analytical scope and sensitivity of forensic toxicological testing of blood in impaired driving investigations. Available from: https:// www. aafs. org/ sites/ defau lt/ files/ media/ docum ents/ 120_ Std_ e1. pdf (2021). Accessed 13 Dec 2024.
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Classified as probably not a substantial claim. May combine two claims.
PremisereasoningScreening via mass spectrometry offers more specificity, and methods can be updated as soon as reference standards are obtained, rather than waiting until analyte-specific immunoassay kits are available.
“Screening via mass spectrometry offers more specificity, and methods can be updated as soon as reference standards are obtained, rather than waiting until analytespecific immunoassay kits are available.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).
PremisereasoningMass spectrometry-based screening is especially helpful for novel drug screening, where a new drug can start and end its life cycle on the market before immunoassay kits are released.
“Mass spectrometry-based screening is especially helpful for novel drug screening, where a new drug can start and end its life cycle on the market before immunoassay kits are released.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
No nitazene-specific ELISA kits are commercially available.
“no nitazene-specific ELISA kits are commercially available”
“Currently, there are no commercially available ELISA kits targeting nitazene analogs.”
The paper relies on this without support.
Developing ELISA kits for novel analytes, such as nitazene analogs, is a time-intensive process.
“Developing ELISA kits for novel analytes, such as nitazene analogs, is a time-intensive process, which can delay their availability for emerging substances.”
The paper relies on this without support.
Mass spectral-based screening is not a viable possibility for every laboratory.
“However, considering that mass spectral-based screening is not a viable possibility for every laboratory, nitazene-specific ELISA kits are also needed to be able to detect nitazene-positive samples.”
The paper relies on this without support.
Cross-reactivity studies for nitazenes remain limited.
“Considering these limitations, ELISA screening favorability for novel substances has decreased and cross-reactivity studies remain limited, particularly for nitazenes.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Immunoassays routinely used for conventional opioids are largely ineffective for most nitazenes due to their distinct chemical structures and insufficient cross-reactivity.
“Immunoassays, routinely used for conventional opioids, are largely ineffective for most nitazenes due to their distinct chemical structures and insufficient cross-reactivity [47,48].”
From Nitazenes: The Emergence of a Potent Synthetic Opioid Threat (2025) · cites this paper as [48]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Methadone kit was not tested at the higher concentrations claimed
MinorThe conclusion says every kit was tested at five and ten times its cutoff. The methods state that the Neogen® methadone kit was tested only at its cutoff, so the higher-concentration conclusion does not apply to that kit.
“None of the tested kits produced a response at tested concentrations (kit cutoff, five times kit cutoff, and ten times kit cutoff).”
“The only exception was for the Neogen® methadone kit, where cross-reactivity testing with nitazenes was limited to the kit cutoff concentration due to insufficient in-house standards to achieve five and ten times the cutoff concentration levels.”
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- Authors
- Pacana, A. L., & Skillman, B. N.
- Affiliations
- Sam Houston State University (US)
- Corresponding author
- Skillman, Britni N.
- ORCID
- Skillman, Britni N.
- Venue
- Journal of Forensic Sciences
- Publisher
- Wiley
- Published
- 2025-04-15
- Volume
- 70
- Issue
- 4
- Pages
- 1609-1614
- ISSN
- 0022-1198 (ISSN-L)
- 1556-4029
- OpenAlex
- W4409458236
- Cited by
- 9
- References
- 15
- Citations by year
- 2026: 5 · 2025: 4
- FWCI
- 4.57
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Journal of Forensic Sciences (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Forensic Sciences Foundation (Lucas Grant (2023‐2024))
- Topics
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Forensic Toxicology and Drug Analysis
- Social Sciences › Psychology › Clinical Psychology › Psychedelics and Drug Studies
- Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Pharmacological Effects and Assays
- Keywords
- new psychoactive substances, cross-reactivity, forensic toxicology, false positives, whole blood, high-resolution mass spectrometry, etonitazene, isotonitazene
- MeSH terms
- Cross Reactions, Enzyme-Linked Immunosorbent Assay, Humans, Psychotropic Drugs, Mass Spectrometry, Substance Abuse Detection, Forensic Toxicology
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