Beyond fentanyl test strips: investigating other urine drug test strips for drug checking applications
Open accessPublished version of a preprint
Abstract
Use of immunoassay test strips for the detection of fentanyl in drug samples has become commonplace in harm reduction, law enforcement, public health, customs, and forensic science settings for testing drug product. With the increase of xylazine in the drug supply in recent years, use of xylazine test strips has also increased. As use of test strips expands, a desire to implement them for other drugs may emerge. However, since these strips are designed for urine testing, it is important to understand their applicability to testing drug product. In this work, we investigate the utility of seven types of urine immunoassay test strips - amphetamine, benzodiazepine, cocaine, methamphetamine, nitazene, opiate, and xylazine - for drug checking applications. Reproducibility, sensitivity, cross-reactivity, and the effect of prolonged exposure to elevated temperatures were studied. Generally, the tests were found to be reproducible, able to detect trace (μg/mL) levels of the analyte of interest, and minimally affected by prolonged storage at elevated temperatures. Nearly all tests showed cross-reactivity with compounds other than the analyte of interest, highlighting the need to better understand these limitations prior to implementation in a drug checking scenario. The viability of expired cocaine, fentanyl, and methamphetamine test strips was also interrogated, and little to no change in sensitivity was found even though the tests were multiple years expired.
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De Vrieze et al. examined the efficacy of the test strips using seized samples in Ghent, composed of either metonitazene, protonitazene, isotonitazene, butonitazene, or N-pyrrolidino etonitazene with an estimated purity of > 90%.
“De Vrieze et al. [22] examined the efficacy of the test strips using seized samples in Ghent. These samples composed of either metonitazene, protonitazene, isotonitazene, butonitazene, or N-pyrrolidino etonitazene with an estimated purity of > 90%.”
From Evaluation of nitazene immunoassay test strips for rapid in-situ detection of nitazene and nitazene analogs in illicit drug samples (2025) · cites this paper as [22]
The Conclusions (page unavailable) explicitly state that this study did not investigate real-world samples or mixtures, contrary to the claim that it tested seized samples in Ghent. Materials & Methods describes prepared analyte solutions, and the paper provides no support for the claimed sample composition or >90% purity. (LLM, conf=0.90)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredIn this study, no cross-reactivity was observed between the test strips and diamorphine, papaverine, acetylcodeine, 6-MAM, noscapine, caffeine, or paracetamol at 100,000 ng/mL.
“This study, and previous studies such as Sisco et al. (2024) found no cross reactivity of the test strips with diamorphine, papaverine, acetylcodeine, 6-monoacetylmorphine (6-MAM), noscapine, caffeine or paracetamol at 100,000 ng/mL.”
From Evaluation of nitazene immunoassay test strips for rapid in-situ detection of nitazene and nitazene analogs in illicit drug samples (2025) · cites this paper as Sisco et al. (2024)
The Results and Discussion (Cross-Reactivity; page unavailable) report that opiate test strips gave positive results for 6-monoacetylmorphine, contrary to the claimed absence of cross-reactivity. Materials & Methods (Cross-Reactivity; page unavailable) also specifies testing at 10,000 and either 500,000 or 1,000,000 ng/mL, not 100,000 ng/mL. (LLM, conf=0.89)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredImmunoassays 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 [47]
The Results and Discussion (Cross-Reactivity; page unavailable) report that opiate test strips detected several conventional opiates, while nitazene test strips detected all three nitazenes tested. The paper discusses structural similarity as a general factor in cross-reactivity, but does not establish that conventional-opioid immunoassays are ineffective for most nitazenes because of their distinct structures; its nitazene panel was limited to three compounds. (LLM, conf=0.78)
Experimental — accuracy not yet measuredThe current study is the first to independently assess, in a laboratory context and using drug standards, the potential of the first commercially available BTNX Rapid ResponseTM NTS for drug checking purposes.
“The current study is the first to independently assess, in a laboratory context and using drug standards, the potential of the first commercially available BTNX Rapid Response™ NTS for drug checking purposes.”
From Nitazene test strips: a laboratory evaluation (2024) · cites this paper as [42]
The Materials & Methods section describes laboratory testing of BTNX strips in solutions containing known analytes, and the Results and Discussion section reports testing nitazene strips against specific nitazenes for drug-checking applications. Our copy does not establish that the strips were the first commercially available BTNX Rapid Response™ NTS, or that this was the first independent assessment of them. (LLM, conf=0.82)
Experimental — accuracy not yet measuredThe discrepancies between this study and De Vrieze et al. may be attributed to differences in sample preparation or potential variability between test strip batches.
“These discrepancies may be attributed to differences in sample preparation or potential variability between test strip batches.”
From Evaluation of nitazene immunoassay test strips for rapid in-situ detection of nitazene and nitazene analogs in illicit drug samples (2025) · cites this paper as [22]
The Results and Discussion section reports that organic solvent used in sample preparation affected some test-strip results, supporting sample preparation as a possible source of differences. Our copy does not connect that effect to discrepancies with De Vrieze et al. or discuss variability between test-strip batches (searched: De Vrieze, batch variability). (LLM, conf=0.76)
Experimental — accuracy not yet measured
The LOD and cross-reactivity results observed in this study were largely consistent with those reported by De Vrieze et al.
“Overall, the LOD and cross-reactivity results observed in this study were largely consistent with those reported by De Vrieze et al. [22]”
From Evaluation of nitazene immunoassay test strips for rapid in-situ detection of nitazene and nitazene analogs in illicit drug samples (2025) · cites this paper as [22]
The Results and Discussion sections on Limit of Detection and Cross-Reactivity report both kinds of test-strip results. But the cited paper is Sisco et al., not De Vrieze et al., and our copy does not compare its results with De Vrieze’s or establish the claimed consistency. No page numbers are available in our copy. (LLM, conf=0.81)
Experimental — accuracy not yet measuredUnderstanding the sensitivity (LOD) of test strips allows estimation of the amount of pure drug powder required to trigger a positive result, thereby allowing prediction of whether the test strips are suitable for bulk analysis and able to detect trace amounts.
“Understanding the sensitivity (LOD) of the test strips allows to estimate the amount of (pure) drug powder required to trigger a positive result, thereby allowing to predict whether the test strips are not only suitable for bulk analysis but are also able to detect trace amounts [42].”
From Nitazene test strips: a laboratory evaluation (2024) · cites this paper as [42]
These findings are generally in line with the limited information available from the manufacturer, the CFSRE, and Sisco et al.
“These findings are generally in line with the limited information available from the manufacturer [40, 41], the CFSRE [41], and Sisco et al. [42].”
From Nitazene test strips: a laboratory evaluation (2024) · cites this paper as [42]
The cited paper’s Results and Discussion report test-strip sensitivity and cross-reactivity, but the citing sentence does not identify what “these findings” are. Without that antecedent, our copy cannot establish whether the findings align with Sisco et al.; a search for manufacturer and CFSRE comparisons did not resolve the ambiguity. (LLM, conf=0.72)
Experimental — accuracy not yet measuredDe Vrieze et al. noted a difference between test strip batches with one lot number providing an LOD for isotonitazene of 2000 ng/mL and the other 3000 ng/mL.
“De Vrieze et al. [22] noted a difference between test strip batches with one lot number providing an LOD for isotonitazene of 2000 ng/mL and the other 3000 ng/mL.”
From Evaluation of nitazene immunoassay test strips for rapid in-situ detection of nitazene and nitazene analogs in illicit drug samples (2025) · cites this paper as [22]
The Results and Discussion, Limit of Detection section reports values for metonitazene, N-piperidinyl etonitazene, and protonitazene, but does not address isotonitazene or differences between test-strip lots. Searches for De Vrieze, isotonitazene, lot numbers, and the claimed 2000 and 3000 ng/mL values found no supporting passage in our copy. Page numbers were not available. (LLM, conf=0.74)
Experimental — accuracy not yet measuredAt the time of the study, only limited independent data on the performance of BTNX NTS was available.
“As only limited independent data is available on the performance of these strips, this lab-based study aimed at evaluating their potential for drug checking applications.”
From Nitazene test strips: a laboratory evaluation (2024) · cites this paper as [42]
The Introduction (page unavailable) says the study evaluates nitazene test strips for drug checking, and Materials & Methods identifies the strips as BTNX products. Neither passage says that independent performance data on BTNX nitazene test strips was limited at the time; searches for prior nitazene-strip research and limited independent data found no such statement in our copy. (LLM, conf=0.70)
Experimental — accuracy not yet measuredDe Vrieze et al. reported cross-reactivity for 5-methyl etazene at 3 μg/mL.
“De Vrieze et al. [22] reported cross-reactivity at 3 µg/mL.”
From Evaluation of nitazene immunoassay test strips for rapid in-situ detection of nitazene and nitazene analogs in illicit drug samples (2025) · cites this paper as [22]
The Cross-Reactivity and Limit of Detection sections discuss nitazene test strips, but do not report a result for 5-methyl etazene at 3 µg/mL. Searches for “De Vrieze,” “5-methyl etazene,” and the stated concentration found no passage addressing the attributed finding; page numbers were unavailable in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measuredThis study corroborates the finding that removal of the 5-nitro group, to form 'desnitazenes' (e.g. metodesnitazene), prevents the detection of these analogs by the test strips.
“this study corroborates their findings that removal of the 5-nitro group, to form ‘desnitazenes’ (e.g. metodesnitazene) prevents the detection of these analogs by the test strips.”
From Evaluation of nitazene immunoassay test strips for rapid in-situ detection of nitazene and nitazene analogs in illicit drug samples (2025) · cites this paper as [22]
The Cross-Reactivity section (page unavailable) reports positive nitazene-strip results for metonitazene, protonitazene, and N-piperidinyl etonitazene. Searches for metodesnitazene, desnitazenes, and the 5-nitro group found no passage addressing whether removal of that group prevents detection; metonitazene is not metodesnitazene. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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References 25 · Cited by 21
- Authors
- Sisco, E., Appley, M. G., Pyfrom, E. M., Banta-Green, C. J., Shover, C. L., Molina, C. A., Biamont, B., & Robinson, E. L.
- Affiliations
- National Institute of Standards and Technology (US)
- University of California, Los Angeles (US)
- University of Washington (US)
- Corresponding author
- Sisco, Edward
- ORCID
- Venue
- Forensic Chemistry
- Publisher
- Elsevier BV
- Published
- 2024-09
- Volume
- 40
- Pages
- 100594
- ISSN
- 2468-1709 (ISSN-L)
- PMC
- PMC11450658
- OpenAlex
- W4400109593
- Cited by
- 21
- References
- 25
- Citations by year
- 2026: 6 · 2025: 14 · 2024: 1
- FWCI
- 4.97
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Forensic Chemistry (publishedVersion)
- ChemRxiv (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- Topics
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Forensic Toxicology and Drug Analysis
- Physical Sciences › Engineering › Biomedical Engineering › Biosensors and Analytical Detection
- Health Sciences › Medicine › Epidemiology › HIV, Drug Use, Sexual Risk
- Keywords
- drug checking, fentanyl, xylazine, cocaine, methamphetamine, benzodiazepines, opiates, nitazenes, amphetamine, cross-reactivity, storage temperature effects
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