Evaluating Updated Fentanyl Immunoassays for Loperamide Interference

FOCUSED REPORT Evaluating Updated Fentanyl Immunoassays for Loperamide Interference Michael E. Walsh , a,b Grace R. Williams, c Paul J. Jannetto, d and K. Aaron Geno a,b, * Background: Loperamide is a µ-opioid receptor agonist that reduces intestinal peristalsis and is used to treat diarrhea. We previously described significant cross-reactivity of loperamide with 2 fentanyl immunoassays. Since then, new fentanyl immunoassays, including a CLIA-waived point-of-care device, have been approved for clinical use. Methods: We evaluated new fentanyl immunoassays for cross-reactivity to loperamide and its major metabolites, N-desmethyl loperamide (dLop) and N-didesmethyl loperamide (ddLop). Previously characterized assays were tested for cross-reactivity to ddLop, which recently became commercially available. Loperamide, dLop, and ddLop were spiked in drug-free urine for analysis by 5 enzyme immunoassays run on automated chemistry analyzers and one lateral flow assay for the detection of fentanyl. Results: Loperamide and its metabolites produced positive results in 3 fentanyl immunoassays. The Immunalysis HEIA was previously determined to be reactive to both loperamide and dLop, but it was not reactive to ddLop. The Immunalysis SEFRIA was reactive to loperamide, dLop, and ddLop at minimum concentrations of 14.7 mg/L, 13.1 mg/L, and 17.0 mg/L. The Thermo Fisher DRI was previously determined to be reactive to loperamide and dLop, and it was reactive to ddLop at a minimum concentration of 33.1 mg/L. The Abbott iCassette, ARK Fentanyl II, and Lin-Zhi LZI II fentanyl assays showed no cross-reactivity to loperamide or its metabolites. Conclusions: The cross-reactivity of loperamide, dLop, and ddLop in several fentanyl immunoassays has the potential to cause false-positive results during urine drug screening. a Department of Pathology and Laboratory Medicine, Dartmouth Health, Lebanon, NH, United States; b Geisel School of Medicine at Dartmouth, Hanover, NH, United States; c Department of Pathology, Virginia Commonwealth University, Richmond, VA, United States; d Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, MN, United States. *Address Correspondence to this author at: Department of Pathology and Laboratory Medicine, Dartmouth Health, One Medical Center Drive, Lebanon, NH 03756, United States. E-mail [email protected]. Received April 24, 2025; accepted July 8, 2025. https://doi.org/10.1093/jalm/jfaf110 © Association for Diagnostics & Laboratory Medicine 2025. All rights reserved. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected]. INTRODUCTION Loperamide (Imodium ® ) is a µ-opioid receptor agonist used as an anti-diarrheal medication. Loperamide has low bioavailability taken orally, often cited at <1% (1), but high, sustained dosing and genetic variation (2) can lead to opioid-like effects on the central nervous system (CNS). Non-medical loperamide use has been reported to mitigate opiate withdrawal symptoms due to loperamide’s ability to reach µ-opioid receptors in the CNS when taken in high doses (3). Online discussions November 2025 | 10:06 | 1607–1613 | JALM 1607 Downloaded from https://academic.oup.com/jalm/article/10/6/1607/8238693 by Dartmouth Libraries user on 11 December 2025 indicate that loperamide doses of 70 to 100 mg, much higher than therapeutic doses (16 mg), are required to alleviate opiate withdrawal symptoms (4). Loperamide overdose can cause life-threatening cardiac arrhythmias (5). Loperamide-associated cardiotoxicity was more likely to lead to death than buprenorphine-associated cardiac events (37% vs 7% of cases) (6). Non-medical use of loperamide was implicated in 12 987 cases, including 59 deaths, reported in the National Poison Data System in the United States from 2010 to 2022 (7). In 2016 to 2018, the FDA issued a Drug Safety Communication, required warnings on the labels of loperamide products, and limited cartons to at most 48 mg of individually packaged doses (8, 9). Since then, the number of cases of loperamide-associated effects has declined both in the United States and worldwide (7, 10). Our previous report evaluated cross-reactivity of loperamide and its major metabolite, N-desmethylloperamide (dLop), in 3 commercial fentanyl immunoassays, 2 of which demonstrated significant cross-reactivity (11). Since then, new formulations and CLIA-waived lateral flow assays for fentanyl have been approved for clinical use. Given the ongoing relevance of loperamide in the setting of the opioid epidemic, as well as the recent commercial availability of N-didsemethylloperamide (ddLop), we sought to update the knowledge surrounding potential false-positive fentanyl assays due to loperamide misuse. MATERIALS AND METHODS Materials and Specimen Handling Loperamide hydrochloride powder (Sigma Aldrich) was weighed on an analytical balance and dissolved into dimethyl sulfoxide (DMSO) to a nominal stock concentration of 1000 mg/L. dLop and ddLop (Cayman Chemical Company) were purchased in 1 mg quantities, and the entirety of the container was dissolved into 1 mL DMSO to a nominal stock concentration 1000 mg/L. Stocks were diluted 10-fold into commercial drugfree urine (UTAK Laboratories) to a nominal concentration of 100 mg/L. Seven samples were made from this solution via 2-fold dilution through drug-free urine to a minimum nominal concentration of 1.6 mg/L. A series containing equivalent amounts of DMSO only was prepared. Samples were tested at our institution prior to distribution to other sites by overnight refrigerated shipment. Assays not reactive at the highest concentration of each compound were not tested further. Samples were tested in at least duplicate apart from the nominal 50 mg/L sample for each drug, which was tested in singlicate for some assays due to an oversight. Assignment of Values by Mass Spectrometry Loperamide and its metabolites were quantified by liquid chromatography with tandem mass IMPACT STATEMENT Immunoassays remain the screening workhorses for drugs of abuse yet are subject to known and unknown interferences. Loperamide misuse as a “poor man’s methadone” increased during the opioid epidemic. As loperamide misuse poses health risks, it is important for providers and laboratories to know whether loperamide cross-reactivity is a significant concern for commercially available fentanyl immunoassays. This study evaluates cross-reactivity of newer fentanyl immunoassays against loperamide and its metabolites, which were shown to cause false-positives in some previous formulations. FOCUSED REPORT 1608 JALM | 1607–1613 | 10:06 | November 2025 Downloaded from https://academic.oup.com/jalm/article/10/6/1607/8238693 by Dartmouth Libraries user on 11 December 2025 spectrometry (LC-MS/MS) as described previously (11), with modifications. Glucuronidase treatment was not performed. Mobile phases consisted of 2 mM ammonium acetate and 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). In lieu of extraction and reconstitution, samples were mixed with an internal standard and diluted into a 1:1 mixture of mobile phases A and B for direct analysis. Internal standards were methadone-D 3 (for loperamide and dLop) and 2-ethylidene-1,5dimethyl-3,3-diphenylpyrrolidine-D 3 (EDDP-D 3 , for ddLop). Samples were separated by reversedphase liquid chromatography using a 100 mm C18 column (Waters Acquity UPLC BEH) in a gradient separation of 2% to 52.5% mobile phase B with multiple reaction monitoring via a Waters Acquity TQS Micro system. Mass transitions are provided in the online Supplemental Tables. A standard curve was generated for loperamide and its metabolites spanning 0 to 1250 ng/mL from the 1000 mg/L stock chemicals described above in independent preparations. Specimens from interference studies were diluted 10-fold or 100-fold in commercial drug-free urine to bring the nominal concentration within the calibrated range of the assay prior to analysis. Fentanyl Assays Commercially available fentanyl immunoassays evaluated include the Abbott iCassette Fentanyl Urine Test Cassette (Abbott Rapid Diagnostics), ARK Fentanyl II (ARK Diagnostics), HEIA Fentanyl Urine Enzyme Immunoassay (Immunalysis Corporation), SEFRIA Fentanyl Urine Enzyme Immunoassay (Immunalysis Corporation), LZI Fentanyl II Enzyme Immunoassay (Lin-Zhi International), and the DRI Fentanyl Enzyme Immunoassay (Thermo Fisher Scientific). See Table 1 for additional information. Data Analysis and Calculations Primary data were communicated to the corresponding author via email and transcribed into Microsoft Excel. Minimum concentrations required for assay positivity and percent crossreactivity were calculated as described previously (11). Units We have utilized ng/mL and mg/L to minimize placeholder zeros (11). To convert mg/L to ng/ mL, multiply by 1000. To convert ng/mL to mg/L, divide by 1000. Table 1. Assays used in this study. Assay (Regulatory status) a Analyzer Cutoff (calibrator) Abbott iCassette Fentanyl Urine Test Cassette (FDA) NA 1 ng/mL (fentanyl) ARK Diagnostics ARK Fentanyl II b (FDA) Abbott Architect c4000 1 ng/mL (fentanyl) Immunalysis HEIA Fentanyl Urine Enzyme Immunoassay c (FUO) Roche cobas c501 2 ng/mL (fentanyl) Immunalysis SEFRIA Fentanyl Urine Enzyme Immunoassay (FDA) Abbott Architect c4000 1 ng/mL (fentanyl) Lin-Zhi International LZI Fentanyl II Enzyme Immunoassay d (FDA) Abbott Architect c4000 5 ng/mL (norfentanyl) Thermo Fisher Scientific DRI Fentanyl Enzyme Immunoassay c (FUO) Roche cobas c502 2 ng/mL (fentanyl) Abbreviation: NA, not applicable. a Regulatory status indicated as follows: FDA, FDA-cleared; FUO, forensic use only. b Also sold as the Thermo Fisher Fentanyl II (12). c These assays were previously characterized against loperamide and dLop (11) and were characterized for reactivity to ddLop in this study. d Also sold as the Roche Fentanyl II Enzyme Immunoassay (13). Loperamide Interference in Updated Fentanyl Assays FOCUSED REPORT November 2025 | 10:06 | 1607–1613 | JALM 1609 Downloaded from https://academic.oup.com/jalm/article/10/6/1607/8238693 by Dartmouth Libraries user on 11 December 2025 RESULTS Cross-reactivity of Loperamide Six fentanyl immunoassays (5 automated assays and one CLIA-waived lateral flow immunoassay) were tested for reactivity with commercial drug-free urine spiked with loperamide, dLop, or ddLop at concentrations ranging from 1.80 to 107.51 mg/L. Minimum reactive concentrations and crossreactivity are presented in Table 2. Each assay was non-reactive against a 10% DMSO solvent blank (full data are available in the Supplemental Tables). Like the Thermo DRI and Immunalysis HEIA evaluated previously (11), the Immunalysis SEFRIA fentanyl assay produced positive results with loperamide. The minimum reactive concentration was 13.7 mg/L with a cross-reactivity of 0.0146%. The Abbott iCassette, ARK Fentanyl II, and Lin-Zhi LZI II fentanyl assays did not produce a positive result at any loperamide concentration tested. Cross-reactivity of N-desmethylloperamide As previously reported (11), dLop displays crossreactivity to the Immunalysis HEIA and Thermo DRI assays. In this study, the Immunalysis SEFRIA fentanyl assay also produced positive results with dLop with a minimum reactive concentration of 13.1 mg/L (0.0180% cross-reactivity). The Abbott iCassette, ARK Fentanyl II, and Lin-Zhi LZI II fentanyl assays did not react with dLop at the maximum concentration (106.7 mg/L). Cross-reactivity of N-didesmethylloperamide Immunalysis SEFRIA and Thermo DRI were reactive to ddLop at minimum concentrations of 16.9 mg/L and 33.1 mg/L. Concentrations of ddLop up to 107.3 mg/L did not produce positive results on any other assay evaluated. While the Immunalysis HEIA did not achieve positivity, it showed a dose-dependent signal on the Roche cobas c501 (see Supplemental Tables). Didesmethyl loperamide was tested for crossreactivity to the other automated urine drug tests in our laboratory described previously (11) and did not exhibit cross-reactivity, including against a buprenorphine assay that was cross-reactive to dLop (Supplemental Table). DISCUSSION As annual deaths from the overdose of synthetic opioids, like fentanyl, remain high in the United Table 2. Cross-reactivities of loperamide and its metabolites to fentanyl immunoassays. Loperamide dLop ddLop Assay Threshold, mg/L a Cross-reactivity, % Threshold, mg/L Cross-reactivity, % Threshold, mg/L Cross-reactivity, % Abbott iCassette NR NR NR NR NR NR ARK Fentanyl II NR NR NR NR NR NR Immunalysis HEIA 23.7 b 0.0084 b 35.7 b 0.0056 b NR NR Immunalysis SEFRIA 13.3 0.0151 11.1 0.180 15.8 0.0127 Lin-Zhi LZI II NR NR NR NR NR NR Thermo DRI 5.72 b 0.035 b 6.90 b 0.029 b 32.0 0.006 Abbreviations: dLop, N-desmethyl loperamide; ddLop, N-didesmethyl loperamide; NR, non-reactive. a Threshold concentration is the minimum concentration required to produce a positive result. b Data from reference 11 were reproduced for comparative purposes. FOCUSED REPORT 1610 JALM | 1607–1613 | 10:06 | November 2025 Downloaded from https://academic.oup.com/jalm/article/10/6/1607/8238693 by Dartmouth Libraries user on 11 December 2025 States (14), it is critical that the laboratory community evaluate the performance of the assays used to detect these compounds. Given regulatory mandates for the availability of fentanyl immunoassays in emergency settings, manufacturers and regulators should carefully consider the design of specificity studies prior to approval for clinical use. Recent additions to fentanyl testing, such as CLIA-waived point-of-care devices for the detection of fentanyl, should be further characterized for cross-reactivity to structurally similar and clinically relevant targets. While 2 of the tested assays are not FDA-cleared assays and would not be expected to be characterized to the same extent as those that are, assay manufacturers varied widely in their testing of fentanyl analogs for crossreactivity, from as few as 2 to over 30. None of the package inserts of the methods in this study indicate evaluation for cross-reactivity with loperamide, whose misuse began appearing online in the early 2010s and became of medical interest no later than a decade ago (3, 15, 16). dLop is the principal metabolite of loperamide in urine and was found in concentrations as high as 44 mg/L in a fatality attributed to loperamide misuse (17, 18). Loperamide was found at 9.2 mg/L in the same fatality, and we observed similar concentrations in a patient specimen (11, 18). It may be further metabolized to ddLop, concentrations of which have not been studied extensively in urine. In a study of one volunteer ingesting 16 mg of loperamide, ddLop was approximately 10% as abundant as dLop to at least 72 h (17). These concentrations are similar to those needed to cross-react with impacted assays in this study. Notably, newer fentanyl immunoassays for use on automated chemistry analyzers, like the Lin-Zhi LZI II, were designed to detect norfentanyl, which is generally more abundant in urine than fentanyl (19). This assay, as well as the ARK Fentanyl II, which also has some cross-reactivity to norfentanyl, did not have cross-reactivity to loperamide or its metabolites. ddLop did not reach positivity on the Immunalysis HEIA, but there was a dose-dependent response in signal, suggesting that positivity could be achieved with higher concentrations of this compound (evaluation of which was precluded by solubility limits). This underscores a limitation of our study. Each compound was evaluated individually; in a real-world scenario, cross-reactive compounds would likely have an additive effect, with contributions from each potentially leading to false-positive screen results. From a practical standpoint, while ddLop alone seems unlikely to evoke a false-positive result from the HEIA assay, the dLop and loperamide present in the same sample plausibly could. A number of studies have reported interfering substances with fentanyl immunoassays (11, 20– 22). While cross-reacting drugs may not always be obvious, given its misuse potential as a “poor man’s methadone” (3, 16), laboratories and clinicians should be aware of the potential for falsepositive results on fentanyl immunoassays caused by cross-reactivity of loperamide. On-label dosage of loperamide (16 mg/day or less) seems unlikely to produce positivity on cross-reactive assays, but this possibility underscores that screens should always be clearly reported in a way that makes it clear that the result is not definitive. While this cross-reactivity could be viewed as advantageous in certain populations, laboratories tempted to use a cross-reactive assay as a screen for loperamide misuse should consider both the relative insensitivity of such an approach, detecting what was presumed to be only more extreme misuse (11), as well as the costs of definitive testing of each positive sample that fails to confirm for fentanyl and how such an algorithm would be constructed. It seems more likely that the possibility of loperamide cross-reactivity should be held in mind by laboratories and clinicians utilizing such an assay. SUPPLEMENTAL MATERIAL Supplemental material is available at The Journal of Applied Laboratory Medicine online. Loperamide Interference in Updated Fentanyl Assays FOCUSED REPORT November 2025 | 10:06 | 1607–1613 | JALM 1611 Downloaded from https://academic.oup.com/jalm/article/10/6/1607/8238693 by Dartmouth Libraries user on 11 December 2025 Nonstandard Abbreviations: dLop, N-desmethylloperamide; ddLop, N-didesmethylloperamide; DMSO, dimethyl sulfoxide. Author Contributions: The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Michael Walsh (Conceptualization-Supporting, Data curation-Equal, Formal analysis-Equal, Investigation-Lead, Methodology-Equal, Project administration-Supporting, Writing—original draft-Equal, Writing—review & editing-Supporting), Grace R Williams (Data curation-Supporting, Investigation-Supporting, Methodology-Supporting, Resources-Equal, Writing—review & editing-Equal), Paul Jannetto (Data curation-Supporting, Investigation-Supporting, Resources-Equal, Writing—review & editing-Supporting), Kimball Geno (Conceptualization-Lead, Data curation-Equal, Formal analysis-Equal, Investigation-Supporting, Methodology-Equal, Project administration-Lead, Resources-Equal, Software-Equal, Supervision-Lead, Visualization-Lead, Writing—original draft-Equal, Writing—review), and & editing-Equal) Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Research Funding: A portion of this work was supported by National Institute on Drug Abuse grant P30 DA033934 (to G.R. Williams). Disclosures: P.J. Jannetto received financial support from Roche Diagnostics, Inc. and Thermo Fisher Scientific, Inc. in the past 36 months, with all support paid directly to Mayo Clinic Labs. P.J. Jannetto is the president-elect and a member of the Board of Directors for the Association for Diagnostics & Laboratory Medicine (ADLM). K.A. Geno received travel, lodging, and registration support from ADLM related to his service on the 2024 ADLM Annual Scientific Meeting Annual Meeting Organizing Committee. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript. REFERENCES 1. Sahi N, Nguyen R, Patel P, Santos C. Loperamide. In: Statpearls. Treasure Island (FL): StatPearls Publishing; 2025. https://www.ncbi.nlm.nih.gov/books/NBK557885/ (Accessed August 2025). 2. Guinchat V, Ansermot N, Ing Lorenzini K, Politis D, Daali Y, Eap CB, et al. Case report: opioid use disorder associated with low/moderate dose of loperamide in an intellectual disability patient with CYP3A and P-glycoprotein reduced activity. Front Psychiatry 2022;13:910684. 3. Stanciu CN, Gnanasegaram SA. Loperamide, the “poor man’s methadone”: brief review. J Psychoactive Drugs 2017;49:18–21. 4. Daniulaityte R, Carlson R, Falck R, Cameron D, Perera S, Chen L, et al. “I just wanted to tell you that loperamide WILL WORK”: a web-based study of extra-medical use of loperamide. Drug Alcohol Depend 2013;130:241–4. 5. Teigeler T, Stahura H, Alimohammad R, Kalahasty G, Koneru JN, Ellenbogen M, et al. Electrocardiographic changes in loperamide toxicity: case report and review of literature. J Cardiovasc Electrophysiol 2019;30: 2618–26. 6. Krantz MJ, Rudo TJ, Haigney MCP, Stockbridge N, Kleiman RB, Klein M, et al. Ventricular arrhythmias associated with over-the-counter and recreational opioids. J Am Coll Cardiol 2023;81:2258–68. 7. Patel A, Rine NI, Spiller HA, Hays H, Badeti J, Zhu M, et al. Loperamide cases reported to United States poison centers, 2010–2022. Inj Epidemiol 2023;10:61. 8. US Food and Drug Administration. FDA limits packaging for anti-diarrhea medicine loperamide (Imodium) to encourage safe use. FDA; 2019. https://www.fda.gov/ drugs/drug-safety-and-availability/fda-limits-packaginganti-diarrhea-medicine-loperamide-imodiumencourage-safe-use (Accessed April 2024). 9. US Food and Drug Administration. FDA Drug Safety Communication: FDA warns about serious heart problems with high doses of the antidiarrheal medicine loperamide (Imodium), including from abuse and misuse. FDA; 2021. https://www.fda.gov/drugs/drug-safety-andavailability/fda-drug-safety-communication-fda-warnsabout-serious-heart-problems-high-doses-antidiarrheal (Accessed April 2025). 10. Ollitrault P, Dolladille C, Chrétien B, Milliez P, Alexandre J. Cardiovascular toxicities associated with loperamide: analysis of the world health organization pharmacovigilance database. Circulation 2021;143: 403–5. 11. Geno KA, Badea A, Lynch KL, Jannetto P, Hubbard JA, Nerenz RD, et al. An opioid hiding in plain sight: loperamide-induced false-positive fentanyl and FOCUSED REPORT 1612 JALM | 1607–1613 | 10:06 | November 2025 Downloaded from https://academic.oup.com/jalm/article/10/6/1607/8238693 by Dartmouth Libraries user on 11 December 2025 buprenorphine immunoassay results. J Appl Lab Med 2022;7:1318–28. 12. Thermo Fisher Scientific. DRI Fentanyl II Assay [package insert]. ARK Diagnostics; 2021. 13. Roche Diagnostics. Fentanyl II Enzyme Immunoassay [package insert]. Lin-Zhi International, Inc; 2023. 14. Garnett MF, Miniño AM. Drug Overdose Deaths in the United States, 2003–2023. Report No. 522. Hyattsville (MD): National Center for Health Statistics; 2024. https://dx.doi. org/10.15620/cdc/170565. (Accessed August 2025). 15. Wu PE, Juurlink DN. Loperamide cardiac toxicity: pathophysiology, presentation, and management. Can J Cardiol 2022;38:1378–83. 16. Dierksen J, Gonsoulin M, Walterscheid JP. Poor man’s methadone: a case report of loperamide toxicity. Am J Forensic Med Pathol 2015;36:268–70. 17. Ganßmann B, Klingmann A, Burhenne J, Tayrouz Y, Aderjan R, Mikus G. Simultaneous determination of loperamide and its desmethylated metabolites in plasma and urine by high-performance liquid chromatography— atmospheric-pressure lonization mass spectrometry. Chromatographia 2001;53:656–60. 18. Baselt RC. Disposition of toxic drugs and chemicals in man. 12th Ed. Seal Beach (CA): Biomedical Publications; 2020. 19. Lam KHB, Menlyadiev M, Buggs V, Parnprome S, Pesce A, Suhandynata RT, et al. A comparative analysis of two commonly used FDA-approved immunoassays for fentanyl detection. J Appl Lab Med 2024;9:905–12. 20. Fitch BA, Lynch KL, Liao H-C. Unexpected fentanyl detection during a liver transplant evaluation. Clin Chem 2023;69:222–5. 21. Lockwood T-LE, Vervoordt A, Lieberman M. High concentrations of illicit stimulants and cutting agents cause false positives on fentanyl test strips. Harm Reduct J 2021;18:30. 22. Wang D, Sun Q, Schneider R, Cunningham SL. Understand the FDA-cleared fentanyl testing: a clinical evaluation of the SEFRIA fentanyl immunoassay. Drug Alcohol Depend 2024;259:111287. Loperamide Interference in Updated Fentanyl Assays FOCUSED REPORT November 2025 | 10:06 | 1607–1613 | JALM 1613 Downloaded from https://academic.oup.com/jalm/article/10/6/1607/8238693 by Dartmouth Libraries user on 11 December 2025