B-279 False positive results for the CEDIA™ Mitragynine Immunoassay caused by methadone and EDDP in clinical specimens

Background Mitragynine is an indole-based opioid-receptor agonist and the most abundant active alkaloid in the plant Mitragyna speciosa, commonly known as kratom. It is consumed for its pain-relieving and euphoric effects, but also known to be an abused and dangerous drug, which causes psychotic addiction and other adverse effects such as respiratory depression, liver toxicity, and death. In recent years, mitragynine has been associated with multiple deaths through illicit use and suspected adulteration with other drugs. Consequently, health care providers are interested in having mitragynine included in comprehensive drug testing panels for patients in treatment programs for substance use disorder and pain management. To evaluate the Thermo Scientific™ CEDIA™ Mitragynine (Kratom) homogeneous enzyme immunoassay (IA, Thermo Fisher Scientific Inc., Fremont, CA) as a presumptive screening tool and to compare results with a definitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for mitragynine and identify possible interferants causing false positive results. Methods Urine specimens submitted between September 2024 and November 2024 were qualitatively assayed by the CEDIA Mitragynine immunoassay on a Beckman Coulter AU 5800 series chemistry analyzer. The instrument was calibrated daily using 20 and 50 ng/mL calibrators with quality control material at 37.5 and 62.5 ng/mL. All presumptive positive screening results at 50 ng/mL cutoff were assayed for mitragynine by LC-MS/MS using CLIA validated method and specimens with discrepant results were identified. The IA was challenged with approximately 150 potentially interfering substances at a concentration of at least 10 µg/mL. Methadone and its metabolite EDDP were quantitatively assayed by LC-MS/MS at 100 ng/mL cutoff. Results The precision of the IA around the cutoff demonstrated a within-run coefficient of variation (CV) of 2.56% at 37.5 ng/mL, 1.71% at 50.0 ng/mL, and 1.70% at 62.5 ng/mL. The following drugs produced positive results for mitragynine: methadone (100 µg/mL), EDDP (100 µg/mL); kratom constituents: speciociliatine (10 µg/mL), speciogynine (25 µg/mL), paynantheine (25 µg/mL); and mitragynine metabolites: 7-hydroxy-mitragynine (10 µg/mL), 16-carboxy-mitragynine (10 µg/mL). No carry over was observed at mitragynine concentrations 200× cutoff. In total, 395 patient urine specimens tested positive by IA. Using LC-MS/MS with a lower reporting limit of 2 ng/mL for mitragynine, 346 (87.6%) specimens were confirmed positive and 49 (12.4%) were negative. Of the 49 discrepant specimens that were false positive by IA, 23 has been ordered for methadone and EDDP: 19 specimens showed methadone and EDDP positive results (82.6%), 4 specimens were methadone and EDDP negative (17.4%). The other 26 has no methadone and EDDP data available. Conclusion High concentrations methadone and its metabolite EDDP may interfere and cause false positive results for mitragynine screening using the CEDIA Mitragynine immunoassay. For qualitative analysis, the CEDIA package insert reports EDDP as an interference causing a false positive mitragynine result at 60 µg/mL but, in contrast with our current findings, methadone at 100 µg/mL was reported to give a true negative mitragynine result. We confirmed the presence of methadone and its metabolite EDDP in clinical random urine specimens with discrepant IA and LC-MS/MS results for mitragynine.