B-323 Evaluating the Detection of 215 Fentanyl Analogs and Synthetic Opioids in Urine Using Four Commercial Immunoassays

Abstract

Background America continues to be plagued by an opioid epidemic, and the third wave, predominated by fentanyl analogs has yet to abate. The detectability of fentanyl analogs and synthetic opioids is of critical concern to clinical and forensic laboratories. The US Centers for Disease Control and Prevention has coordinated production of a Traceable Opioid Material® Kits (TOM Kits®) to support laboratory detection of current and potentially emerging opioids as well as common co-drugs found in fentanyl-containing samples. Previously published studies by this group have focused on analyzing compound structures in relation to assay detectability to predict the likely target epitope. The final data analysis of these over 1,800 measurements will also include this prediction. Methods From the TOM Kits®, 215 opioids were evaluated. These were analyzed using four commercially available fentanyl immunoassays: ARK™ Fentanyl II, Immunalysis Fentanyl Urine HEIA®, Immunalysis Fentanyl Urine SEFRIA® Drug Screening Kit, and the Lin-Zhi Fentanyl Enzyme Immunoassay. The detectability of the opioids was initially evaluated by preparing each opioid in certified drug-free human urine at 1 ng/mL, then analyzing the sample using the four immunoassays to determine if the analog screened positive in singlicate. Testing of that opioid was discontinued if the initial results was positive. If the result was negative, the resultant value was evaluated to determine whether a concentration of 10 ng/mL or 100 ng/mL would be appropriate for the second analysis. Opioids that did not screen positive at a concentration of 100 ng/mL were considered undetected by that immunoassay. Results The difference in reactivity of the immunoassay’s reagents was evaluated in conjunction with the chemical structure of each opioid. All four immunoassays detected 106 of the opioids at the concentrations tested. Eighty-two opioids had variable cross-reactivity with the four immunoassays determined by the unique epitope for each reagent antibody. 28 opioids were not detected by any assay and 17 of these were the emerging synthetic opioids. At the lowest concentration tested (1 ng/mL), the ARK II assay detected 36 compounds, the SEFRIA detected 74, LZI detected 5, and the HEIA detected 18. Higher concentration testing was conducted on the other compounds. The undetected opioids included the surgical anesthetic parent compounds remifentanil and alfentanil, and sufentanil metabolite norsufentanil. Additionally, three carfentanil analogs were not detected by the four immunoassays. Of the 34 compounds only detected by one assay, 27 of those positive results were from the Lin-Zhi assay. Conclusion This information will be of use in both clinical and forensic settings in evaluation of the potential false-positive or false negatives in urine fentanyl screening. The detectability of fentanyl analogs and synthetic opioids varies by assay and it may be possible to predict the detection or lack thereof for a particular assay based on a pattern elucidated by the analysis of the TOMs Opioid compound set. This data may be used to evaluate the potential false negative or false positive results of commercially available fentanyl and norfentanyl homogeneous immunoassays and to predict the likely target epitope of each assay.