B-286 Fentanyl immunoassays on the line: a comparative study among three different assays for fentanyl detection

Abstract Background Fentanyl is a synthetic opioid drug that is widely used as a potent analgesic and anesthetic. Due to the recent fentanyl epidemic and spike in overdose death, fentanyl screening in urine has become more critical. The most common screening methodology for fentanyl is immunoassay and choosing the right immunoassay is essential to avoid consequences from false negative or false positive results. The commonly used FDA approved fentanyl immunoassays available are Immunalysis, ARK II from Ark Diagnostics, and FEN2 by Lin-Zhi (Roche). In this study we aimed to compare their performance with Liquid Chromatography Mass Spectrophotometry (LC-MS/MS) confirmatory assay and evaluated their specificity and sensitivity rates. Furthermore, we investigated the presence of fentanyl analogs which could result in a positive result in immunoassays but could be missed in a confirmatory assay that only detects fentanyl or the primary metabolite norfentanyl. Methods Fifty remnant patient urine samples were analyzed for fentanyl across 3 different immunoassay systems: the Immunalysis (cutoff 2ng/mL), Lin-Zhi FEN2 (cutoff 5 ng/mL), and ARK II (cutoff 1?ng/mL) and all were performed on Beckman AU5800 platforms. Samples were either analyzed right away or frozen for further analysis. All the samples were tested on a LC-MS/MS confirmatory method with a limit of detection of 0.5 ng/mL for both fentanyl and the primary metabolite norfentanyl. In addition, all samples were tested for fentanyl analogs on a second LC-MS/MS method. Those analogs include: 3-Methylfentanyl, 4-Fluorobutyrylfentanyl, 4-Fluoroisobutyrylfentanyl, Acetylfentanyl, Acetylnorfentanyl, Acrylfentanyl, Alfentanil, Butyryl fentanyl, Carfentanil, Cyclopropyl fentanyl, Despropionyl fentanyl, Furanyl fentanyl, Isobutyryl fentanyl, Methoxyacetyl fentanyl, O/P-fluorofentanyl, Sufentanil, Tetrahydrofuranyl fentanyl, Valeryl fentanyl. Results Out of 50 patient urine samples, there were 27 positive and 23 negative samples reported by LC-MS/MS. The Immunalysis assay had 5 false positive and 5 false negatives, ARK II had 0 false positives and 2 false negatives, and the Lin-Zhi FEN2 had 0 false positives and 1 false negative. The 5 false positive samples in Immunalysis assay were consistently negative in other immunoassays and LC-MS/MS confirmatory assays. The false negative results in Immunalysis assay were due to lack of assay cross reactivity for norfentanyl. The sensitivity and specificity of all the immunoassays in this study were determined against LC-MS/MS confirmatory results. Out of all 50 samples that were tested for fentanyl analogs on LC-MS/MS only 3 samples across all assays tested positive for the analog Despropionyl Fentanyl as well as fentanyl and norfentanyl. No other fentanyl analogs were detected in our samples. Conclusion Our data set indicates that ARK II and Lin-Zhi assays have a comparable performance in detection of fentanyl or norfentanyl, while Lin-Zhi shows a slightly better sensitivity. Immunalysis assay, however, demonstrates significantly lower sensitivity and specificity. The false positives in Immunalysis assay occurred near the assay’s detection cutoff. This high rate of false positive could be due to intrinsic features of this assay and potential cross reaction with other drugs/medications in the sample. Finally, our data indicates that fentanyl analogs were not the reason behind any of the false positives.