Performance Evaluation of Two FDA-Approved Fentanyl Immunoassays against LC-MS/MS

• Published in: American journal of clinical pathology 2022-11, Vol.158 (Supplement_1), p.S45-S46
• Main Authors: Militello, L, Zhang, V
• Format: Article
• Language: English
• Subjects: Fentanyl; Immunoassay; Liquid chromatography; Mass spectroscopy; Performance evaluation
• ISSN: 0002-9173; 1943-7722
• DOI: 10.1093/ajcp/aqac126.088

Abstract Introduction/Objective The increased number of deaths caused by Fentanyl in recent years created the need for accurate, automated assays for fentanyl detection. Many products have been brought to market but there is concern over the sensitivity and specificity of the available immunoassays. Here we evaluate the performance of our current assay, the ARK Fentanyl II Assay (ARK), against the newly FDA-approved Fentanyl Immunoassay for Roche Cobas (Roche), using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the gold standard. To our knowledge, this is the first study to evaluate the performance of the Roche fentanyl immunoassay. Methods/Case Report We collected discarded patient urine samples screened for fentanyl using ARK over 3 months to get a total of 100 positive and 100 negative results. All samples were stored at -20 °C and later tested again using ARK, Roche and in-house LC-MS/MS assays. Both immunoassays were performed on Roche Cobas c501 chemistry analyzer. In-house LC-MS/MS assay gave quantitative measurements for both fentanyl and norfentanyl with a limit of quantification of 1 ng/mL for both. Samples with results above 1 ng/mL of either analyte were considered positive for the presence of fentanyl. The upper limit of quantification of the LC-MS/MS assay was 2000 ng/mL. The results obtained from LC-MS/MS were considered as reference values to determine the sensitivity and specificity of the immunoassays, which were calculated by dividing the number of positive and negative samples correctly identified by immunoassay by total positive and negative samples based on LC-MS/MS results, respectively. ARK is designed to detect fentanyl with a cutoff of 1 ng/mL. Roche is designed to detect norfentanyl with a cutoff of 5 ng/mL. Results (if a Case Study enter NA) Analytical sensitivity and specificity were 91.9% and 99.3% for ARK and 96.8 and 98.6% for Roche. Fentanyl and norfentanyl levels by LC-MS/MS ranged from 2000 ng/mL with a mean of 57 and 232 and standard deviation of 235 and 578, respectively. False positives were seen in 1 sample for ARK and 2 samples for Roche. False negatives were seen in 5 samples for ARK and 2 samples for Roche. Roche accurately identified 6 positive samples that had norfentanyl concentrations less than the cutoff of 5 ng/mL, indicating a potential greater sensitivity than stated. Conclusion Both immunoassays performed reasonably well, showing great specificity. Roche displayed greater sensitivity