Utility of detecting fentanyl analogs during LC-MS/MS confirmation for positive fentanyl urine drug screens
Authors: Omosule Catherine , Roper Stephen , Farnsworth Christopher 
Published: November 2022

Abstract

Introduction
Fentanyl immunoassays have considerable crossreactvitiy with fentanyl derivatives such as acetylfentanyl, acrylfentanyl, furanyfentanyl and the primary fentanyl metabolite norfentanyl. This poses substantial analytical challenges to clinical laboratories that confirm fentanyl by mass spectrometry and may lead to false negative confirmations in patients that have analogs or primarily metabolites in their urine. As a result, some laboratories have developed targeted liquid chromatography tandem mass spectrometry (LS-MS/MS) methods to detect analogs and/or metabolites. However, there is no data in the literature demonstrating the frequency of analog detection in urine drugs screens, and the potential false positive immunoassay rate as a result of analogs and metabolites that are undetected is not known. Of note, acetyl-, acryl- and furanyl-fentanyl analogs have been confirmed in our testing region, and they cross-react with the Ark Fentanyl I and II immunoassays.

Methods
Results from 6,192 urine drugs screens positive for fentanyl by immunoassay [ARK I fentanyl assay and ARK II fentanyl immunoassays between January 1, 2020 to December 31, 2021 performed on a Roche cobas c502 chemistry analyzer with confirmatory LC-MS/MS (Waters Xevo TLD) at the Barnes Jewish Hospital Laboratory (St. Louis, MO) were retrospectively extracted from the laboratory information system (Cerner). The confirmatory assay was clinically validated to detect fentanyl (0.3 ng/mL), norfentanyl (5 ng/mL), acetylfentanyl (1 ng/mL), acrylfentanyl (1 ng/mL), and furanylfentanyl (1 ng/mL). An internal standard mix was added to patient samples followed by vortexing, centrifugation, and separation of the supernatant over a 6min linear gradient using a 2.1 × 150 mm 1.8µm C18 column. Data were acquired in positive ion mode and resulted as positive if predefined criteria for relative retention time, product ion ratio, peak shape/resolution, and signal intensity were met.

Results
84.8% of the samples (n= 5247) confirmed positive for fentanyl, norfentanyl or a fentanyl analog, whereas 15.2% (n=944) did not confirm by LC-MS/MS. Of the 5247 positive results, fentanyl alone was present in 1,279 (24.4%) specimens while norfentanyl alone was present in 235 (4.48%) specimens. Both fentanyl and norfentanyl were present in 3968 (75.6%) specimens. 239 (4.55%) specimens confirmed positive for acetylfentanyl, all of which (100%) had fentanyl and/or norfentanyl concurrently detected. Among 61 (1.16%) specimens which were positive for acrylfentanyl, fentanyl and/or norfentanyl were concurrently detected in 60 specimens, and 1 had neither fentanyl nor norfentanyl detected. Furanylfentanyl was undetected in all specimens.

Conclusions
The fentanyl analogs acetylfentanyl, acrylfentanyl, and furnaylfentanyl are infrequently detected in our patient population. Furthermore, the assessed analogs are almost exclusively detected in the presence of fentanyl and/or norfentanyl by LC-MS/MS confirmatory assays. This implies that fentanyl analogs may not be a common source of false positive results by fentanyl immunoassays.