Academy of Diagnostics & Laboratory Medicine - Scientific Short

Should clinical laboratories confirm fentanyl analogs by LC/MS-MS in urine drug tests?

Catherine L. Omosule

This manuscript is currently under review at the Journal of Applied Laboratory Medicine: “Omosule CL, Roper SR, Farnsworth CW. Frequency of fentanyl analogs and metabolites detected by LC/MS-MS in clinical specimens.”

The opioid epidemic has left no urban or rural town untouched. Fentanyl overdoses constitute a substantial portion of the opioid-related deaths in the U.S. with over 100,000 casualties documented in 2021 (1). The majority of fentanyl-involved overdose fatalities involve illicitly synthesized fentanyl (2). While fentanyl derivatives like alfentanil, sufentanil, and remifentanil are medically used for sedation and pain management, acetylfentanyl, acrylfentanyl, carfentanil, and several other analogs are used in designer drugs due to potency that exceeds that of parent fentanyl. In fact, a recent analysis of urine drug screens (UDS) submitted for testing by health care professionals as part of routine care implied that 40.6% of fentanyl-positive UDS from non-prescribed fentanyl users contained fentanyl analogs (3), making their detection necessary for initiating treatment and guiding clinician-patient dialog regarding illicit use.

To detect fentanyl in clinical laboratories, random urine specimens are routinely tested with fentanyl immunoassays (IAs). Fentanyl IAs are cost-effective, accessible, and have a rapid turnaround time. However, they have considerable cross-reactivity of the available IAs with fentanyl analogs and other structurally similar compounds. Thus, many laboratories perform confirmatory testing on presumptively positive UDS using targeted mass spectrometry-based approaches; often considered the gold standard for fentanyl detection. There is a concern clinically that illicitly used fentanyl analogs may be positive by IA but not confirm by targeted mass spectrometry, yielding a falsely negative confirmatory test. Importantly, limited data currently exists assessing the frequency of fentanyl analog detection via mass spectrometry in urine specimens that are positive for fentanyl via IAs.

To assess the frequency of fentanyl analog detection clinically, we utilized a liquid chromatography tandem mass spectrometry (LC/MS-MS) method clinically validated to detect acrylfentanyl, acetylfentanyl, and furanylfentanyl analogs at positivity cutoffs of 1ng/mL each (4). We also tested for the primary fentanyl metabolite, Norfentanyl at a cutoff of 5 ng/ml and fentanyl at a cutoff of 0.3 ng/ml. We retrospectively analyzed data from 2020-to 2021 obtained from the Barnes Jewish Hospital Laboratory and identified 6191 presumptive positive UDS samples. Of these, 88.5% (n=5483) of the samples confirmed positive by LC/MS-MS for fentanyl, norfentanyl, or an analog. Of the specimens that were positive, parent fentanyl and/or norfentanyl, were present in 95.7% (n=5247) of the samples. Most positive samples were concurrently positive for fentanyl and norfentanyl (67.4%, n= 3698). Further, 5.2% (n=286) of the samples contained acrylfentanyl or acetylfentanyl analogs. Fentanyl and norfentanyl were concurrently detected in 99.7% of these samples (n= 285). Furanylfentanyl was undetected in all specimens.

This study has important implications for clinical laboratories that utilize LC/MS-MS confirmatory testing for urine drug tests. First, fentanyl and norfentanyl are almost always concurrently detected when the fentanyl analogs acetylfentanyl and acrylfentanyl are present. Thus, LC/MS-MS confirmation for acetylfentanyl and acrylfentanyl  analogs may be unnecessary for most laboratories since confirming fentanyl and/or norfentanyl alone is sufficient. Secondly, acetylfentanyl, acrylfentanyl, and furanylfentanyl are unlikely contributors to presumptive positive IAs that do fail to confirm by LC/MS-MS. Likely culprits include fentanyl analogs that were not detected in this study, or cross-reactivity of the ARK II fentanyl assay with interfering substances. 


  1. Ahmad F, Cisewski J, Rossen L, Sutton P. Provisional drug overdose death counts, (n.d.).
  2. Centers for Disease Control and Prevention, National Center for Injury Prevention and Control, 2021. Fentanyl. Accessed 03/24/2022, (n.d.).
  3. Stanton JD, Whitley P, LaRue L, Bundy WL, Dawson E, Huskey A. Fentanyl analog positivity among near-real-time urine drug test results in patients seeking health care. Drug Alcohol Depend 2020; 217: 108264.
  4. Budelier MM, Franks CE, Logsdon N, Jannetto PJ, Scott MG, Roper SM, Farnsworth CW. Comparison of two commercially available fentanyl screening immunoassays for clinical use. J Appl Lab Med 2020; 5:1277-1286.

Academy of Diagnostics & Laboratory Medicine Designation

Fellows of the Academy use the designation of FADLM. This designation is equivalent to FACB and FAACC, the previous designations used by fellows of the National Academy of Clinical Biochemistry and AACC Academy. Those groups were rebranded as Academy of Diagnostics & Laboratory Medicine in 2023.