CLINICAL ISSUES :: TOXICOLOGY
to develop rules from the MS spectra of existing fentanyl standards that allow the identification of unknown fentanyl ana- logs in urine, helping laboratory scientists obtain information on all the constituents of a sample – including retrospectively. The advent of real-time, MS-based
Training Fast
confident results
Support Figure 1: LC-MS screening workflow utilizing an extensive compound database and library
an ultra-high-performance LC system coupled with HRAM MS to screen and semi-quantify fentanyl and its analogs in a single analytical run. When you com- pare this to immunoassay workflows, which require a series of incubation and wash steps, the use of HRAM MS can make testing much more efficient and by reducing manual tasks, frees up analysts to perform other tasks (see Figure 1). An alternative emerging workflow for
targeted fentanyl analysis is the use of paper spray tandem MS. In this workflow, the sample is deposited as a fluid (urine) on a paper sampling substrate contain- ing previously loaded labeled standards. This paper substrate is then dried and analyzed quantitatively with tandem MS. No other sample treatment is needed between the spotting of the sample on the paper cartridge and analysis. This method allows for rapid analysis without an LC step and offers a significantly simplified process for biological sample collection. This workflow is being tested in real-time at substance use disorder clinics and has already shown comparable quantitative results to LC-MS for identifying fentanyl analogs at low levels.7
Detecting the unknowns Importantly, high-resolution MS has potential for untargeted analysis, which gives you information about everything in the sample, not just known compounds of interest. In an experiment to identify novel unknown fentanyl opioids, an MS2 spectra library generated for the CDC FAS panel of 213 fentanyl analogs was used to establish a set of class- and struc- tural similarity-based rules to identify unknown fentanyls from urine samples.8 The workflow for this experiment
comprised three key steps. First, it used multiple search tools to find class-based common fragments
among the reference spectra. Eight com- mon fragments of fentanyl compounds were identified through this search. Second, it conducted a similarity search using spectral data found in reference libraries, and an internal in-house spec- tral library of 213 fentanyls generated using the FAS Kit v1-3 was searched for similarity patterns. In the third and final step, the workflow investigated molecu- lar networks to identify compounds that differed by transformations, such as methylation, hydration, dealkylation and so on. These rules were used to cor- rectly identify a set of ‘unknown’ fentanyl standards from the CDC Emergent Panels spiked into urine. This ability to perform untargeted
analysis is arguably one of the great- est advantages of high-resolution MS for fentanyl screening. Its potential for providing unbiased information on all constituents of the sample, and in turn identifying the presence of unknown fentanyl analogs, provides a rapid view on the emerging and changing illicit drug landscape. It can also allow the ret- rospective analysis of previous samples to see if newly identified fentanyls were present in past overdose cases.
Conclusion Forensic laboratories face an increasing workload trying to analyze the growing number of illicit opioid analogs in circu- lation. The higher potencies of fentanyls and the large number of structurally similar fentanyl isomers found in illicit drugs present a unique analytical chal- lenge that requires highly sensitive and accurate techniques that work quickly on biological samples. HRAM MS coupled with LC can
accurately distinguish between structur- ally similar isomers of fentanyls at low concentrations. Moreover, it is possible
approaches, installed at harm-checking facilities alongside high-resolution clinical laboratory tools, will be powerful weapons in our arsenal in the war against drugs, helping to turn the tide against the rise in opioid-related overdoses and deaths.
REFERENCES
1. CDC WONDER, Atlanta, GA: Centers for Dis- ease Control and Prevention. 2020. http://wonder.
cdc.gov/. Updated October 29, 2021. Accessed November 30, 2021.
2. Centers for Disease Control and Preven- tion. Synthetic Opioid Overdose Data. https://
www.cdc.gov/drugoverdose/deaths/synthetic/ index.html. Updated March 25, 2021. Accessed November 30, 2021.
3. O’Donnell JK, Halpin J, Mattson CL, et al. Deaths involving fentanyl, fentanyl analogs, and U-47700 – 10 States, July-December 2016. MMWR Morb Mortal Wkly Rep. 2017;66(43):1197-1202. doi:10.15585/
mmwr.mm6643e1.
4. Wilson N, Kariisa M, Seth P, et al. Drug and Opioid-Involved Overdose Deaths – United States, 2017-2018. MMWR Morb Mortal Wkly Rep. 2020;69(11):290-297. doi:10.15585/
mmwr.mm6911a4.
5. Mattson CL, Tanz LJ, Quinn K, Kariisa M, Patel P, Davis NL. Trends and Geographic Patterns in Drug and Synthetic Opioid Overdose Deaths – United States, 2013–2019. MMWR Morb Mortal Wkly Rep 2021; 70:202–207.
6. Samra S, Hassell K, and Van Natta, K. Screening and semi-quantitation of 212 fentanyl analog compounds by the Orbitrap Exploris 120 mass spectrometer. 2021 Technical Note 66050. Thermo Fisher Scientific.
https://assets.thermo-
fisher.com/TFS-Assets/CMD/Technical-Notes/ tn-66050-lc-ms-orbitrap-exploris-fentanyl- analog-compounds-tn66050-en.pdf. Accessed November 30, 2021.
7. Kennedy JH, Palaty J, Gill CG, et al. Rapid analysis of fentanyls and other novel psychoac- tive substances in substance use disorder patient urine using paper spray mass spectrometry. Rapid Commun Mass Spectrom. 2018;32(15):1280-1286. doi:10.1002/rcm.8164.
8. Samra S. Advanced search tools for unknown discovery of novel fentanyl opioids detected by HRAM Orbitrap mass spectrometry using CDC Fentanyl Analog Screening (FAS) Kit. Poster 058 presented at: Society Forensic Toxicologists Annual Meeting September 30,2021. Thermo Fisher Scientific. https://www.
thermofisher.com/uk/en/home/about-us/events/ industrial/society-of-forensic-toxicologists-soft. html. Accessed November 30, 2021.
Stephanie Samra, MS, is an Applications Manager for Clinical Research and Toxicology at Thermo Fisher Scientific.
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