CLINICAL ISSUES :: TOXICOLOGY


Detecting new and emerging fentanyls with confidence using LC-MS


By Stephanie Samra, MS T


he opioid crisis is a public health emergency in the United States and an emerging problem in other coun-


tries. In 2019, nearly 50,000 people in the United States died from opioid-involved overdoses.1


To keep ahead of the manufac-


turing and trafficking of illicit substances, drug enforcement agencies rely on insights from forensic laboratories to track the rise of new forms of opioids in circulation. Yet emerging synthetic opioids, such as fentanyl and fentanyl analogs, are chal- lenging to detect with conventional drug detection assays. To meet the changing demands of forensic drug analysis, new mass spectrometry (MS)-based methods that allow more sensitive, accurate and efficient detection of opioid analogs are coming to the forefront.


The challenge of analyzing fentanyls Around 73% of opioid-involved deaths are due to synthetic opioids, such as fen- tanyl and fentanyl analogs.2


Carfentanil,


the most potent fentanyl analog detected in the U.S., is estimated to be 10,000 times more potent than morphine.3 According to the National Forensics Laboratory Information System, the number of reported fentanyl-related overdose cases increased from 945 in 2013 to 71,341 in 2017.4


More recent


numbers from the Centers for Disease Control and Prevention (CDC) show that overdose deaths involving synthetic opioids, such as fentanyl and fentanyl analogs, were nearly 12 times higher in 2019 than in 2013.5 Forensic and clinical toxicology labora-


tories must be able to confidently identify fentanyls in clinical samples from an increasing caseload of overdoses, and rapidly provide results to health profes- sionals and law enforcement agencies. Yet with more than 200 different fentanyl ana- logs in circulation and many more rapidly emerging, this presents a challenge both in terms of efficiency and accuracy. The increased potency of fentanyl ana-


logs relative to morphine means they are often present in the blood at much lower levels and can be below the sensitivity thresholds of conventional drug testing methods. Moreover, although forensic laboratories have established methods for known fentanyls and other opioids,


16 JANUARY 2022 MLO-ONLINE.COM


the rapidly expanding list of new illicit fentanyls means standard techniques and reference reagents are not fit- for-purpose. The chemical similarity between different fentanyl analogs also pushes the capabilities of current forensic analytical chemistry tools to the limit in terms of their sensitivity and specificity.


Current analytical methods for fentanyls Most


forensic laboratories perform


immunoassays to detect specific drugs. But these assays are limited by the speci- ficities of the antibodies used and the availability of antibodies that are vali- dated against different opioid analogs. As new drugs emerge, antibodies are not readily available to detect them, and developing and validating new antibod- ies for immunoassays can take up to 12 months. By this time, new opioid analogs may well be in circulation. Most conventional immunoassays


are relatively low throughput, involving multiple wash steps and only suitable for detecting a few analytes simultaneously. More advanced analytical methods, such as liquid chromatography (LC) coupled with high-resolution accurate mass (HRAM) MS (e.g., Orbitrap mass spectrometry), enable parallel analysis through their multichannel liquid chro- matography capability. These alterna- tive approaches are quickly becoming the new gold-standard method in many forensic laboratories. However, there is still a challenge.


LC-MS techniques require libraries of standards against which fentanyls can be correctly identified. But there are so many new analogs emerging with no existing standards that it is difficult for laboratory analysts to feel confident they understand the limits of detection for these new compounds, and that they have accurately and comprehensively detected every isomer in a sample.


More sensitive analytical solutions To help forensic laboratories confirm the presence of new fentanyl analogs, the Centers for Disease Control and Prevention (CDC) released a Fentanyl Analog Screening Kit (FAS kit) and four Emergent Panels (FAS V1-4) in 2019 in


response to the rapid emergence of further analogs. In total, these reference materi- als support the screening for 250 opioids, including over 210 fentanyl analogs. To demonstrate the potential of LC-MS


methods for identifying closely related synthetic opioids, reference panels were used to develop optimized workflows based on Orbitrap mass spectrometry for detecting new and emerging fentanyl analogs. By creating an MS spectral library using the 213 fentanyl analogs, it tested the potential of HRAM MS for its ability to detect closely related fentanyls from biological samples. It was found that HRAM MS can identify fentanyls in urine with high sensitivity. The limit of detection for a majority of the com- pounds tested was 0.5 ng/mL and ≥75% of the fentanyl standards had a limit of detection of 1.0 ng/mL or better in a synthetic biological matrix, surine.6 One key advantage of LC-MS over


immunoassays is that LC-MS is more specific and can identify individual compounds within a compound class. The addition of LC to high-resolution MS means fentanyl isomers with identical mass spectra can still be differentiated because they are well separated by LC. Similarly, because some fentanyl iso- mers co-elute chromatographically, but have different MS2 spectra and unique product ions from other isomers, these can accurately be distinguished from each other. Although there are some scenarios where analogs have similar retention times and spectra, and cannot be distinguished, these high-resolution MS protocols and the spectral library make it possible to narrow down the analytes to one of two isomers.


More efficient workflows A further advantage of using high- resolution MS for fentanyl analysis is the potential for more efficient analysis to help meet the demands of increas- ing caseloads in forensic laboratories. Although it might seem that optimizing samples for analysis in an LC-MS work- flow might require more time-consuming and laborious sample preparation steps than traditional immunoassays, sample preparation with HRAM MS is surpris- ingly straightforward. Diluted urine samples can be directly injected into


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