The opioid crisis in the United States remains a serious public health problem as the number of re-ported opioid overdose deaths continues to increase (1). Many chemical fentanyl derivatives have been introduced into the drug-using community and distributed through the street heroin supply—a concern due to their high potency (2). Although these analogs are structurally similar to fentanyl, they often go undetected since they are not part of routine testing (3). We have found that developing analytical methods for analyzing fentanyl analogs and derivatives is a difficult but necessary task due to their growing prevalence.

During our method development, we did not experience many pre-analytical concerns. Overall, we encountered few problems with stability, extraction procedures, and in vitro breakdown. However, we observed many inactive metabolites and impurities for many of the targeted analytes.

We also wanted to differentiate stereoisomers (same chemical formula but different three-dimensional configuration) since their analgesic potencies can differ. For example, the trans isomer of 3-methylfentanyl is approximately 400 times as potent as morphine while the cis isomer is approximately 6,000 times as potent (4).

Meeting Analytical Challenges

Fentanyl analogs present many analytical challenges. The structural similarities and isomeric nature of these compounds create the first limitation (5). Separation in a lab’s gas chromatography (GC) or liquid chromatography (LC) method is the only way to achieve specificity of isomers since they can-not be distinguished in a mass spectrometer (MS) (6).

However, chromatographic separation can be difficult when adding these compounds to established broad screening assays. Fentanyl analogs typically elute within the same portion of the gradient or temperature program, leading to chromato-graphic overlap. Even for high-resolution MS, accurately identifying isobaric compounds with identical masses remains problematic.

For GC-MS, automated mass spectral deconvolution and identification system (AMDIS) software is paramount. Developed by the National Institute of Standards and Technology, AMDIS extracts clean mass spectra from complex matrix that laboratories can use to search a mass spectral library for compound identification. When collecting spectra for the library, we try to encompass as many minor fragments as possible, as AMDIS will incorporate subtle retention time differences and associated fragments to help differentiate isomers and structurally similar compounds.

Using LC-tandem MS (MS/MS) with multiple reaction monitoring (MRM) can be complicated for analyzing fentanyl analogs due to the resolution limits of a triple quadrupole instrument. Using chromatographic conditions that achieve baseline separation of true isobars is important as their precursor and product ions are often identical. Chromatographic separation of structural analogs with slight differences in molecular mass is also important, since the [M+H]+ of one analyte may show interference due to contribution from other compounds and their isotopes.

In LC-quadrupole time-of-flight (QTOF) MS screening, the parent ion is always present as the base peak. This can be advantageous because it may be the only discerning ion, as most fentanyl-related compounds have 188.14380 and 105.06988 as fragment ions. However, this can become a disadvantage when differentiating isomers. In these instances, we recommend relying on retention time-based standard material rather than a library match for identification.

As an added dimension of specificity, we sometimes also re-inject on a different gradient using the same column and mobile phases as part of a broader screen. Although the fragment ions are usually the same mass-to-charge ratio (m/z), it is common for the qualifier ratios to differ significantly. Be aware, some inactive fentanyl metabolites and fentanyl impurities also are isobars of novel fentanyl analogs.

Another major challenge in keeping up with fentanyl derivatives is that when they first appear in the illicit drug community, not many isotopically labeled internal standards are available. Under re-verse phase chromatographic conditions, labs have observed matrix effects for some of the more nonpolar compounds. When the novel synthetic opioid compound library was small, laboratories could overcome these matrix effects with hydrophilic interaction chromatography (HILIC). The prob-lematic matrix components eluted prior to the analytes of interest, allowing a laboratory to use analog internal standards to achieve adequate quantitation.

However, as this class of fentanyl analogs increased, chromatographic separation of isomer pairs and other structurally similar compounds became a major challenge. Since HILIC columns tend to require longer equilibration times than reverse-phase columns (7), laboratories could not implement the necessary gradient for chromatographic separation. As a result, laboratories used reverse phase chromatography as more isotopically labeled internal standards became available.

Putting Result in Context

Once result validation is complete, laboratories should review historical fentanyl-related data with the updated mass spectral libraries to identify potential false positives. In our laboratory, we reprocess previous LC-QTOF/MS data with a library containing the recently validated compounds.

We decided to use LC-QTOF/MS because the data collected for each sample is full scan mass spectra. We investigate compounds with unexpectedly high positivity rates as potential fentanyl metabolites or impurities. The investigation includes cross-referencing against isobars that have been previously identified and looking further back in time if needed. We look for an emerging trend over time as op-posed to a steady state of positive findings.

Preparing calibration and control material is also difficult. Because fentanyl derivatives are very potent, reporting limits are low (8). When preparing working stock standards in organic solvents at low concentrations, we routinely experienced loss of analyte, most likely due to the adsorption to glass and/or plastic. To prevent this loss, we prepared a serial dilution curve in matrix at ten times the target standard concentrations, then diluted these intermediate standard tenfold in the preferred calibration matrix to achieve the required low concentration calibrators.

Remaining Relevant

Although pre-analytical issues have been associated with the detection of fentanyl analogs, most of the challenges that we observed fell into the analytical or post-analytical phases of testing. Due to the nature of these compounds, labs should carefully consider the limitations of the instrumentation and techniques when deciding on a platform for method development. The benefits of retrospective data analysis and the ability to update method scope rapidly are also of tremendous value.

The evolving opioid drug market is a significant challenge facing laboratories in ensuring that their testing procedures keep pace. Dedicated resources, technical expertise, and close coordination between research and development teams and operational laboratory staff are essential to remain relevant and provide responsible and effective testing that truly helps efforts to curb the opioid epidemic.

Stephanie Diaz, MS, is a scientist, analytical R&D at Johnson & Johnson in Spring House, Pennsylvania.
Brian Holsey, BS, is a laboratory supervisor at NMS Labs in Willow Grove, Pennsylvania. Email:


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CLN's Focus on Mass Spectrometry is supported by Waters Corporation.

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