Investigating the effect of substitution location on fentanyl analog identification for methyl-substituted fentanyl analogs using GC-EI-MS

Fentanyl and fentanyl analogs are key figures at the center of the ongoing opioid epidemic in the United States. Currently, gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) instrumentation is one of the main analytical tools used for the identification of fentanyl analogs in forensic laboratories. However, the resulting retention times and EI mass spectra for many fentanyl analogs are quite similar given that there are only subtle differences in their structures. This research investigates the effect of substitution location on fentanyl analog identification using GC-EI-MS and the National Institute of Standards and Technology (NIST) Simple Similarity Search (SSS) and Hybrid Similarity Search (HSS) algorithms through the analysis of a series of 11 methyl-substituted fentanyl analogs.

The 95% confidence interval retention times for each isomer were never more than ±0.009 min and a statistically significant (p < 0.001) difference was observed for all pairwise comparisons. Careful examination of the EI mass spectra revealed several general trends about the influence of substitution location on the observed EI mass spectra. The combination of unique fragmentation patterns, formed through shifts in m/z values due to the location of substitution, and the retention time enabled the identification of the 11 methyl-substituted fentanyl analogs analyzed in this study. The SSS was able to correctly identify all methyl-substituted fentanyl analogs within the top five hits for compounds that were present in the library. In comparison, the HSS was used to identify compounds absent from the mass spectral libraries and provided drastically different results depending on the location of substitution.