Identification of Unique Fragmentation Patterns of Fentanyl Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry

Abstract

The opioid crisis in the United States is being fueled by the rapid emergence of new fentanyl analogs and precursors that can elude traditional library-based screening methods, which require data from known reference compounds. Since reference compounds are unavailable for new fentanyl analogs, we examined if fentanyls (fentanyl + fentanyl analogs) could be identified in a reference-free manner using a combination of electrospray ionization (ESI), highresolution ion mobility (IM) spectrometry, high-resolution mass spectrometry (MS), and higher-energy collision-induced dissociation (MS/MS). We analyzed a mixture containing nine fentanyls and W-15 (a structurally similar molecule) and found that the protonated forms of all fentanyls exhibited two baseline-separated IM distributions that produced different MS/MS patterns. Upon fragmentation, both IM distributions of all fentanyls produced two high intensity fragments, resulting from amine site cleavages. The higher mobility distributions of all fentanyls also produced several low intensity fragments, but surprisingly, these same fragments exhibited much greater intensities in the lower mobility distributions. This observation demonstrates that many fragments of fentanyls predominantly originate from one of two different gas-phase structures (suggestive of protomers). Furthermore, increasing the water concentration in the ESI solution increased the intensity of the lower mobility distribution relative to the higher mobility distribution, which further supports that fentanyls exist as two gas-phase protomers. Our observations on the IM and MS/MS properties of fentanyls can be exploited to positively differentiate fentanyls from other compounds without requiring reference libraries and will hopefully assist first responders and law enforcement in combating new and emerging fentanyls.

Elucidating the Gas-Phase Behavior of Nitazene Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry

“…It is not entirely unexpected that the intensities of the two IM distributions of nitazenes depends on the presence of water in the ESI solvent, though the extent to which a nitro group affects the signal intensities of the two IMS distributions was surprising. Hollerbach et al recently demonstrated that fentanyls (fentanyl + fentanyl analogs) also exhibit two gas-phase protomers whose two IMS distributions depend on the water concentration in the ESI solvent . However, previously observations showed that water caused the second IMS distributions of fentanyls to exhibit higher signal intensities compared to the first.…”

“…Since all fourteen nitazenes exhibited at least two IMS distributions, an explorative study was performed to determine the cause of these two peaks, such as them being rotational conformers (rotomers), protonation site specific conformers (protomers), or another type of gas-phase conformer. In a previous study involving fentanyl and fentanyl analogs, Hollerbach et al examined how the concentration of water in the ESI solvent affected the IMS distributions and noticed large differences in the intensity ratios of the two IM distributions that all fentanyls exhibited . The same logic was used in this study and the fourteen nitazenes (and five TAAs) were prepared in a mixture of 1:1 methanol:water.…”

“…This is where high-resolution hybrid measurements, like those from a recently developed SLIM-Orbitrap platform, can play a critical role. Hollerbach et al recently demonstrated the potential of SLIM-Orbitrap MS for finding novel chemical signatures of fentanyls . The authors proposed that their technology could be extended to find unique chemical signatures of other NSOs, and nitazenes are a logical extension from fentanyls since they are becoming increasingly prevalent and are concerning from a public health standpoint.…”

“…In 2019, Zaknoun and co-workers were working with an atmospheric pressure DT-IMS and a radioactive nickel source and discovered that fentanyl and fentanyl analogs exhibit multiple IMS distributions instead of just one, whereas other opioids like heroin, morphine, codeine, hydrocodone, and methadone only exhibit single IMS distributions. , Butler and Baker later proposed that the two IMS distributions of fentanyl they observed using electrospray ionization and a low pressure drift tube were either gas-phase conformers (e.g., rotomers) or protomers (i.e., gas-phase conformations adopted due to differences in the protonation site) . Hollerbach et al later performed a comprehensive set of experiments demonstrating that the intensities of the two IMS distributions of all fentanyls change as a function of water concentration in the electrospray solvent, which is known behavior of other compounds that exhibit protomers (e.g., para-aminobenzoic acid) . Additionally, both Hollerbach et al and Aderorho et al found that the two IMS distributions produce different fragmentation spectra, which is also expected behavior of protomers. , It is worth noting that the reduced mobility values obtained with atmospheric pressure IMS systems are sensitive to impurities in the buffer gas (e.g., water) and clustering effects (e.g., from mixtures of analytes). , Large mobility shifts can occur if both are not properly controlled, and this can lead to incorrect reduced mobility values (and hence CCS).…”

Rapid differentiation of xylazine metabolites using SLIM IM-MS

“…10–16 This powerful technique has seen increased utility in clinical, forensic, and toxicological applications. Our group has reported on a recently commercialized high-resolution IM platform, Structures for Lossless Ion Manipulations (SLIM), 17–19 for the rapid differentiation of synthetic cannabinoid isomers. 20…”