Chromatographic separation of the isobaric compounds cyclopropylfentanyl, crotonylfentanyl, methacrylfentanyl, and para-methylacrylfentanyl for specific confirmation by LC-MS/MS.

Cyclopropylfentanyl has been encountered by law enforcement and public health officials beginning in mid-2017 and has been associated with numerous overdoses and fatalities. The detection and identification of cyclopropylfentanyl has become more challenging with the subsequent emergence of multiple structural isomeric fentanyl species, some of which have been detected in seized drug casework. These include crotonylfentanyl, methacrylfentanyl, para-methylacrylfentanyl, and ortho-methylacrylfentanyl; all of which have the same exact mass and similar fragmentation patterns. Since these compounds may be scheduled differently and pharmacologically act differently, it is important to be able to differentiate them analytically. Our method was developed and validated for the analysis and differentiation of cyclopropylfentanyl from its isomers by liquid chromatography tandem mass spectrometry (LC-MS/MS), in order to confirm the identify of cyclopropylfentanyl in biological specimens from death investigation casework; 36 postmortem blood samples were submitted for analysis. Cyclopropylfentanyl, crotonylfentanyl, methacrylfentanyl, and para-methylacrylfentanyl were successfully resolved using a 13-minute run time and a simple gradient elution. Ortho-methacrylfentanyl was resolved only in a 20-minute chromatographic run. The assay met validation performance characteristics, with an analytical range of 1-100 ng/mL and limits of detection of 0.1 ng/mL for all analytes. Analysis of commonly encountered substances showed no interferences. All samples previously reported positive for cyclopropylfentanyl were confirmed positive for cyclopropylfentanyl in the absence of its isomers. To our knowledge to date, no positive toxicological specimens have been encountered for any cyclopropylfentanyl isomers.