LAUSR

BACKGROUND The opioid analgesic fentanyl and its analogues pose a major health concern due to its high potency and the increasing number of overdose deaths worldwide. The analogues of fentanyl may differ in potency, toxicity and legal status and it is therefore important to develop analytical methods for their correct identification. This can be challenging since many fentanyl analogues are structural isomers. Two fentanyl isomers that have been in the spotlight lately due to difficulties regarding separation and identification are cyclopropylfentanyl and crotonylfentanyl, which have been reported to display nearly identical fragmentation patterns and chromatographic behavior. METHODS Chromatographic separation of cyclopropylfentanyl and crotonylfentanyl by UHPLC was investigated using three different stationary phases (HSS T3, BEH C18 and Kinetex biphenyl) employing gradient elution with a mobile phase consisting of 10 mM ammonium formate pH 3.1 and MeOH. Detection was performed by MS/MS. In addition, the major metabolites of the two compounds formed upon incubation with human liver microsomes (HLM) were identified by UHPLC-QTOF-MS analysis. RESULTS Baseline separation of cyclopropylfentanyl and crotonylfentanyl was achieved on the BEH C18 column with retention times of 6.79 and 7.35 minutes, respectively. The major metabolites of the two analogues formed by HLM differed, with the main biotransformation being N-dealkylation and carboxylation for cyclopropylfentanyl and crotonylfentanyl, respectively. We demonstrated the usefulness of the two approaches by unambiguously identifying cyclopropylfentanyl, as well as its metabolites, in two authentic post mortem blood samples. CONCLUSIONS In the present study, we successfully demonstrated that cyclopropylfentanyl and crotonylfentanyl can be distinguished by methods commonly available in forensic laboratories.