LC-QTOF-MS Identification of Major Urinary Cyclopropylfentanyl Metabolites Using Synthesized Standards

Cyclopropylfentanyl is a fentanyl analog implicated in 78 deaths in Europe and over 100 deaths in the United States, but toxicological information including metabolism data about this drug is scarce. The aim of this study was to provide the exact structure of abundant and unique metabolites of cyclopropylfentanyl along with synthesis routes. In this study, metabolites were identified in 13 post-mortem urine samples using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Samples were analyzed with and without enzymatic hydrolysis, and seven potential metabolites were synthesized in-house to provide the identity of major metabolites. Cyclopropylfentanyl was detected in all samples, and the most abundant metabolite was norcyclopropylfentanyl (M1) that was detected in 12 out of 13 samples. Reference materials were synthesized (synthesis routes provided) to identify the exact structure of the major metabolites 4-hydroxyphenethyl cyclopropylfentanyl (M8), 3,4-dihydroxyphenethyl cyclopropylfentanyl (M5) and 4-hydroxy-3-methoxyphenethyl cyclopropylfentanyl (M9). These metabolites are suitable urinary markers of cyclopropylfentanyl intake as they are unique and detected in a majority of hydrolyzed urine samples. Minor metabolites included two quinone metabolites (M6 and M7), not previously reported for fentanyl analogs. Interestingly, with the exception of norcyclopropylfentanyl (M1), the metabolites appeared to be between 40% and 90% conjugated in urine. In total, 11 metabolites of cyclopropylfentanyl were identified, including most metabolites previously reported after hepatocyte incubation.