Extension of the Temporal Window for the Determination of Alpha-Methylthiofentanyl and Thiofentanyl in Rat Urine by Monitoring the Metabolite Norfentanyl Using Online Solid-Phase Extraction (SPE) Coupled with Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS)

Alpha-methylthiofentanyl and thiofentanyl are psychoactive substances that have been abused in recent years, strongly threatening social and public security. Because they are rapidly metabolized in vivo, their determination presents a great challenge for forensic science. An automated method of online solid-phase extraction coupled with ultra-high performance liquid chromatography—tandem mass spectrometry was developed for the determination of alpha-methylthiofentanyl, thiofentanyl, and the metabolite norfentanyl in urine. The analytes were first enriched and purified by an Oasis HLB online SPE column and eluted on a Hypersil GOLD C18 analytical column. Chromatographic separation was achieved with mobile phase components of 2 mM ammonium formate and 0.1% formic acid in water and same composition in methanol in gradient elution mode. The limit of detection and lower limit of quantitation of the method for the three analytes were 0.2 and 2 pg/ml, respectively. Acceptable linearity was obtained from 2 to 800 pg/ml. The accuracy was in the range of 87.98–108.95%; and precision, calculated as the relative standard deviation, was less than 14.63%. The method recoveries were from 65.56 to 113.72%. This highly sensitive method was applied to study the detection window of the precursor compounds and metabolites in rat urine. Both alpha-methylthiofentanyl and thiofentanyl are metabolized to norfentanyl, which was first revealed in this study. With the aid of norfentanyl, the detection windows of alpha-methylthiofentanyl and thiofentanyl were extended to 18–26 days and 20–27 days, respectively. This study provides important auxiliary tools for the forensic identification of fentanyl analogs.