MSACL 2024 Abstract

INTRODUCTION: Fentanyl presents a growing problem in today’s illicit drug market, where it has been identified laced into several recreational drugs. Appearance of fentanyl analogs has also raised concerns over their wide range of potency (some as might as 100X that of fentanyl). However, the similarity in their molecular structure creates ambiguity in field deployable and rapid clinical screening methods. Herein we present an ion mobility-tandem mass spectrometry (IM-MS/MS) workflow developed to differentiate between more than a dozen fentanyl analogs, many of which are isomers. We have applied this workflow to the analysis of spiked urine samples to demonstrate the separation and quantification capabilities. Lastly, we have applied these methods to other new psychoactive substances (NPS) including xylazine and synthetic cannabinoid metabolite isomers.

OBJECTIVES: The primary objective of this study was to develop an LC-IM-MS/MS method for identification of isomeric fentanyl analogs, xylazine, and synthetic cannabinoids and their metabolites in human urine.

METHODS: A targeted LC-IM-MS/MS method was developed for quantitation of 17 fentanyl analogs/metabolites. Both traditional drift tube IMS (Agilent 6560 IM-QTOF) and more recently developed structures for lossless ion manipulations (MOBILion Systems MOBIE SLIM) methods were used to optimize resolution and sensitivity. Spiked urine samples were prepared using solid phase extraction (SPE) prior to LC-IM-MS/MS analysis. Targeted quantification using Skyline was based on retention time, collision cross section (CCS), and MS/MS transition(s) from known standards. A further group of nine synthetic cannabinoid metabolites and several xylazine metabolites were also quantified.

RESULTS: IM-based separations were achieved for five classes of fentanyl analog isomers, across a total of 17 compounds. Mobility-aligned MS/MS analysis also revealed unique fragments that could be correlated with differences in CCS. Various experimental parameters including sample preparation and instrument conditions were optimized for resolution and sensitivity. Protonated fentanyl analogs presented unique CCS over a range from 172.7-194.9 Å2, and the improved resolving power achieved using high-resolution demultiplexing and/or SLIM’s 13-meter pathlength provided sufficient separation of these compounds. Limits of detection for fentanyl were in the low pg/mL range, which far exceeds the typical 1 ng/mL reporting cutoff of most clinical assays. Furthermore, differentiation of various metabolites (e.g., norfentanyl) also yielded important information that could be representative of other unique analogs.

CONCLUSION: As more fentanyl analogs and other new psychoactive substances (NPS) appear in the recreational drug community, often leading to health emergencies, there is a tremendous need for developing rapid, high-resolution methods that can unambiguously identify these different substances to assess risk and inform treatment. IM-MS/MS methods present a powerful opportunity for providing comprehensive characterization of this complex molecular class. Our developed methods are a promising first step towards the routine implementation of ion mobility in the clinical lab.