Detection of Fentanyl Analogs and Novel Synthetic Opioids in Hair

Significance of the Topic

The proliferation of novel synthetic opioids including fentanyl analogs has intensified the opioid crisis by introducing potent, unregulated substances into recreational drug markets. Hair analysis offers a non-invasive and stable matrix for long-term monitoring of drug exposure, providing a retrospective window that exceeds the diagnostic capabilities of urine, blood, or saliva. There is an urgent need for robust, high-sensitivity screening methods capable of detecting trace levels of diverse NSOs in hair for forensic and clinical toxicology.

Study Objectives and Overview

This work presents a comprehensive workflow that integrates a simplified hair extraction protocol with SWATH® acquisition on the SCIEX X500R QTOF system. The method targets selective isolation and quantification of 15 fentanyl analogs and synthetic opioids at sub-pg/mg concentration levels in head hair samples. Real-world applicability was demonstrated by analyzing hair from individuals reporting non-medical opioid or heroin use within the past year.

Methodology

• Hair Preparation and Extraction: Approximately 50 mg of head hair was cleaned, ground, and incubated in methanol with internal standards, followed by overnight extraction at 55 °C and collection of the organic phase.
• UHPLC Separation: Chromatographic separation was performed on a Phenomenex C18 column (100 × 2.1 mm, 1.7 μm) at 45 °C with a water–acetonitrile gradient, 0.5 mL/min flow rate, 11.5 min runtime and 3 μL injection volume.
• Mass Spectrometry and SWATH Acquisition: Data were acquired on the QTOF system using 12 variable Q1 windows (230–450 m/z) and a 0.555 sec cycle time, generating high-resolution TOF MS and MS/MS spectra.
• Data Analysis: SCIEX OS Software 1.5 facilitated targeted processing of SWATH data against a library of 15 analytes and 3 internal standards, applying mass error, retention time, isotope ratio, and library match criteria to compute combined confidence scores.

Instrumentation

• SCIEX ExionLC™ AC System with Phenomenex C18 column
• SCIEX X500R QTOF Mass Spectrometer with SWATH acquisition
• SCIEX OS Software 1.5 for data acquisition and processing

Main Results and Discussion

The method achieved near-baseline separation of all 15 target compounds within an 11.5 min LC run. Calibration curves spanning three orders of magnitude yielded R2 > 0.99 for fentanyl and furanylfentanyl. Intra- and inter-day precision and accuracy remained below 25 % and 20 % respectively, with limits of detection in the sub-pg/mg range. Analysis of authentic hair samples confirmed multiple NSOs at concentrations as low as 0.97 pg/mg, with MS/MS library match scores exceeding 99 % and combined confidence scores above 90 %.

Benefits and Practical Applications

• Non-invasive collection and simple storage requirements for hair samples.
• High sensitivity enabling trace-level quantification for forensic investigations.
• Comprehensive digital archiving of full-scan MS/MS data supports retrospective screening as new analogs emerge.
• Flexible panel expansion allows rapid inclusion of newly identified compounds without reconfiguring acquisition parameters.

Future Trends and Opportunities

Further developments in high-resolution MS and data-independent acquisition are expected to increase screening depth and throughput. Integration of machine learning and advanced data mining techniques may streamline identification of unknown analogs within large SWATH datasets. Expanded hair testing libraries and multiplexed workflows could enable population-level monitoring of evolving NSO use patterns in public health and forensic contexts.

Conclusion

This workflow, combining targeted hair extraction with SWATH acquisition on the SCIEX X500R QTOF system, delivers a robust, sensitive, and adaptable platform for comprehensive detection of fentanyl analogs and synthetic opioids in hair. Its high confidence identification and quantification at sub-pg/mg levels makes it a valuable tool for forensic and toxicology laboratories, supporting both immediate casework and retrospective data mining.

References

1. vMethod™ Application – Single-Injection Screening of 664 Forensic Toxicology Compounds on a SCIEX X500R QTOF System