A Comprehensive Approach to Targeted and Untargeted Screening Methodology for Emerging Synthetic Fentanyl Analogues

Importance of the topic

Opioid overdose remains a major public health concern driven by the rapid emergence of new and highly potent synthetic fentanyl analogues. Traditional analytical workflows struggle to keep pace with the introduction of hundreds of novel analogues that have nearly identical structures and m/z values. A robust, dual strategy combining targeted screening and untargeted discovery is essential to detect known fentanyl derivatives and uncover previously unreported compounds in clinical, forensic, and regulatory laboratories.

Objectives and study overview

This application note describes a comprehensive workflow to screen for approximately 150 synthetic fentanyl analogues plus 4-ANPP (an important precursor). Key goals include:

• Developing a chromatographic separation capable of resolving isobaric fentanyl compounds.
• Implementing high-resolution accurate mass (HRAM) LC-Q-TOF acquisition for both targeted and untargeted analyses.
• Building spectral and formula libraries for confident identification and retrospective data mining.

Methodology and used instrumentation

The workflow integrates both targeted Find by Formula (FBF) and untargeted Molecular Feature Extractor (MFE) approaches, supplemented by neutral loss scanning, reporter ion detection, variable mass defect filtering, and molecular structure correlation.

Used instrumentation:

• Agilent 1290 Infinity II LC System: binary pump, multisampler (6 °C), column thermostat set at 55 °C.
• Agilent Poroshell 120 Phenyl Hexyl column (2.1×100 mm, 2.7 μm).
• Agilent 6546 LC/Q-TOF Mass Spectrometer with Agilent Jet Stream ESI, positive mode.

Key LC conditions included a gradient from 30% to 98% organic over 14 min, 0.35 mL/min flow, and 2 µL injection. MS parameters featured stable mass accuracy, MS scan rates up to 6 spectra/s, auto MS/MS at collision energies of 10, 20, and 40 V.

Main results and discussion

Targeted analysis using FBF with a customized personal compound database library (PCDL) enabled reliable detection of all 150 analogues by accurate mass, retention time, and isotope score. The Poroshell column achieved baseline separation of isobaric pairs that share identical formulae but differ in structure. MS/MS spectra were added to the PCDL, allowing Find by Auto MS/MS to confirm identities through spectral matching.

In untargeted mode, MFE identified molecular features across the full data set, while variable mass defect filtering efficiently isolated fentanyl-type compounds by applying a linear mass-defect regression. Neutral loss and reporter ion scans revealed recurring losses (e.g., 149.0800 Da) and fragment ions (m/z 105, 188) characteristic of the fentanyl backbone. Agilent Molecular Structure Correlator (MSC) further supported structural hypotheses and prediction of fragment ions.

Benefits and practical applications

This dual screening strategy provides laboratories with:

• Comprehensive coverage for both known and unknown synthetic opioids.
• High throughput analysis capable of handling an expanding compound library.
• Confidence in identifications through orthogonal criteria: retention time, accurate mass, isotope pattern, and MS/MS matching.
• Retrospective data mining to discover analogues not originally targeted.

Future trends and opportunities

As novel fentanyl analogues continue to emerge, integrating machine learning for pattern recognition in untargeted data and expanding spectral libraries with community-shared entries will further enhance detection capabilities. Advances in ultra-high-resolution instrumentation and automated data processing will also accelerate response times. The approach can be extended to other classes of synthetic drugs, supporting public health, forensic investigations, and regulatory control.

Conclusion

The described HRAM LC/Q-TOF workflow effectively addresses the analytical challenges posed by diverse and isobaric synthetic fentanyl analogues. By combining targeted and untargeted data acquisition, advanced algorithms, and robust chromatographic separation, laboratories can achieve comprehensive screening, confident identifications, and the flexibility to adapt to emerging threats.

References

• Seth P.; et al. Overdose Deaths Involving Opioids, Cocaine, and Psychostimulants – United States, 2015–2016. MMWR Morb Mortal Wkly Rep. 2018;67(12):349–358.
• Krajewski L. C.; et al. Application of the Fentanyl Analog Screening Kit Toward the Identification of Emerging Synthetic Opioids in Human Plasma and Urine by LC-Q-TOF. Toxicology Letters. 2020;320:87–94.