Screening and semi-quantitation of 212 fentanyl analog compounds by the Orbitrap Exploris 120 mass spectrometer

Significance of the Topic

The proliferation of illicit fentanyl analogs has significantly contributed to the current opioid overdose crisis. Accurate, high-throughput screening in biological matrices such as urine is essential for forensic investigations, clinical research, and public health monitoring. High-resolution accurate mass (HRAM) spectrometry offers unparalleled sensitivity and specificity for detecting a broad panel of synthetic opioids.

Objectives and Study Overview

This study demonstrates the capability of the Thermo Scientific Orbitrap Exploris 120 mass spectrometer, coupled with Vanquish Flex UHPLC, to screen and semi-quantitate 212 fentanyl-related compounds—including fentanyl itself—in human urine. The method integrates a targeted MS2 spectral library for confident identifications and supports retrospective analysis of emerging analogs.

Methodology

Sample preparation was limited to a 20× dilution of urine in water to highlight screening performance. A 15.5-minute reversed-phase gradient was run on an Accucore Phenyl Hexyl column using 2 mM ammonium formate with 0.1% formic acid (mobile phase A) and a 50:50 mixture of methanol/acetonitrile with 2 mM ammonium formate and 0.1% formic acid (mobile phase B). Full MS scans (m/z 100–1000) at 60,000 resolution were followed by data-dependent MS2 acquisition targeting a list of 212 precursor ions. A custom MS2 library of 213 spectra—including carfentanil—was built in mzVault and deployed in TraceFinder for spectral matching.

Instrumentation

• UHPLC: Thermo Scientific Vanquish Flex system with binary pump, column compartment, and autosampler
• Column: Thermo Scientific Accucore Phenyl Hexyl, 2.6 μm, 100 × 2.1 mm, 40 °C
• Mass spectrometer: Thermo Scientific Orbitrap Exploris 120 with OptaMax NG H-ESI probe
• Data processing: Thermo Scientific TraceFinder 5.1 using mzVault library

Main Results and Discussion

Limits of detection (LOD), quantitation (LOQ), and identification (LOI) were determined for all 212 analogs using nine-point calibration curves (0.25–100 ng/mL) diluted 20× prior to analysis. Key findings include:

• For >75% of analogs, LOD ≤2.5 ng/mL; a majority achieved LOD ≤0.5 ng/mL
• LOQ and LOI were ≤10 ng/mL for >75% of compounds, with many as low as 2.5 ng/mL
• Retention time reproducibility ±0.2 min; mass accuracy <5 ppm without spectral averaging
• Isotope pattern match score ≥80% and MS2 match score ≥90% ensured high confidence

Benefits and Practical Applications

This streamlined workflow enables one-run screening and semi-quantitation of 212 fentanyl analogs with minimal sample preparation. The HRAM data support retrospective mining for newly encountered analogs, critical for forensic laboratories facing rapidly evolving illicit drug landscapes. The approach reduces dependence on multiple targeted assays and enhances laboratory throughput.

Future Trends and Applications

Emerging directions include expanding MS2 libraries to encompass novel analogs, integrating supported liquid extraction for deeper sensitivity, and automating data-processing pipelines. Advances in machine learning for spectral interpretation may further enhance retrospective discovery of unknown opioids. Coupling HRAM screening with quantitative assays can streamline workflows in clinical toxicology and public health surveillance.

Conclusion

The described HRAM UHPLC-MS/MS method on the Orbitrap Exploris 120 enables rapid, sensitive, and confident screening of 212 fentanyl analogs in urine. Custom MS2 libraries and data-dependent acquisition ensure robust identifications, while simple sample dilution underscores the method’s practicality for high-throughput forensic and clinical laboratories.

References

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3. Cerilliant Corporation. Opioid Certified Reference Material Kit. Round Rock, Texas.
4. Cayman Chemical. Fentanyl Analog Screening Kit and Emergent Panels. Ann Arbor, Michigan.
5. Xie X, Van Natta K, Hassell K. LC-MS/MS toxicology platform and method for high-resolution, accurate mass detection of drugs. Thermo Fisher Scientific Technical Note 73771; 2020.