LC-MS/MS detection of fentanyl and related synthetic opioids in biological matrices

Significance of the Topic

The emergence of fentanyl and its analogs as ultra-potent synthetic opioids has led to a surge in overdose incidents worldwide. Detecting these compounds at trace levels in biological samples is critical for forensic toxicology and clinical diagnostics. Advanced analytical methods that offer high sensitivity, specificity and throughput are essential to support public health and safety efforts.

Objectives and Study Overview

This study aimed to develop a single liquid chromatography tandem mass spectrometry (LC-MS/MS) protocol capable of identifying fentanyl, a range of fentanyl analogues and key metabolites in blood and urine. A secondary goal was to establish limits of detection for each target compound under realistic sample preparation conditions.

Methodology and Instrumentation

Sample preparation employed a solid-phase extraction workflow to isolate analytes from certified blank whole blood and urine. Calibration curves were generated via serial dilutions from 0.32 to 1000 pg/mL using fentanyl-d5 as an internal standard. Multiple reaction monitoring transitions for each analyte were optimized through flow injection analysis. Four different reversed-phase columns were screened under a five-minute gradient to identify the column providing the best combination of peak shape, separation efficiency and sensitivity.

Instrumentation Used

• Shimadzu LCMS-8060 triple quadrupole mass spectrometer with heated dual ionization source (DUIS combining ESI and APCI)
• Restek Raptor Biphenyl LC column (2.1×50 mm, 2.7 µm particle size)
• LabSolutions software for MRM optimization and data acquisition

Results and Discussion

Restek Raptor Biphenyl provided superior chromatographic performance and sensitivity compared to three other tested columns. Limits of detection in blood ranged from 0.32 to 8 pg/mL for most analytes, with naloxone at 200 pg/mL. Urine LODs were between 1.6 and 40 pg/mL. Calibration curves exhibited linearity (R2 > 0.998) across the full range. Representative chromatograms confirmed baseline separation of up to 14 targets within a rapid five-minute run time.

Benefits and Practical Applications

• High sensitivity enables reliable detection at sub-ng/mL levels
• Rapid analysis supports high sample throughput in forensic and clinical laboratories
• Single-run method reduces instrument time and resource consumption

Future Trends and Opportunities

Further work will focus on extending the method to include quantitative validation (precision, accuracy and limits of quantitation), expanding the target list to emerging analogues and integrating automation for high-volume screening. Coupling with high-resolution mass spectrometry may enhance non-targeted surveillance of novel synthetic opioids.

Conclusion

The developed LC-MS/MS method on the Shimadzu LCMS-8060 with DUIS successfully identifies fentanyl, its analogues and metabolites in blood and urine at picogram per milliliter levels. The combination of optimized MRM transitions, efficient sample preparation and a fast chromatographic gradient delivers a robust solution for forensic toxicology.

References

• Lockwood R, Pryor K, Parks M, Gilles C. LC-MS/MS detection of fentanyl and related synthetic opioids in biological matrices. Shimadzu Application Note PO-CON1816E; 2018.