LC/MS/MS Analysis of Fentanyl and Related Analogs Using Biocompatible Solid Phase Microextraction

Significance of the topic

The growing abuse of fentanyl and its analogs poses serious public health and forensic challenges. These synthetic opioids are highly potent, responsible for a surge in overdose deaths, and require rapid, sensitive analytical methods for quantitation in biological samples. A streamlined workflow supports law enforcement, clinical diagnostics, and quality control in pharmaceutical settings.

Objectives and overview of the study

This work aimed to develop a fast, reproducible LC/MS/MS assay for nine fentanyl-related compounds in urine. By integrating biocompatible solid phase microextraction (BioSPME) with a high-efficiency fused-core biphenyl column, the study sought to achieve sub-ng/mL detection limits, minimize matrix effects, and enable high-throughput screening.

Methodology and instrumentation

A mixed-mode C8/strong cation exchanger BioSPME fiber was conditioned, rinsed, and used to extract spiked urine samples under controlled agitation. After a brief rinse, analytes were desorbed in 90:10 methanol:water with 0.1% ammonium hydroxide.
Chromatographic separation employed an Ascentis Express Biphenyl column (5 cm x 2.1 mm, 2.7 µm). Mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B), with a gradient from 40% to 80% B over three minutes and a total run time of 5.5 minutes. The flow rate was 0.6 mL/min at 50 °C. Detection was performed using an Agilent 1290 Infinity II coupled to a 6460 QQQ mass spectrometer in positive-ion ESI multiple reaction monitoring mode.

Main results and discussion

The fused-core biphenyl column provided baseline resolution of nine fentanyl analogs in under three minutes. Calibration curves were linear from 0.05 to 50 ng/mL for most analytes, except remifentanil (1–10 ng/mL). Limits of quantitation reached 0.05 ng/mL for seven compounds, with remifentanil and alfentanil at 1 ng/mL. Recoveries ranged from 66.7% to 111%, with most above 70% at 0.05 ng/mL. Method precision was excellent, with percent relative standard deviations below 10% for all but two analytes lacking matched internal standards. Matrix effects were under 10% for all compounds, demonstrating effective suppression of nonspecific binding by the BioSPME coating.

Benefits and practical applications

• Reduced sample preparation steps compared to conventional SPE.
• Fast turnaround with total analysis time under six minutes.
• High sensitivity suitable for trace-level detection in urine.
• Minimal matrix interference enabling direct analysis of complex biological fluids.
• Applicable to forensic toxicology, clinical monitoring, and pharmaceutical quality control.

Future trends and possibilities for application

Advances may include expansion to additional opioid analogs and other drug classes, integration with high-resolution mass spectrometry for non-targeted screening, and automation of BioSPME sampling for routine lab workflows. Portable or field-deployable devices leveraging biocompatible microextraction could support on-site forensic or clinical testing.

Conclusion

The combination of BioSPME and fused-core biphenyl LC/MS/MS delivers a rapid, sensitive, and robust method for quantifying fentanyl and related analogs in urine. The procedure offers sub-ng/mL detection limits, strong reproducibility, and minimal matrix effects, making it a valuable tool for high-throughput analytical laboratories.

References

No specific literature references were provided in the source document.