Sample Preparation for Fentanyl Analogs in Whole Blood

Significance of the Topic

The rise of fentanyl analogs has intensified the opioid crisis, driving demand for sensitive and reliable blood testing methods. Accurate quantitation of these potent opioids in whole blood is essential for clinical toxicology, forensic investigations, workplace screening and regulatory compliance.

Objectives and Study Overview

This work evaluates and compares multiple sample-preparation techniques to isolate a broad panel of fentanyl analogs from whole blood. The study aims to achieve a limit of quantitation of 0.1 ng/mL, maximize analyte recovery and minimize matrix effects within efficient workflows.

Methodology and Instrumentation

The study examined three extraction approaches:

• Supported Liquid Extraction (SLE+) using diatomaceous earth sorbent with various organic elution solvents (DCM, ethyl acetate, MTBE).
• Silica-based Mixed-Mode SPE (ISOLUTE HCX) requiring conditioning, equilibration and elution with DCM/IPA/NH4OH or EA/ACN/NH4OH.
• Polymeric Mixed-Mode SPE (EVOLUTE EXPRESS CX) omitting conditioning and equilibration, eluted under similar solvent conditions.

All protocols used 100 µL blood samples pre-treated with dilute aqueous base or acid, followed by elution, evaporation and reconstitution in 50:50 mobile phase. LC-MS/MS analysis was performed on a Shimadzu Nexera X2 UHPLC coupled to a SCIEX 5500 triple quadrupole. Chromatography employed a Restek Raptor Biphenyl column (100 × 2.1 mm, 2.7 µm) at 40 °C, isocratic 50:50 formic acid in water/methanol, 0.4 mL/min, 7 min run time. MS settings included 600 °C source, curtain gas 20, collision gas 8, positive ion mode with compound-specific transitions.

Results and Discussion

All methods achieved the target LOQ of 0.1 ng/mL. EVOLUTE EXPRESS CX with DCM/IPA/NH4OH elution provided the highest recoveries across fentanyl analogs but showed 10–40 % signal suppression. SLE+ with MTBE elution offered a balance of moderate to high recoveries (>50 %) and lower matrix interference. ISOLUTE HCX eluted with EA/ACN/NH4OH demonstrated the least matrix effects but slightly reduced recoveries. The polymeric SPE reduced preparation time by eliminating conditioning and equilibration steps compared to silica-based SPE.

Benefits and Practical Applications

The evaluated workflows support robust quantitation of multiple fentanyl analogs in small blood volumes. Rapid SLE+ and polymeric SPE approaches suit high-throughput toxicology labs. Selection of elution solvents allows tuning of recovery versus cleanliness according to clinical, forensic or QA/QC requirements.

Used Instrumentation

• Shimadzu Nexera X2 UHPLC system
• SCIEX 5500 Triple Quadrupole MS
• ISOLUTE SLE+ 96-well plates (400 µL)
• ISOLUTE HCX and EVOLUTE EXPRESS CX SPE plates
• Biotage SPE Dry 96 evaporator

Future Trends and Opportunities

Advances in automated sample handling, novel sorbent chemistries and micro-extraction techniques will further streamline opioid analysis. Integration with high-resolution and ambient ionization MS approaches can expand panels and improve turnaround. Continued development of low-volume, point-of-care devices holds promise for real-time monitoring.

Conclusion

Optimizing sample-preparation for fentanyl analogs requires balancing recovery, matrix suppression and workflow efficiency. SLE+ with MTBE, polymeric mixed-mode SPE and silica mixed-mode SPE each offer distinct advantages. Laboratories should select methods aligned with sensitivity needs, sample throughput and instrumentation.

References

1. National Survey on Drug Use and Health. National Survey Data.
2. Florence CS, et al. The Economic Burden of Prescription Opioid Overdose, Abuse, and Dependence in the United States, 2013. Med Care. 2016 Oct;54(10):901–906.