Direct Analysis of Opioids and Metabolites from Whole Blood Using Ostro Sample Preparation Plates Combined with UPLC-MS/MS for Forensic Toxicology

Significance of the topic

The misuse and abuse of natural and synthetic opioids remain a critical concern in forensic toxicology. Accurate identification and quantification of opioids and their glucuronide metabolites in whole blood are essential for determining cause of death, impairment, or involvement in criminal cases. Traditional methods often require enzymatic or chemical hydrolysis, increasing analysis time and cost while introducing variability.

Objectives and study overview

This study aimed to develop a rapid, reliable workflow for direct analysis of 22 opioid drugs and metabolites in whole blood. By combining Ostro sample preparation plates with UPLC-MS/MS, the method eliminates enzymatic hydrolysis, reduces sample handling steps, and achieves high throughput suitable for forensic laboratories.

Methodology and sample preparation

Whole blood samples were pretreated directly in Ostro Pass-through 96-well plates. Cells were lysed with ZnSO₄/NH₄CH₃COOH solution, followed by protein precipitation using acetonitrile containing deuterated internal standards. After brief mixing, the extract was eluted, evaporated under nitrogen, and reconstituted in aqueous formic acid solution.

UPLC-MS/MS conditions

Analysis was performed on an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with a gradient elution from 2% to 52.8% acetonitrile over 6 minutes. A Xevo TQD mass spectrometer operating in positive-ion MRM mode monitored two transitions per compound. Total cycle time was 8 minutes, with all analytes eluted in under 5.5 minutes.

Instrumentation

• Ostro Pass-through 96-well Sample Preparation Plate
• ACQUITY UPLC BEH C18 Column (2.1 × 100 mm, 1.7 μm)
• ACQUITY UPLC System
• Xevo TQD Triple Quadrupole Mass Spectrometer
• MassLynx Software v4.1

Main results and discussion

Chromatographic separation achieved baseline resolution of critical isomeric pairs (e.g., morphine vs. hydromorphone and their glucuronides). Mean recoveries exceeded 60% for all compounds, with most above 80%. Calibration curves (5–500 ng/mL; 1.25–125 ng/mL for fentanyl) showed linearity (R² > 0.973), accuracy within 2% deviation, and precision (CV < 10% for all but three minor metabolites). Sensitivity allowed low-ng/mL detection with signal-to-blank ratios exceeding 20×.

Benefits and practical applications

• Direct analysis of glucuronide metabolites without hydrolysis
• Rapid, in-well sample preparation in a 96-well format increases throughput
• Removal of phospholipid matrix interferences improves MS performance
• Short analysis time (8 minutes total) enables high sample throughput
• Robust linearity, accuracy, and sensitivity meet forensic QA/QC requirements

Future trends and applications

Integration of this workflow with automated liquid-handling platforms could further increase throughput. Expansion to additional drug classes and conjugates, coupled with high-resolution MS, may enhance screening capabilities. Applications in clinical toxicology, workplace drug testing, and therapeutic drug monitoring are foreseeable. Ongoing improvements in sample prep materials and column chemistries will continue to streamline forensic analyses.

Conclusion

The described Ostro-UPLC-MS/MS method provides a fast, sensitive, and reliable approach for direct quantification of a comprehensive panel of opioids and metabolites in whole blood. By eliminating hydrolysis and simplifying sample cleanup, the workflow meets the demanding throughput and performance standards of modern forensic laboratories.

References

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