A Fully Automated On-line Forensic Toxicology Solution for the Determination of Opioids in Blood by Smart SPE Clean-Up and GC-MS

Importance of the Topic

Forensic toxicology laboratories increasingly struggle with high sample volumes and limited personnel, yet must deliver reliable results admissible in court. Automation of sample preparation and analysis accelerates turnaround times, improves repeatability and allows skilled staff to concentrate on data interpretation, method development and reporting.

Study Objectives and Overview

This application note describes the development and validation of a fully automated on-line workflow for the quantification of four opioid compounds (morphine, 6-monoacetylmorphine, codeine and dihydrocodeine) in blood. The method couples smart on-line micro solid-phase extraction (smart SPE) with gas chromatography–mass spectrometry (GC-MS) using a GERSTEL MultiPurpose Sampler (MPS) and an Agilent single quadrupole GC-MS system.

Methodology

Samples of defibrinated horse blood were spiked with target analytes and deuterated internal standards. After buffer addition to promote analyte protonation, the MPS performed automated vortex mixing and centrifugation. The clear supernatant was processed through miniaturized Instrument Top Sample Prep (ITSP) cartridges packed with custom cation-exchange sorbent. Conditioning, sample loading, washing (water, acetate buffer pH 4.5 and methanol) and elution (dichloromethane/isopropanol/ammonium hydroxide) were fully automated. The eluate was evaporated to dryness, derivatized with BSTFA + 1% TMCS and injected directly into the GC-MS.

Instrumentation

The workflow employs a GERSTEL MPS 2 XT Dual Rail autosampler equipped with solvent filling station, wash stations, trays (VT-98 and VT-40), smart SPE ITSP kit, multi-position vortexer (mVorx), robotic centrifuge (CF-200) and evaporation station (mVAP). GC-MS analysis uses an Agilent 7890B gas chromatograph coupled to a 5975C mass selective detector.

Results and Discussion

Calibration curves for all four analytes displayed excellent linearity (R2 ≥ 0.997) using both 10 mg and 30 mg SPE bed sizes, comparable to derivatization-only samples. Recoveries exceeded 90% for all compounds with relative standard deviations below 10%. The lowest calibration levels yielded signal-to-noise ratios above 10 for all analytes except 6-monoacetylmorphine (S/N ≈ 8), yet the precision at this level remained within 2%. Method limits of detection ranged from 3 µg/L to 9 µg/L. Ion ratio precision across 30 measurements showed %RSD values mostly under 20%, meeting forensic guidelines.

Benefits and Practical Applications

• Substantial reduction in solvent consumption (up to 90%), lowering reagent and waste disposal costs.
• Improved reproducibility and consistent calibration over multiple days.
• Streamlined workflow with parallel sample preparation and GC-MS analysis using the PrepAhead function.
• Freeing of analyst time for high-value tasks.

Future Trends and Opportunities

The automated on-line SPE-GC-MS approach can be extended to post-mortem whole blood and other biological matrices. Implementation of triple-quadrupole MS detection would enhance selectivity and sensitivity for low-abundance analytes. Integration with laboratory information management systems (LIMS) and expansion to additional forensic targets promise further efficiency gains.

Conclusion

A robust, fully automated on-line sample preparation and GC-MS method was established for four forensic opiates in blood. The approach delivers high recoveries, excellent precision and significant savings in time and solvents. Ongoing work will focus on method adaptation for complex post-mortem matrices and higher-resolution MS detection.

Reference

• ITSP Solutions Inc. Automated Online μSPE-LC/MS/MS for the Measurement of Basic Drugs in Blood.
• UKIAFT. Forensic Toxicology Laboratory Guidelines. Science and Justice 50:166–176 (2010).