Automated liquid-liquid extraction (LLE) of forensic drugs from plasma

Significance of the Topic

The automated liquid–liquid extraction (LLE) of drugs from plasma addresses key challenges in bioanalytical workflows by reducing manual handling errors, improving throughput, and ensuring consistent recovery. This method is particularly relevant for forensic and clinical applications where high sensitivity, reproducibility, and minimal matrix interference are essential.

Objectives and Study Overview

This study demonstrates an automated LLE technique for screening optimal extraction conditions for forensic drugs in plasma. It aims to:

• Evaluate different solvent and buffer combinations using an autosampler workbench.
• Couple the extraction with high-performance LC–MS/MS to assess sensitivity and linearity.
• Verify reproducibility across large sample batches.

Methodology and Instrumentation

Instrumentation

• Agilent 7696A Sample Prep WorkBench for automated LLE and serial dilutions.
• Agilent 1290 Infinity LC System for chromatographic separation.
• Agilent 6490 Triple Quadrupole LC/MS System with Jet Stream ESI for detection.

Automated Extraction Workflow

• Buffers tested: pH 3.0 (1% formic acid), pH 7.0, and pH 10.0 (1% ammonium hydroxide) in 50 mM ammonium acetate.
• Organic solvents: 11 ratios of ethyl acetate/hexane (100:0 to 0:100, v/v), yielding 33 solvent–buffer combinations.
• Automated mixing, vortexing, phase separation, pipetting of the organic layer, and dilutions performed on the WorkBench; centrifugation and evaporation handled offline.
• Injection volume of 15 µL; column: Zorbax Eclipse Plus 2.1×50 mm, 1.8 µm, maintained at 40 °C.

Main Results and Discussion

Optimal Extraction Conditions

• The highest analyte peak areas were observed at pH 10.0 with 90% ethyl acetate/10% hexane.

Analytical Performance

• Calibration curve prepared by automated serial dilutions across nine levels (1–2000 pg/mL) showed excellent linearity (R² > 0.99) with a lower limit of quantification at 1 pg/mL.
• Reproducibility test on 50 plasma samples yielded an RSD of 6.4% for target analytes.

Impact of Vial Material

• Comparative analysis of glass vs. polypropylene vials revealed significantly fewer leachable contaminants when using glass vials in the Sample Prep WorkBench.

Benefits and Practical Applications

The automated LLE approach offers:

• Reduced manual labor and pipetting errors, enabling high sample throughput.
• Improved consistency and reproducibility, critical for large-scale forensic and clinical studies.
• Enhanced sensitivity, supporting detection at sub-picogram levels.
• Lower risk of contamination by using glass vials for extractions.

Future Trends and Applications

Automation and miniaturization will continue to shape bioanalytical sample preparation. Emerging trends include:

• Integration of online LLE with LC–MS/MS for real-time analysis.
• Development of more versatile workbench platforms capable of handling multiple extraction modes.
• Application to broader analyte classes, including polar metabolites and biomolecules.

Conclusion

This workbench-automated LLE method, combined with ultra-sensitive LC–MS/MS, delivers robust, high-throughput extraction of forensic drugs from plasma. The optimized protocol ensures minimal matrix effects, excellent reproducibility, and sub-picogram sensitivity, illustrating its value for bioanalytical and forensic laboratories.

References

1. Guowen Liu et al. Strategy of Accelerated Method Development for High-Throughput Bioanalytical Assays Using Ultra High-Performance Liquid Chromatography Coupled with Mass Spectrometry. Anal. Chem. 2009, 81, 9225–9232. For Forensic Use.