Fully Automated Method using Dispersive Liquid Liquid Micro Extraction (DLLME) for Extractable and Leachable studies

Significance of Automated DLLME for Extractables and Leachables Studies

Dispersive Liquid–Liquid Microextraction (DLLME) is a rapid, high-enrichment sample preparation technique used to isolate trace organic compounds from complex pharmaceutical matrices. Automating this workflow reduces manual handling, lowers solvent consumption and boosts laboratory throughput—critical factors for extractables and leachables testing in drug development and quality control.

Study Objectives and Overview

This technical note outlines a fully automated DLLME protocol developed jointly by GlaxoSmithKline (GSK) and Anatune Ltd. Its aim is to integrate mixing, centrifugation and micro-extraction steps on a robotic platform, enabling overnight operation with minimal analyst intervention.

Methodology

A biopharmaceutical drug product (2 mL) was spiked with a panel of hydrophobic analytes and a deuterated internal standard. Protein precipitation was achieved by adding 4.5 mL isopropanol and vortexing. After an initial centrifugation step to remove precipitated protein, 1.5 mL of supernatant was transferred to a high-recovery vial. Chloroform (75 µL) and water (4 mL) were added sequentially, each followed by vigorous mixing to form a fine emulsion. A final centrifugation separated the chlorinated solvent layer, from which 35 µL was withdrawn for GC/Q-TOF analysis.

Used Instrumentation

• Dual Head GERSTEL MPS robotics
• GERSTEL mVORX automated vortex mixer
• Anatune CF-200 centrifuge module
• Agilent 7200 GC-Q-TOF system

Main Results and Discussion

Precision studies at 0.33 µg/mL yielded relative standard deviations below 10% for all target analytes. Linearity experiments demonstrated correlation coefficients (r) above 0.98 across a three-order dynamic range. Recoveries ranged from approximately 92% to 132%, confirming negligible matrix effects. Typical chromatograms exhibited sharp, baseline-resolved peaks even at trace concentration levels.

Practical Benefits of the Automated DLLME Method

• Significant reduction in hands-on time, allowing analysts to perform multiple tasks or schedule unattended overnight runs.
• Minimized solvent and sample consumption compared to manual protocols.
• Improved reproducibility through standardized robotic handling and centrifugation.
• Scalable throughput by processing multiple vials in parallel.

Future Trends and Potential Applications

Ongoing developments may include the adoption of greener dispersive solvents, integration with laboratory information management systems for data tracking, and the extension of automated DLLME to other complex matrices such as food, environmental or biological samples. Miniaturization of vial formats and coupling with high-resolution mass spectrometry could further enhance sensitivity and reduce resource usage.

Conclusion

The fully automated DLLME workflow described here combines robust sample cleanup with high-efficiency microextraction on a single robotic platform. It addresses key challenges in extractables and leachables analysis by delivering reproducible, high-throughput results with minimal solvent consumption and analyst involvement.