Automation Of Liquid-liquid Extractions In A 96-well Format Using The Packard Multiprobe #3415

S. Sitaraman, T. Grever and R. Hurd
Bioanalytical Systems, Inc. ¢ 2701 Kent Avenue ¢ West Lafayette, IN 47906 USA

ABSTRACT

Purpose

To assess the feasibility of automating liquid-liquid extractions using a Packard MultiPROBE.

Methods

Two manual liquid-liquid extractions were transferred to the 96-well format using the Packard MultiPROBE. To test the general utility of this approach, assays based on extraction solvents more dense (theophylline) and less dense (naproxen) than water were chosen. After a single extraction and evaporation, the samples were reconstituted and injected onto an HPLC system using a 96-well autosampler.

Results

Both compounds yielded successfully validated methods. The resulting assays were sensitive, selective, accurate and precise. Inter-assay precision and accuracy for theophylline ranged from 3.1% to 7.5%, and - 0.3% to 1.3% respectively. Inter-assay precision and accuracy for naproxen ranged from 3.7% to 10.7%, and - 5.3% to 7.2% respectively.

Conclusions

General strategies for automating liquid-liquid extractions have been achieved using the 96-well format. The resulting assays drastically reduce sample preparation time (at least 3-fold faster when compared to manual extraction), decrease material and labor costs and increase throughput in clinical studies.

PURPOSE - Designing the Feasibility and Ease of Automation

  • Two commonly available drugs (naproxen and theophylline) used to test feasibilty and ease of automating liquid-liquid extraction
  • Test general utility of approach in using solvents more and less dense than water
  • Decrease the cumbersome nature of traditional liquid-liquid extractions
  • Automation will help reduce sample preparation time, decrease material and labor costs and increase throughput in clinical studies

METHOD - Validation Scheme

  • Fresh Calibration Line
  • QCs at the limits of detection (n=6)
  • 3 day Inter-assay precision and accuracy

General Assay Procedure-Naproxen

  • Load Samples (calibrators, QCs, unknowns) onto 96-well plate
  • Add buffer, ISTD, and extraction solvent to each well
  • Vortex plate, centrifuge and aspirate lower aqueous layer to waste
  • Evaporate the organic and reconstitute before injecting onto HPLC system using a 96-well autosampler

Calibration Standard Statistics - Naproxen

Representative Low Calibrator Chromatogram for Naproxen

Representative Blank Chromatogram for Naproxen

Inter-Assay Quality Control Sample Statistics - Naproxen

Typical Calibration Line for Naproxen

General Assay Procedure- Theophylline

  • Load Samples (calibrators, QCs, unknowns) onto 96-well plate
  • Add buffer, ISTD, and extraction solvent to each well
  • Vortex plate, centrifuge and aspirate lower organic layer to another 96-well plate
  • Evaporate the organic and reconstitute before injecting onto HPLC system using a 96-well autosampler

Calibration Standard Statistics- Theophylline

Representative Low Calibrator Chromatogram for Theophylline

Representative Blank Chromatogram for Theophylline

Inter-Assay Quality Control Sample Statistics - Theophylline

Typical Calibration Line for Theophylline

Conclusions

  • Faster - At least 3 - fold faster when compared to manual extraction
  • Analyst time used more effectively
  • Increased throughput of clinical samples
  • Traditionally liquid-liquid extractions associated with being cumbersome, automation reduces many repetitive activities
  • Decreased material and labor costs
  • Proves the utility of this approach for solvents that are either more or less dense than water