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Automated solid-phase microextraction and thin-film microextraction for high-throughput analysis of biological fluids and ligand–receptor binding studies

Nature Protocols volume 5, pages 140–161 (2010)Cite this article

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Abstract

This protocol describes how to perform automated solid-phase microextraction (SPME) and thin-film microextraction (TFME) in a 96-well plate format for high-throughput analysis of drugs, metabolites and any other analytes of interest in biological fluids using liquid chromatography–electrospray tandem mass spectrometry. Sample preparation time required is typically 1 min per sample; hence, the throughput achievable with automated SPME/TFME is comparable with automated 96-well liquid–liquid extraction and solid-phase extraction methods, but greater than most online solid-phase extraction methods. The technique is applicable to complex samples such as whole blood without additional pretreatment. The amount of analyte extracted by SPME/TFME is proportional to the free (unbound) concentration of the analyte; hence, SPME/TFME can be used to determine both total and free concentrations of analytes from a single biofluid sample and to perform automated ligand–receptor binding studies in order to determine binding affinity and/or overall extent of ligand binding to a complex biofluid.

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Figure 1: The Concept 96 autosampler from PAS Technology.
Figure 2: Automated SPME workflow.
Figure 3
Figure 4: Development of high-throughput SPME bioanalytical methods.
Figure 5: Evaluation of the success of coating procedure.
Figure 6: Example linear regression calibration curves for determination of free and total concentration using automated SPME.
Figure 7: Example chromatograms obtained using automated SPME for the analysis of benzodiazepines in whole blood.
Figure 8: Example binding isotherm obtained for the binding between diazepam and human serum albumin.

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Acknowledgements

The authors thank the Natural Sciences and Engineering Research Council of Canada and Supelco for financial support. The authors thank Dietmar Hein (PAS Technology) for collaboration and the design and development of the Concept 96 autosampler.

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Authors and Affiliations

  1. Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada

    Dajana Vuckovic, Erasmus Cudjoe & Janusz Pawliszyn

  2. Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA

    Florin Marcel Musteata

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  1. Dajana Vuckovic
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  2. Erasmus Cudjoe
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  3. Florin Marcel Musteata
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  4. Janusz Pawliszyn
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Contributions

D.V. developed fiber coatings and evaluated performance of Concept 96 using fiber geometry for LC-MS applications, developed high-throughput applications, performed first automated ligand-binding study and wrote the manuscript. E.C. developed and evaluated thin-film configuration and corresponding coatings of the device. F.M.M. developed the theory and experimental workflow for ligand-receptor binding studies regardless of the degree of depletion. J.P. developed the initial idea for this project and supervised the activities in all stages of instrument and experimental design.

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Correspondence to Janusz Pawliszyn.

Supplementary information

Supplementary Methods

A detailed step-by-step procedure to generate in vitro multi-point ligand-receptor binding isotherms using SPME/TFME and a PAS Concept 96 (PDF 59 kb)

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Vuckovic, D., Cudjoe, E., Musteata, F. et al. Automated solid-phase microextraction and thin-film microextraction for high-throughput analysis of biological fluids and ligand–receptor binding studies. Nat Protoc 5, 140–161 (2010). https://doi.org/10.1038/nprot.2009.180

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