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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