Radiometric solvent-partitioning assay for screening cocaine hydrolases and measuring cocaine levels in milligram tissue samples

To permit rapid screening and characterization of novel cocaine hydrolases, as well as accurate measurement of cocaine levels in small samples of tissue, a radiometric assay was developed. The assay is based on selective, organic solvent partition of [H-3]benzene-labeled cocaine or of [H-3]benzoic acid liberated during enzymatic hydrolysis. With dilute samples the assay can be conducted entirely in scintillation vials and quantitated by addition of appropriate aqueous buffer and toluene-based fluor, making phase separation unnecessary. In this way, several hundred samples can be assayed in an afternoon, nanogram quantities of enzyme can be characterized without prior purification, and cocaine concentrations can be accurately measured in milligram samples of tissue after administration of [H-3]cocaine in vivo. (C) 2002 Elsevier Science (USA). All rights reserved.