TY - JOUR
T1 - Radiometric solvent-partitioning assay for screening cocaine hydrolases and measuring cocaine levels in milligram tissue samples
AU - Brimijoin, Stephen
AU - Shen, Maryann L.
AU - Sun, Hong
N1 - Funding Information:
This work received financial support from the Mayo Foundation for Medical Research. We thank Dr. Oksana Lockridge, Eppley Institute, University of Nebraska, for the gift of purified human BChE. We also thank Thomas Kollmeyer and Giewn J. Park for suggestions regarding alkaline extraction of cocaine.
PY - 2002/10/15
Y1 - 2002/10/15
N2 - 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 [3H]benzene-labeled cocaine or of [3H]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 [3H]cocaine in vivo.
AB - 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 [3H]benzene-labeled cocaine or of [3H]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 [3H]cocaine in vivo.
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U2 - 10.1016/S0003-2697(02)00238-5
DO - 10.1016/S0003-2697(02)00238-5
M3 - Article
C2 - 12413452
AN - SCOPUS:6444244966
SN - 0003-2697
VL - 309
SP - 200
EP - 205
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -