A rapid, sensitive assay for cocaine and its metabolites in biological fluids using solid-phase extraction and high-performance liquid chromatography.

An improved method for the simultaneous determination of cocaine and its metabolites, benzoylecgonine (BE), norcocaine, and ecgoninemethylester (EME), in rat plasma and urine is described. Following derivatization of EME to p-fluorococaine, chromatography was performed on two high-performance liquid chromatography (HPLC) columns in series (5-microm spheric C8 and 5-microm cyanopropyl) using a mobile phase containing acetonitrile/HPLC water/trifluoroacetic acid (28:72:0.1) with bupivacaine as an internal standard. Quantitation limits were 25 ng/mL for cocaine, BE, and norcocaine and 50 ng/mL for EME using 300-500 microL rat plasma and 500 microL of rat urine. The assay was linear from the limit of quantitation to 2000 ng/mL for cocaine and its metabolites in both plasma and urine samples. Because this method uses a small amount of sample (300 microL plasma or 500 microL of urine), it is applicable to study of the pharmacokinetics and disposition of cocaine and its major metabolites.