Determination of cocaine, norcocaine, benzoylecgonine and ecgonine methyl ester in rat plasma by high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic method with ultraviolet detection at 235 nm is described for the determination of cocaine and its metabolites benzoylecgonine, norcocaine and ecgonine methyl ester in rat plasma, collected during toxicity studies. Following simultaneous solid-phase extraction of all analytes and the internal standard tropacocaine, cocaine, benzoylecgonine and norcocaine were separated on a C₁₈ column. Ecgonine methyl ester and cocaine were separated on coupled cyanopropyl and silica columns, following derivatization of ecgonine methyl ester to p-fluorococaine. The extraction efficiencies of these compounds from plasma ranged from 78 to 87%, while the limits of detection ranged from 35 to 90 ng/ml. The assay was linear from 300 to 5000 ng/ml, and the within-day precision 2 to 8% over this concentration range.