Determination of Cocaine in Biological Matrices Using Reversed Phase HPLC: Application to Plasma and Brain Tissue

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Abstract
An HPLC assay using UV detection has been developed for the analysis of cocaine in biological matrices. Samples were precipitated with perchloric acid, extracted in 5% chloroform/pentane at pH 10, and back-extracted into 0.1 N hydrochloric acid. Samples were chromatographed using a Zorbax RX-C18®, 4.6 × 250 mm, 5 μm, column with the absorbance monitored at 235 nm. The method was applied to quantification of cocaine in plasma and brain tissue of Sprague-Dawley rats following intraperitoneal administration of 15 mg/kg cocaine. The method was characterized by its extreme ease of sample preparation and ruggedness over time. The average recovery for the method was marginal; 52 and 38% for cocaine and the internal standard, respectively, in plasma and 42 and 35% of the cocaine and the internal standard, respectively, in brain. However, due to the extreme cleanliness of the sample after preparation, the limit of detection of the method for cocaine was less than 5 ng/ml in plasma and less than 5 ng/g in brain. The limit of quantification was 25 ng/ml in plasma and 25 ng/g in brain. The method was linear from 50–4000 ng/ml in both plasma and brain. Spiked quality control samples were assayed over a three month period. The inter-day coefficient of variation was 10.2% in plasma at 200 ng/ml (n=22), 11.6% at 200 ng/ml in brain (n=38) and 0.7% at 2500 ng/ml in both plasma and brain (n=23, n=43, respectively). The method would appear to be acceptable for the analysis of cocaine in both saliva and urine.

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