High-performance liquid chromatographic determination of cocaine and its metabolites in serum microsamples with fluorimetric detection and its application to pharmacokinetics in rats

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Abstract

A sensitive, selective and simple HPLC method with fluorimetric detection is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). Chromatographic separation is achieved on a Hypersil BDS C18 column (100×2.1 mm, 5 μm) with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.6, containing 1.0·10−4 M tetrabutylammonium phosphate (14:10:76, v/v/v). The detection limit (0.5 ng/ml) for all the compounds, using direct fluorometric detection operated at excitation and emission wavelengths of 230 and 315 nm, respectively, was approximately five-times lower than that of using a UV detector operated at 235 nm. The effects of ratio of 2-propanol to chloroform in extraction solvents on the recovery and precision for cocaine and its metabolites were systematically examined. The method was used to study the pharmacokinetics of cocaine after administration of intravenous 2 mg/kg and oral 20 mg/kg doses.

Introduction

Cocaine is a potent psychomotor stimulant and a major drug of abuse in the USA [1]. Methods for analyzing cocaine and its metabolites are of increasing importance for the purposes of defining the level of toxicity and lethality, or screening for cocaine use. Cocaine is rapidly distributed, deactivated by extensive metabolism, and then excreted (Fig. 1). Benzoylecgonine, one of the main metabolites, is formed by hepatic carboxylesterase or spontaneous hydrolysis [2]. Although benzoylecgonine is not a pharmacologically active metabolite, it is of great forensic and analytical interest because of its long half-life. Norcocaine is formed by N-demethylation of cocaine and is the only cocaine metabolite found to have in vivo pharmacological activity in animals and humans [3], [4]. Benzoylnorecgonine is the hydrolysis product of norcocaine.

It is essential to determine the concentration of a drug and its active metabolites with precision before investigating concentration–effect relations to partition drug effects into pharmacokinetic and pharmacodynamic components [5], [6], [7]. High-performance liquid chromatography (HPLC) has been used extensively for the determination of cocaine and its metabolites in biological samples [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. Most HPLC assays for cocaine with 4.6 mm I.D. columns required a large sample size (e.g., 0.2–2 ml) to attain a desired sensitivity [8], [9], [10], [11], [12], [13]; these methods are more suitable for clinical evaluation, where the sample volume is not a limiting factor. However, in research with small laboratory animals (e.g., rats), sample size is a critical consideration, especially in pharmacokinetic studies where repeated blood sampling is necessary to trace temporal changes in blood levels of the drug and its metabolites. In past research, to determine cocaine and its metabolites in rats [14], [15], [16], we developed the most simple and sensitive HPLC–UV methods possible for serum microsamples (50 μl) to accommodate repeated sampling using commercially available 2 mm I.D. columns. These smaller bore columns enable one to not only increase sensitivity, but also reduce solvent consumption by up to 80%. The first aim of the present study was to increase detection sensitivity further for cocaine and its metabolites by modifying our previous HPLC–UV method [16] with a fluorescence detector. The fluorimetric detection based on the weak native fluorescence of the benzene ring present in the molecules of cocaine and its metabolites has been successfully used for determining cocaine concentrations in human plasma and hair samples [9].

In a previous study [14], we found 1 ml of extraction solvent [ethanol–chloroform (17.5:82.5, v/v)] was sufficient for recovery of cocaine and its metabolites from serum samples. The ratio of ethanol to chloroform was known to be critical in determining the extent of recovery for benzoylecgonine and benzoylnorecgonine, which are insoluble in chloroform, but soluble in ethanol due to their amphoteric nature. Therefore, the second aim of the study was to systematically investigate the effects of the ratio of alcohol to chloroform in an extraction solvent on the recovery and precision for each of the four compounds, thus, facilitating the choice of an optimal solvent for extracting compounds of interest. This method was hereby applied to evaluate the pharmacokinetics of cocaine after intravenous (i.v.) and oral (p.o.) cocaine administration.

Section snippets

Instrumentation

The HPLC system consisted of a Perkin-Elmer (PE) 200 LC pump coupled to a Perkin-Elmer autosampler ISS-200 (Norwalk, CT, USA); both units were controlled by a PE Nelson 600 Series Link interface. A Hewlett-Packard HP 1100 fluorescence detector (Waldbronn, Germany) was operated at the excitation and the emission wavelengths of 230 and 315 nm, respectively. The separation was performed on a Hypersil BDS C18 column, 100 mm×2.1 mm I.D., 5 μm particle size (Hypersil, MA, USA) with a 2-μm Rheodyne

Method evaluation

During method development, a Brownlee C18 column, 100 mm×2.1 mm I.D., 5 μm particle size (Applied Biosystems, Foster City, CA, USA) employed in our previous HPLC method [16] was used to compare the sensitivity of fluorimetric and UV detection for cocaine and its metabolite. Fig. 2A and B showed peak height for the four compounds increased markedly with fluorescence detector compared to UV detector operated at 235 nm. The performance of the Hypersil column described in the Experimental section

Acknowledgements

This research was supported by Grant R01 DA05305, from the National Institute on Drug Abuse, USA.

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