Quantitative determination of cocaine, cocaethylene (ethylcocaine), and metabolites in plasma and urine by high‐performance liquid chromatography

A sensitive HPLC assay was developed to quantitate the relatively nonpolar compounds cocaine, cocaethylene (ethylcocaine), norcocaine, and norcocaethylene, as well as the relatively polar metabolites benzoylecgonine and benzoylnorecgonine, in rat plasma and urine. The assay in plasma employed two successive liquid–liquid extractions and separate injections onto two different columns (C₈ and C₈) to separate and quantitate the relatively polar and nonpolar compounds. In urine, the procedure employed a liquid–liquid extraction followed by solid‐phase extraction (C₈ light–load cartridges) and two separate injections as for plasma. The UV absorbance of the effluent was monitored at 235 nm. Linear standard curves were obtained over the concentration ranges of 25 to 1000 ng/mL and 5 to 250 ng/mL in plasma and urine, respectively. The inter‐ and intraday coefficients of variation for the assay of all compounds in plasma and urine were <18% at low concentrations (12.5–100 ng/mL) and <12% at high concentrations (125–250 ng/mL). There was no degradation of these compounds during the extraction procedure or during 2 months of storage at −20°C. The quantitation limits for the assay of the relatively nonpolar and polar compounds in plasma were 25 (2.5 ng in 0.1 mL) and 50 ng/mL (5 ng in 0.1 mL), respectively. For the assay in urine, the quantitation limits were 5 (2.5 ng in 0.5 mL) and 12.5 ng/mL (6.25 ng in 0.5 mL) for the assay of the relatively nonpolar and polar compounds, respectively. The methods have been applied to quantitate those compounds in rat plasma.