Analysis of cocaine and cocaine metabolites by high pressure liquid chromatography

A high pressure liquid chromatography procedure was developed for analysis of cocaine and two metabolites (norcocaine and benzoylecgonine) in biological fluid. Following extraction with chloroform/Isopropanol (3/2), chromatography is performed with a mu-Bondapak C-18 reverse phase column eluted with water/acetonitrile/methanol (8/1/1) containing 1% acetic acid and 0.3 M EDTA. Ultraviolet detection of individual compounds was made at 235 nm and quantitation was based on peak height ratio to internal standard. Detection limit based on 0.5 mL of sample is less than 1 microgram/mL. Confirmation of the individual compounds was made by direct probe mass spectral analysis of the eluted peaks and comparison to known standards.