Photolytic derivatization for improved LCEC determinations of pharmaceuticals in biological fluids

Although electrochemical (EC) methods have been demonstrated to be sensitive and selective, wide use of EC detection in high performance liquid chromatographic (HPLC) assays in forensic and clinical toxicology laboratories has not been forthcoming. This fact is due to the general difficulty involved with the use of reductive EC detection methods, as well as to the lack of EC response in either the oxidative or reductive mode, for a number of classes of drugs having substantial clinical and forensic importance. The use of an on-line, post-column, continuous photolytic derivatization step, followed by conventional oxidative amperometric detection, alleviates many of these problems. In this report, the use of HPLC-photolysis-EC (HPLC-h nu-EC) for the trace determination of a number of controlled substances in biological fluids is presented. Following system optimization, the determination of phenobarbital, cocaine, methylphenidate, and several 1,4-benzodiazepines (and metabolites) is linear over three orders of magnitude. In addition, HPLC-h nu-EC offers a sensitive approach for these compounds, in that limits of detection (LODs) are all below 1 microgram/ml, ranging from 1 ng/ml to 750 ng/ml. The validity of this newer method is demonstrated in collaborative studies involving the trace determinations of phenobarbital in human serum, and chlordiazepoxide and its major metabolite, norchlordiazepoxide, in human urine. Finally, the authors' view of the role of HPLC-h nu-EC in the clinical and forensic toxicology laboratory is presented.