Determination of melatonin in biological fluids in the presence of the melatonin agonist S 20098: comparison of immunological techniques and GC-MS methods.

Immunoassays were investigated for the determination of melatonin in biological samples in the presence of a naphthalenic structural analogue S 20098, which is currently under development as a melatonin agonist. The lack of specificity of commercially available antibodies in the presence of closely related molecules led us to develop an LC-RIA procedure with a quantification limit set at 15 pg/ml(-1). Because this technique was not sensitive enough and difficult to use on a routine basis, a more sensitive GC-MS technique was developed. This method involved automated solid-phase extraction (plasma) or liquid-liquid extraction (saliva), derivatization of the indolic moiety and GC separation with an automated switching device before MS detection. The method was validated over the range 1-100 pg/ml(-1), with a quantification limit set at 1 pg/ml(-1) in human plasma and saliva. Intra-assay and inter-assay precision and accuracy were within 16% for all concentrations investigated and each biological matrix. The stability of melatonin in plasma and saliva under various storage conditions was also determined. The specificity of the assay for the analysis of melatonin in the presence of S 20098 and its metabolises was demonstrated. The method was subsequently applied for the determination of endogenous melatonin concentrations in plasma and saliva samples from clinical studies performed with S 20098 to provide pharmacodynamic data.