Development of a fluorescence polarization immunoassay for the routine detection of N-desmethylzopiclone in urine samples

Abstract

A polarization fluoroimmunoassay was developed for the detection of N-desmethylzopiclone in urine and the reagents were adapted for use on the Vitalab Eclair analyser. Therefore, N-fluoresceinthiocarbamyldes-methylzopiclone was synthesized as a fluorescent tracer and used in combination with an existing pool of antibodies, raised against the hemisuccinyl derivative of N-desmethylzopiclone. The optimization of the assay was performed on a semi-quantitative basis, relative to a cut-off value of 300 ng ml^–1. No significant interference was observed from a selection of existing drugs and endogenous compounds. The minimum detectable dose of the immunoassay was calculated to be 30 ng ml^–1(pooled-variance t-distribution, p= 0.01, degrees of freedom = 10). Intra-and inter-assay relative standard deviations were <10 and <12%, respectively. For the confirmation of positive samples, an established reversed-phase HPLC technique, in combination with fluorescence detection, was used. The combined screening/confirmation procedure was applied to cumulative excretion samples, after ingestion of one tablet of Imovane (zopiclone, 7.5 mg).