Development of a stereospecific radioimmunoassay for the analysis of zopiclone and metabolites in urine

Abstract

A sensitive and specific radioimmunoassay has been developed, allowing the stereospecific detection of nanogram amounts of (+)- and (−)-enantiomers of zopiclone and its major metabolites in urine, without prior extraction or purification. Antisera were obtained from two series of four rabbits, immunized with optically pure (+)- and (−)- N-hemisuccinyldesmethylzopiclone, conjugated to bovine serum albumin according to the active ester method. The assay was stereospecific, allowing discrimination between the two enantiomers of N-desmethylzopiclone with mutual cross-reactivities below 2%. Substantial cross-reaction was observed with the parent compound, although lower than expected, and to a lesser extent with the N-oxide metabolite. A selection of hypnotics, anxiolytics, antidepressants and some other widely used drugs did not interfere with the assay (<0.1%), when tested at a concentration level of 10 μg/ml. The (<0.1%), when tested at a concentration level of 10 μg/ml. The sensitivity of the assay was 50 pg/ml and 10 pg/ml for the (+)- and (−)-enantiomers, respectively. The binding assay described here was used to evaluate the stereoselective excretion pattern of zopiclone. Analysis of cumulative excretion samples from a volunteer revealed a mean metabolic excretion ratio (+)/(−) of 2.2, ranging from 1.7 (7 h) to 4.4 (36 h). A mean excretion ratio (+)/(−) of 2.5 ± 1 was calculated after analysis of urine samples from 20 patients receiving zopiclone as a hypnotic daily.