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March 3, 2026Open Access

Evaluation of the stability and cross-reactivity of zopiclone and eszopiclone using immunoassay kits

Key Points

The study evaluates the stability and cross-reactivity of zopiclone and eszopiclone in urine samples.
Used immunoassay urine screening kit for ZOP analysis.
Conducted liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS).
Analyzed spiked urine samples for cross-reactivity confirmation.
Compared quantitative results from clinical urine samples.
Long-term storage of urine samples can lead to false-negative results for ZOP.
Metabolites of ZOP and EZOP were identified, indicating their impact on testing validity.
Cross-reactivity confirmed for both zopiclone and eszopiclone in urine tests.

Abstract

Zopiclone (ZOP) and eszopiclone (EZOP) are hypnotics belonging to the cyclopyrrolone class, which are metabolized to zopiclone-N-oxide (ZOPNO) and N-desmethylzopiclone (NDZOP), respectively, and finally to 2-amino-5-chloropyridine (2A5C) after long-term storage, freezing, and conservation. This study aimed to examine the stability of ZOP and EZOP using a commercially available immunoassay urine screening kit for ZOP. ZOP and EZOP, as well as their metabolites and 2A5C, were analyzed in urine using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Spiked urine samples were used to confirm cross-reactivity for the immunoassay test and to compare the quantitative results of the clinical urine samples. The results indicate that urine samples stored for an extended period or altered postmortem may lead to false-negative results for ZOP, providing important information for interpreting results in cases involving ZOP.

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