Quantitation of zopiclone and desmethylzopiclone in human plasma by high-performance liquid chromatography using fluorescence detection

Abstract

A simple, reliable HPLC method using fluorescence detection (excitation 307 and emission 483 nm) was developed and validated for simultaneous quantitation of zopiclone and its metabolite desmethylzopiclone in human plasma. Following a single-step liquid-liquid extraction, the analytes and internal standard (zaleplon) were separated using an isocratic mobile phase on a reversed-phase C 18 column. The lower limit of quantitation was 3 ng/mL for zopiclone and 6 ng/mL for desmethylzopiclone with a relative standard deviation of less than 5%. A linear dynamic range of 3-300 ng/mL for zopiclone and of 6-500 ng/mL for desmethylzopiclone was established. This HPLC method was validated with between-batch precision of 1.7-4.2% and 3.2-7.5% for zopiclone and desmethylzopiclone respectively. The between-batch accuracy was 99.4-111.5% and 101.6-104.8% for zopiclone and desmethylzopiclone, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zopiclone and desmethylzopiclone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days' storage in a freezer. This validated method is simple and repeatable enough to be used in pharmacokinetic studies.

Zopiclone degradation in biological samples : Characteristics and consequences in forensic toxicology

“…Freeze-thaw stability tests verified that ZOP was stable in spiked human plasma for three freeze-thaw cycles [118,129] but stability for five freezethaw cycles has also been reported [131]. Short-term or in process stability tests give no evidence of degradation in plasma when quality control samples were stored at room temperature for 12 h [131] or 24 h [129]. No loss of ZOP was detected when blood samples (plasma) was stored at 4-8°C for less than 6 h. The concentration of ZOP in blood was reduced by 25% and 29% for the (-)-and (+)-enantiomers, respectively, after 20 h storage at ambient temperature [119].…”

“…Several analytical methods, including high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS), have been developed for the identification and quantification of ZOP in whole blood, serum and/or plasma [118,[120][121][122][123][124][125][126][127][128][129][130][131][132][133][134][135][136]. Some assays (HPLC, LC-MS, LC-MS/MS) have also been developed to separate and determine ZOP [127,[137][138][139] and/or its metabolites (NDZOP and ZOPNO) in urine [140][141][142].…”

“…A couple of methods have been developed to include also the analysis of ZOP metabolites NDZOP and/or ZOPNO in plasma. One method (HPLC) has been reported to detect ZOP and its major metabolite NDZOP [129], and the other both of metabolites together with the parent drug [131]. Stereo specific methods [LC, capillary electrophoresis (CE), radioimmunoassay (RIA), HPLC, LC-MS/MS] have been developed to separate the enantiomers in urine [143][144][145] and in plasma [119,[146][147][148].…”

“…Studies of the stability of ZOP have also been carried out from stability experiments in connection with method development and validation. Long-term stability tests with spiked human plasma showed that ZOP was stable for one month [118,129], for 74 days [131] and for six months [119] when stored at -20°C or lower. Freeze-thaw stability tests verified that ZOP was stable in spiked human plasma for three freeze-thaw cycles [118,129] but stability for five freezethaw cycles has also been reported [131].…”

“…Long-term stability tests with spiked human plasma showed that ZOP was stable for one month [118,129], for 74 days [131] and for six months [119] when stored at -20°C or lower. Freeze-thaw stability tests verified that ZOP was stable in spiked human plasma for three freeze-thaw cycles [118,129] but stability for five freezethaw cycles has also been reported [131]. Short-term or in process stability tests give no evidence of degradation in plasma when quality control samples were stored at room temperature for 12 h [131] or 24 h [129].…”