Simultaneous Determination of Zopiclone and Its Two Major Metabolites (N-Oxide and N-Desmethyl) in Human Biological Fluids by Column Liquid Chromatography After Solid-Phase Extraction

A reverse phase liquid chromatographic procedure with fluorescence detection for the simultaneous determination of zopiclone and its main metabolites, N-desmethylzopiclone and zopiclone-N-oxide, in serum, blood and urine is described. An aliquot (0.5–1 mL) of the sample after the addition of 0.25 mL of 250 ng/mL solution of harmane in 0.2 M NaH2PO4 as the internal standard is passed through a 1-mL BondElut C18 silica extraction column. The column is selectively washed with water and acetonitrile to remove polar, neutral and acidic compounds. The desired compounds are eluted with a 0.25 mL aliquot of a mixture of methanol + 35% perchloric acid (100:1 v/v). A 10–25 μL aliquot of the eluate is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm C18 silica particles which is eluted at ambient temperature with a mobile phase of acetonitrile - 0.1% tetramethylammonium perchlorate (17:83 v/v) adjusted to pH 3.8 with 10% perchloric acid at a flow rate of 1.8 mL/min. The peaks are detected with a fluorescence detector (ex = 320 nm, em = 520 nm). The extraction recovery of all the compounds is in the range of 90–95%. The chromatogram is clean and the desired peaks are well separated from each other and from extraneous peaks.