LIQUID-CHROMATOGRAPHIC DETERMINATION OF CLOZAPINE AND N-DESMETHYLCLOZAPINE IN HUMAN SERUM USING SOLID-PHASE EXTRACTION

A 0.5-ml aliquot of a serum sample, after the addition of a 50-mu l aliquot of a 5 mu g/ml solution of amoxapine as the internal standard, is vortex-mixed with 0.5 mi of acetonitrile and centrifuged. The supernatant is applied to a 1-ml BondElut C-18 silica extraction column-conditioned with subsequent washings with 1 M HCl, methanol and water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 15-mu l aliquot of the eluate is injected onto a 150 x 4.6 mm I.D. column packed with 5-mu m C-8 silica particles and eluted at ambient temperature with a mobile phase of 0.1% tetramethylammonium perchlorate-acetonitrile (73:27, v/v) adjusted to pH 4.2 with 10% perchloric acid. The peaks are detected with an absorbance detector at 245 nm.