Determination of clozapine and its metabolite, norclozapine in various biological matrices using high-performance...

To develop and validate an HPLC method for the quantitation of clozapine and its metabolite, norclozapine in human plasma, rat plasma, and the various human plasma lipoprotein fractions.

Using liquid-liquid extraction, clozapine, and norclozapine are extracted from the biological matrix with MTBE. After concentration, the residue was reconstituted in 1mM HCl and injected on to a C6 Phenyl column (3 microm, 2.0 x 150 mm). Mobile phase was 10 mM ammonium acetate, pH 5--acetonitrile--methanol (5:3:2, v/v/v). Loxapine served as the internal standard. Absorbance was measured at 254 nm.

Quantitation limits for clozapine and norclozapine was 100 ng/mL and 50 ng/mL, respectively. Recovery for both clozapine and norclozapine was near 100%. Percent accuracy for intraday variability in human plasma, rat plasma, and human TRL, HDL, LDL, and LPDP lipoprotein fraction was between 92 to 113% for both analytes. Intraday precision for the same matrices was less than 9% CV for both analytes. Percent accuracy and precision for interday variability in human plasma was 97 to 103% and less than 10% CV, respectively. Samples were stabile in the autosampler for 80 h at 10 degrees C and on the benchtop for 2 h. It should be noted, Clozapine-N-oxide, which is a known metabolite of Clozapine, was not determined since it is not clinically active.

This method is a simple, fast and robust HPLC assay for the determination of clozapine and norclozapine in various matrices and lipoprotein fractions.