Quantitative determination of clozapine in serum by instrumental planar chromatography

An instrumental planar chromatographic (HPTLC) method for quantitative analysis of clozapine in human serum was developed and validated. Clozapine was extracted with n-hexane-isoamyl alcohol (75:25 v/v). The chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of chloroform and methanol (9:1 v/v) as mobile phase. Quantitative analyses were carried out by densitometry at a wavelength of 290 nm. The method was linear between 10 and 100 ng/spot, corresponding to 0.10 and 1.00 ng/microL of clozapine in human serum after extraction process and applying 10 microL to the chromatographic plates. The method correlation coefficient was 0.999. The intra-assay variation was between 2.10 and 3.33% (n = 5) and the interassay was between 2.67 and 4.44% (n = 9). The detection limit was 0.03 ng/microL, and the quantification limit was 0.05 ng/microL. The method proved to be accurate, with a recovery between 97.00 and 99.00%, with an RSD not higher than 7.22%, and was selective for the active principle tested. This method was successfully applied to quantify clozapine in patient serum samples. In conclusion, the method is useful for the quantitative determination of clozapine in serum.