Determination of clozapine and its two major metabolites in human serum by liquid chromatography using ultraviolet detection

A new, simple, reverse phase, and highly reproducible HPLC-UV method that has all the comprehensive features of good column, mobile phase, internal standard and extraction has been developed for the assay of clozapine (CLZ), norclozapine (NCLZ) and clozapine-N-oxide (CLZNO) in human serum. The method is very easy to adapt, overcomes the problems of earlier methods and is very economical. Amoxapine is the internal standard. All three analytes are extracted from alkaline serum using ethyl acetate and the absolute extraction efficiency is similar to 95%. The UV detector is set at 230 nm. This mobile phase is a mixture of phosphate buffer (0.05M, pH 2.7), acetonitrile and methanol (62:20:18 v/v) and contains 2.5 mt triethylamine per liter of solution. The detection limit of the method is 2 ng/mL for CLZ and NCLZ and 4 ng/mL for CLZNO. The mean CV for the method is 5%.