Development of a High-Performance Liquid Chromatography Assay Using Relative Molar Sensitivity for the Simultaneous Quantification of Clozapine and <i>N</i>-Desmethylclozapine Concentrations in Human Plasma

The LM1010 high-performance liquid chromatography (HPLC) system determines the plasma concentrations of clozapine using an absolute calibration method. In this study, the LM1010 HPLC method using relative molar sensitivity (RMS) was developed to quantify the plasma concentrations of clozapine and N-desmethylclozapine. The RMS values were calculated from the ratio of the peak area for clozapine or N-desmethylclozapine to that of highly pure and stable carbamazepine used as an RMS reference. The relative standard deviation of the RMS determined by three different laboratories was 1.723％ for clozapine and 1.821％ for N-desmethylclozapine, suggesting that the mean RMS values of 1.843 and 1.639, respectively, can be used for quantitative analysis in different laboratories. In a retrospective analysis of data obtained from a previous study using a conventional LM1010 method, Deming regression analyses showed a high correlation between the data calculated based on RMS values and the data obtained by HPLC using an internal standard method (slope = 0.942 and 0.974 for clozapine and N-desmethylclozapine, respectively). This result was equivalent to the regression between the results obtained from the LM1010 method using absolute calibration and the HPLC method. A prospective analysis of the RMS method showed strong correlations between RMS and HPLC results (slope = 0.922 and 1.037 for clozapine and N-desmethylclozapine, respectively) and was equivalent to correlations between results obtained from the LM1010 using absolute calibration and the HPLC methods. These findings suggest that the RMS method is suitable for the quantitative assay of plasma concentrations of clozapine and N-desmethylclozapine and offers an alternative to the current absolute calibration method.