High throughput therapeutic drug monitoring of clozapine and metabolites in serum by on-line coupling of solid phase extraction with liquid chromatography–mass spectrometry

The characteristics of automated on-line solid phase extraction with liquid chromatography–mass spectrometry (SPE-LC–MS) are very amenable for flexibility and throughput in therapeutic drug monitoring (TDM). We demonstrate this concept of automated, on-line SPE-LC–MS for the analysis of clozapine and metabolites (desmethylclozapine and clozapine-N-oxide) in serum. Method development, optimisation and validation are described and a comparison with previously published methods for the determination of clozapine and metabolites in serum and plasma is made. Optimisation of chromatographic and SPE conditions for increased throughput resulted in SPE-LC–MS cycle times of only about 2.2 min, demonstrating the great potential of automated on-line SPE-LC–MS for TDM. The new method is shown to be clearly favourable, in particular in terms of ease of sample handling, throughput and detection limits. Recovery is essentially quantitative. Detection limits are at about 0.15–0.3 ng ml−1, depending on the ionisation source used. Calibration follows a quadratic model for clozapine and its N-oxide and a linear model for the desmethyl metabolite (all cases: R > 0.99). Accuracy, evaluated at three concentration levels spanning the whole therapeutic range, shows that bias is less than 10%. Precision (intra – and inter assay) ranges from about 5% R.S.D. at the high end of the therapeutic range (700–1000 ng ml−1) to about 20% R.S.D. (OECD2 defined limit) at the lower limit of quantitation (∼50 ng ml−1). The lower limit of quantitation is well below the low end of the therapeutic range at 350 ng ml−1.