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 (700-1,000 ng ml(-1)) to about 20% R.S.D. (OECD defined limit) at the lower limit of quantitation ( approximately 50 ng ml(-1)). The lower limit of quantitation is well below the low end of the therapeutic range at 350 ng ml(-1).

On-fiber derivatization for direct immersion solid-phase microextraction. Part II. Acylation of amphetamine with pentafluorobenzoyl chloride for urine analysis.

On-fiber derivatization was used for solid-phase microextraction (SPME) in order to increase the detectability and extractability of drugs in biological samples. Amphetamine, which was used as a model compound, was derivatized with pentafluorobenzoyl chloride (PFBCl) and subjected to gas chromatography with electron capture or mass spectrometric detection. Extraction was performed by direct immersion of a 100 microm polydimethylsiloxane-coated fiber into buffered human urine. On-fiber derivatization was performed either after or simultaneously with extraction. The former procedure gave cleaner chromatograms but the latter turned out to be superior with respect to linearity and repeatability. For the on-fiber derivatization of amphetamine an excess of reagent is required. Because a considerable part of the PFBCl loaded on to the fiber is used up by reaction with matrix compounds and water, a reagent loading time of 5 min was needed to obtain a linear range (r = 0.9756) from 250 pg mL(-1) to 15 ng mL(-1). Due to an interfering matrix compound, the limit of detection was also found to be dependent on the reagent loading time, i.e., the limit of detection for a PFBCl loading time of 5 min is 250 pg mL(-1) whereas that for a 1 min loading time it is 100 pg mL(-1). The relative standard deviation (n = 7) of the method was about 11% at an amphetamine concentration of 1 ng mL(-1). The applicability of the method for the determination of drugs in biological samples is shown.