GC-MS identification and UHPLC determination of clozapine in blood samples

The main purpose of the work was to optimize a GC-MS screening procedure for identification of low blood clozapine levels and to develop and validate a simple HPLC technique for its determination in biological samples. Gas chromatography in tandem with mass spectrometry, combined with pre-analytical purification (mixed-mode solid-phase extraction) and derivatization (PFPA acylation) has been chosen as the method for analytical identification. It has been shown that derivatization procedure is vital component of clozapine and its metabolites identification by GC-MS. Reverse-phase liquid chromatography has been used as a quantitative instrument. HPLC determination has been done under isocratic conditions on Zorbax Extend-C18 column (150×4.6 mm, 5 μm); mobile phase – acetonitrile: phosphate buffer pH 5.2 = 30:70 v/v; flow-rate of 1.0 mL/min and UV detection at 254 nm. The method has been validated in human plasma/serum samples (75% recovery). Limit of quantitation (LOQ) for clozapine in blood samples has been estimated to 25 ng/mL, along with excellent linearity (Pearson\u0027s r = 0.9999 over 25 to 2000 ng mL–1 range) and precision (RSD 2.9%). A clinical case reported demonstrates the importance of clozapine confirmation and level determination, as in cases of combined acute intoxication it is possible to have a major discrepancy between anamnestic data and clinical picture. A possible drug interaction between clozapine, sertraline and mirtazapine, resulting in toxicity escalation due to their concomitant use has been reported.