Simultaneous Determination of Thioridazine and Its Metabolites by UHPLC–MS/MS With Application to a Pharmacokinetic Study in Rats

To develop an LC-MS/MS method for simultaneous quantification of thioridazine and its metabolites in rat plasma, samples were prepared by protein precipitation and filtration using thioridazine-d3 as an internal standard. Chromatographic separation was achieved on a reversed-phase C18 UPLC column with 0.1% formic acid in acetonitrile and 10-mM ammonium acetate in water at 0.7 mL/min. Quantification was performed using multiple reaction monitoring in positive ion mode with the following precursor-product ion transitions: m/z 371.1 → 126.1 (thioridazine), m/z 387.1 → 126.1 (mesoridazine), m/z 403.1 → 126.1 (thioridazine-2-sulfone), m/z 387.2 → 126.1 (thioridazine-5-sulfoxide), and 374.1 → 129.1 (thioridazine-d3). This method showed good linearity: 0.1-1000 ng/mL for thioridazine (r² = 0.9980) and 0.5-1000 ng/mL for mesoridazine (r² = 0.9981), thioridazine-2-sulfone (r² = 0.9976), and thioridazine-5-sulfoxide (r² = 0.9967). The accuracy of the LLOQ and QC samples for thioridazine and its metabolites was within 85%-115%, with intrabatch and interbatch precisions (CV%) below 15%. The average recovery ranged from 98% to 109%, and no significant matrix effect was observed, with values ranging from 93% to 110%. All analytes were stable under the conditions used in this study. This method was applied in a pharmacokinetic study following the intravenous administration of thioridazine to rats.