A simple, selective, and reliable bioanalytical method was developed for the quantitation of paroxetine in human plasma by LC-MS/MS. Paroxetine, a unique small molecule with a single secondary amine, posed significant challenges in the development of reproducible chromatography. Substantial retention time shift was observed for paroxetine using acidic mobile phases with either new or used columns. Buffer strength played an essential role in reducing the observed column-to-column retention time variation. A 96-well plate-based liquid-liquid extraction was developed. The method was validated and used to support clinical studies for the measurement of paroxetine in human plasma in the range of 0.250-50.0 ng/mL using paroxetine-d6 as its internal standard. This article discusses factors impacting the retention time of analytes in liquid chromatography.

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