QbD-Driven Development and Validation of a Bioanalytical LC-MS Method for Quantification of Fluoxetine in Human Plasma.

The current studies describe the Quality by Design (QbD)-based development and validation of a LC-MS-MS method for quantification of fluoxetine in human plasma using fluoxetine-D5 as an internal standard (IS). Solid-phase extraction was employed for sample preparation, and linearity was observed for drug concentrations ranging between 2 and 30 ng/mL. Systematic optimization of the method was carried out by employing Box-Behnken design with mobile phase flow rate (X1), pH (X2) and mobile phase composition (X3) as the method variables, followed by evaluating retention time (Rt) (Y1) and peak area (Y2) as the responses. The optimization studies revealed reduction in the variability associated with the method variables for improving the method robustness. Validation studies of the developed method revealed good linearity, accuracy, precision, selectivity and sensitivity of fluoxetine in human plasma. Stability studies performed in human plasma through freeze-thaw, bench-top, short-term and long-term cycles, and autosampler stability revealed lack of any change in the percent recovery of the drug. In a nutshell, the developed method demonstrated satisfactory results for analysis of fluoxetine in human plasma with plausible utility in pharmacokinetic and bioequivalence studies.