A sensitive and high-throughput LC/MS/MS method using a silica column and an aqueous-organic mobile phase for the analysis of fluoxetine and norfluoxetine in human plasma

A sensitive and high-throughput LC/MS/MS method was developed and validated for the determination of fluoxetine and its metabolite norfluoxetine in human plasma. Analytes of interest were extracted from alkalized human plasma using liquid/liquid extraction. The extract was injected onto a silica column with an aqueous-organic mobile phase consisting of acetonitrile, water, trifluoroacetic acid (TFA), and ammonium acetate. The chromatographic run time was 2.0 min per injection, with retention time of 1.1 min for both fluoxetine and norfluoxetine, and 1.2 min for the internal standard (IS), fentanyl-d(5). Fluoxetine was monitored at m/z 310 --> 44, norfluoxetine at 296 --> 134, and IS at m/z 342 --> 188, respectively. The standard curve range was 0.5-250 ng/mL and low limit of quantitation (LLOQ) was 0.5 ng/mL for both fluoxetine and norfluoxetine. The inter-day precision and accuracy of the quality control (QC) samples were <5.1% relative standard deviation (RSD) and <7.3% relative error (RE).