Sensitive and high-throughput bioanalysis of fluoxetine and nor-fluoxetine in rabbit and human plasma using SPE-LC-MS/MS

Abstract

Fluoxetine is a commonly prescribed antidepressant agent in psychotherapy. A rapid, selective and sensitive LC-MS/MS method was developed and validated for simultaneous estimation of fluoxetine and its metabolite nor-fluoxetine in rabbit and human plasma. The assay was linear over the concentration range of 0.048–100 ng mL⁻¹ with a lower limit of detection of 32 pg mL⁻¹ (0.032 ng mL⁻¹) for both fluoxetine and nor-fluoxetine. Separation and detection of analytes were achieved on a reversed phase Waters Symmetry Shield™ C₁₈ column, with an isocratic mobile phase consisting of methanol and 0.5% formic acid (80 : 20, v/v) at a flow rate of 0.75 mL min⁻¹. A turnover rate of 2.5 min per sample enables the high-throughput bioanalysis of fluoxetine. An automated solid phase extraction method was employed for efficient extraction of analytes from the matrix. Thereafter, analytes were monitored by using MS/MS with an electrospray ionization source in positive multiple reaction monitoring mode. The method was successfully applied to in vivo pharmacokinetic studies in rabbit and in vitro protein binding studies in human plasma. Due to high sensitivity and low requirement of sample volume, the method could be applicable for preclinical and clinical applications such as therapeutic drug monitoring in special population (children and geriatric patients) using only 0.03 mL of plasma.