Development and Validation of an HPLC Method, with Fluorescence Detection, for Simultaneous Determination of Paroxetine and its Metabolites in Plasma

Abstract

An isocratic reversed‐phase high performance liquid chromatography (HPLC) method for simultaneous determination of paroxetine, a potent selective serotonin reuptake inhibitor and its metabolites {(‐)‐trans‐4‐(4‐fluorophenyl)‐3‐(4‐hydroxy‐3‐methoxyphenoxymethyl) piperidine, BRL 36610 A} (metabolite A) and {(‐)‐trans 4‐(4‐fluorophenyl)‐3‐(3‐hydroxy‐4‐methoxyphenoxymethyl) piperidine, BRL 36583 A} (metabolite B) in plasma has been developed. The method utilizes a Zorbax Eclipse XDB‐C18 5‐µm column, a mobile phase composed of acetonitrile‐phosphate buffer (KH₂PO₄ 0.04 M; pH=3.5) (30:70, v/v) at a flow rate of 1.0 mL/min and protriptyline as internal standard. The total analysis time was 12 min and paroxetine eluted at 10 min. A fluorescence detector was used with excitation and emission wavelength adjusted at 295 and 350 nm, respectively. All the experiments were performed at room temperature.

The isolation of paroxetine and its metabolites from plasma samples has been achieved from pH=12 with liquid‐liquid extraction using ether. The organic layer was removed, evaporated at 40°C under a nitrogen steam, and the residue was dissolved in 200 µL mobile phase and injected into the HPLC system.

The method was linear over the concentration range of 7–200 ng/mL for paroxetine, 12–200 ng/mL for metabolite A, and 27–200 ng/mL for metabolite B. The quantification limits were 7, 12, and 27 ng/mL, respectively, proving the suitability of the method for paroxetine drug level monitoring in the therapeutic range. The extraction recovery was about 84% for paroxetine. Within day precision of the method (RSD%) for paroxetine, metabolite A, and metabolite B were 0.26–7.5, 3.4–13, and 5.7–15%, respectively, while between day precision of the method was 4.9, 5.5, and 6.0%, correspondingly. No endogenous compounds were found to interfere with the analysis. Twenty six substances were studied for interference and none of them interfered in the determination.

The method was applied successfully in the quantitative determination of paroxetine in plasma samples of patients receiving 20–40 mg of Seroxat^R (paroxetine) and the concentrations were in the therapeutic range. Metabolites A and B were not detected in the plasma samples of the patients.

Keywords:

• Paroxetine
• Paroxetine metabolites
• Plasma
• HPLC

Acknowledgment

We are grateful to the Special Research Account of the University of Athens for financial support.