Automated HPLC assay of fluoxetine and norfluoxetine in serum

Abstract

This automated assay determines the concentration of the antidepressant fluoxetine (Prozac®) and its active metabolite norfluoxetine in serum by reversed-phase HPLC with spectrophotometric detection. Extraction, injection, and quantification are performed by the Gilson Aspec® automated sample handler. The patient's specimen, with added protriptyline as internal standard, is extracted with solid-phase and liquid-liquid methods. Separation is conducted isocratically on a 5-μm (particle size) Supelcosil LC-8-DB reversed-phase column with a triethylamine acetate:acetonitrile mobile phase. The detection limit is 10 μg/L and absorbance varies linearly with concentration between 20 and 1000 μg/L for both compounds. Mean recovery was 62% for fluoxetine and 70% for norfluoxetine over linear limits. Within-run and day-to-day imprecision (CV), evaluated at three concentrations (50, 200, and 500 μg/L), ranged from 2% to 7%. An extensive interference study of 108 drugs was conducted. Results (n = 58) by the automated method (y) correlated well with those by a manual HPLC method (x): y = 0.96x + 10.20 (r = 0.951, S(y/x) = 42.9) for fluoxetine, and y = 0.95x - 1.37 (r = 0.917, S(y/x) = 47.2) for norfluoxetine.