Automated HPLC assay of fluoxetine and norfluoxetine in serum

Abstract

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-microns (particle size) Supelcosil LC-8-DB reversed-phase column with a triethylamine acetate:acetonitrile mobile phase. The detection limit is 10 micrograms/L and absorbance varies linearly with concentration between 20 and 1,000 micrograms/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 micrograms/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, Sylx = 42.9) for fluoxetine, and y = 0.95x - 1.37 (r = 0.917, Sylx = 47.2) for norfluoxetine.