AUTOMATED HPLC ASSAY OF FLUOXETINE AND NORFLUOXETINE IN SERUM

This automated assay determines the concentration of the antidepressan t fluoxetine (Prozac(R)) and its active metabolite norfluoxetine in se rum by reversed-phase HPLC with spectrophotometric detection. Extracti on, injection, and quantification are performed by the Gilson Aspec(R) automated sample handler. The patient's specimen, with added protript yline as internal standard, is extracted with solid-phase and liquid-l iquid methods. Separation is conducted isocratically on a 5-mu m (part icle size) Supelcosil LC-8-DB reversed-phase column with a triethylami ne acetate:acetonitrile mobile phase. The detection limit is 10 mu g/L and absorbance varies linearly with concentration between 20 and 1000 mu g/L for both compounds. Mean recovery was 62% for fluoxetine and 7 0% for norfluoxetine over linear limits. Within-run and day-to-day imp recision (CV), evaluated at three concentrations (50, 200, and 500 mu g/L), ranged from 2% to 7%. An extensive interference study of 108 dru gs was conducted. Results (n = 58) by the automated method (y) correla ted well with those by a manual HPLC method (x): y = 0.96x + 10.20 (r = 0.951, S-ylx, = 42.9) for fluoxetine, and y = 0.95x - 1.37 (r = 0.91 7, S-ylx = 47.2) for norfluoxetine.