Determination of fluoxetine and norfluoxetine in plasma by a modified high performance reversed phase liquid chromatographic method with fluorescence detection

A simple and rapid reversed phase liquid chromatographic method for the determination of fluoxetine (F) and norfluoxetine (NF) in plasma has been developed and validated. Protriptyline (P) was used as internal standard. Plasma samples were extracted from alkaline pH with hexane-isoamyl alcohol 98: 2 v/v on a rotator drive for 10 min. The organic phase was back extracted with H3PO4 0.1 M and the aqueous phase was injected directly onto a 5 mum Kromasil 100-C8 column (25 cm x 4 mm i.d.), using acetonitrile/0.05 M KH2PO4 45:55 v/v as the mobile phase. The fluorescence detector was set at 310 nm (lambda(em)=231 nm). Retention times were 4.6, and 6.4 min for internal standard, NF, and F, respectively. Peak height ratios and plasma F and NF concentrations were linearly related from 8 and 40 to 800 ng/mL (r>0.995 and 0.998, respectively). The detection limits for F and NF were 2.5 and 13 ng/mL, respectively. Precision and accuracy were evaluated from spiked plasma samples and were, for F:CV=5.4-20% and E-r=0.6-9.4% and for NF:CV=4.7-18% and E-r=0.9-10%. The developed method is simple, accurate, and of enough detectability to be used for F and NF monitoring in plasma samples of patients under antidepressant therapy with F or in bioavailability studies.