Simultaneous determination of fluoxetine and norfluoxetine in dried blood spots using high-performance liquid chromatography-tandem mass spectrometry

Publication date: Available online 5 October 2017
Source:Clinical Biochemistry
Author(s): Anne Caroline Cezimbra da Silva, Juliana Raquel Raasch, Tainara Gomes Vargas, Giovana Piva Peteffi, Roberta Zilles Hahn, Marina Venzon Antunes, Magda Susana Perassolo, Rafael Linden
BackgroundTherapeutic drug monitoring (TDM) of the widely prescribed antidepressant fluoxetine (FLU) is recommended in certain situations, such as occurrence of toxicity, inadequate response or suspect of poor adherence. Dried blood spot (DBS) sampling is an increasingly studied alternative for TDM, particularly for outpatients, due to its ease of collection and inherent stability.ObjectivesThe aim of this study was to develop and validate an LC-MS/MS assay for the simultaneous quantification of FLU and norfluoxetine (NFLU) in DBS.Design and methodsThe assay is based on a liquid extraction of single DBS with 8mm of diameter, using FLU-D6 as the internal standard, followed by reversed phase separation in an Accucore® C18 column (100×2.1mm, 2.6μm). Mobile phase was composed of water and acetonitrile (gradient from 80:20 to 50:50, v/v), both containing formic acid 0.1%. The assay was validated and applied to 30 patients under FLU pharmacotherapy.ResultsThe assay was linear in the range 10–750ngmL⁻¹. Precision assays presented CV% of 3.13–9.61 and 3.54–7.99 for FLU and NFLU, respectively, and accuracy in the range of 97.98–110.44% and 100.25–105.8%. FLU and NFLU were stable at 25 and 45°C for 7days. The assay was evaluated in 30 patients under FLU treatment. Concentrations of both compounds were higher in DBS than in plasma, and the use of the multiplying factors 0.71 and 0.68 for FLU and NFLU, respectively, allowed acceptable estimation of plasma concentrations, with median prediction bias of −0.55 to 0.55% and mean differences of 0.4 to 2.2ngmL⁻¹.ConclusionsThe presented data support the clinical use of DBS for therapeutic drug monitoring of FLU.

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