Development and validation of a rapid LC-ESI-MS/MS method for quantification of fluoxetine and its application to MS binding assays

Abstract

In the present study, a rapid and sensitive LC-ESI-MS/MS method for quantification of (S)-fluoxetine as a native marker in mass spectrometry (MS) binding assays addressing the human serotonin transporter (hSERT) was developed and validated. The concept of MS binding assays based on mass spectrometric quantification of a nonlabeled marker recently introduced by us represents a promising alternative to conventional radioligand binding without the drawbacks inherently connected with radioisotope labeling. For high-performance liquid chromatography (HPLC), a 20 × 2-mm RP-18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L(-1), pH 9.5) at a ratio of 80:20 (v/v) and a flow rate of 800 μL min(-1) in an isocratic mode were used, resulting in a chromatographic cycle time of 60 s. Employing [(2)H(5)]fluoxetine as internal standard enabled ESI-MS/MS quantification of (S)-fluoxetine between 3 nmol L(-1) and 50 pmol L(-1) (LLOQ) in matrix obtained from binding experiments without the need of any sample preparation. Validation of the method showed that linearity, intra-, and inter-batch accuracy as well as precision meet the requirements of the FDA guidance for bioanalytical method validation. Considering sensitivity and speed, the established method is clearly superior to those published for biological matrices so far. Furthermore, the method was transferred to other RP-18 columns of different lengths and respective validation experiments demonstrated its versatility and chromatographic robustness. Finally, the newly developed method was successfully applied to MS binding assays for hSERT. The affinity determined for (S)-fluoxetine in saturation experiments was in good agreement with literature data obtained in respective radioligand binding assays.

(S)‐ and (R)‐Fluoxetine as Native Markers in Mass Spectrometry (MS) Binding Assays Addressing the Serotonin Transporter

“…[29] Here, we aimed to fully characterize (S)-fluoxetine binding to hSERT by association and dissociation experiments. The developed MS binding assay using (S)-fluoxetine as a marker [16] seemed best suited for this purpose-to directly determine the association (k + 1 ) and dissociation (k À1 ) rate constants. In addition, the direct determination of those parameters has the benefit of offering independent access to the K d value (K d = k À1 /k + 1 ) of (S)-fluoxetine, which is commonly obtained from saturation experiments.…”

“…[16] Based on this LC-MS quantification method, we demonstrate the feasibility of saturation experiments for SERT based on (S)-fluoxetine as a native marker. For this purpose, membrane preparations of HEK293 cells stably expressing the target were used [16] under incubation conditions as commonly applied in radioligand binding studies.…”

“…[16] Based on this LC-MS quantification method, we demonstrate the feasibility of saturation experiments for SERT based on (S)-fluoxetine as a native marker. For this purpose, membrane preparations of HEK293 cells stably expressing the target were used [16] under incubation conditions as commonly applied in radioligand binding studies. [21] The aim of the present study was to characterize (S)-fluoxetine binding to hSERT in kinetic experiments and to establish competitive experiments, thus enabling the determination of the affinity of test compounds for the target.…”

“…The concept of MS binding assays is based on a nonlabeled reporter ligand, also referred to as native marker. [16][17][18] The assay can be carried out in analogy to conventional radioligand binding assays in a 96-well plate format. Quantification of the target bound marker in the native, nonlabeled form is achieved by LC-ESI-MS/MS.…”

“…17 cently established a LC-MS quantification method for (S)-fluoxetine as a native SERT marker. [16] Fluoxetine (5) is an SSRI and one of the most frequently prescribed antidepressant drugs worldwide, and as such, it is of great therapeutic relevance. [19,20] Radioligand binding employing [ 3 H]fluoxetine is poorly described probably due to the radiochemical instability mentioned by Wong et al; [19] this might also explain why it is not commercially available.…”