Development and validation of an LC‐ESI‐MS/MS method for the quantification of D‐84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT, hSERT and hNET

Abstract

MS Binding Assays represent a label-free alternative to radioligand binding assays. In this study, we present an LC-ESI-MS/MS method for the quantification of (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol [(R,R)-D-84, (R,R)-1], (S,S)-reboxetine [(S,S)-2], and (S)-citalopram [(S)-3] employed as highly selective nonlabeled reporter ligands in MS Binding Assays addressing the dopamine [DAT, (R,R)-D-84], norepinephrine [NET, (S,S)-reboxetine] and serotonin transporter [SERT, (S)-citalopram], respectively. The developed LC-ESI-MS/MS method uses a pentafluorphenyl stationary phase in combination with a mobile phase composed of acetonitrile and ammonium formate buffer for chromatography and a triple quadrupole mass spectrometer in the multiple reaction monitoring mode for mass spectrometric detection. Quantification is based on deuterated derivatives of all three analytes serving as internal standards. The established LC-ESI-MS/MS method enables fast, robust, selective and highly sensitive quantification of all three reporter ligands in a single chromatographic run. The method was validated according to the Center for Drug Evaluation and Research (CDER) guideline for bioanalytical method validation regarding selectivity, accuracy, precision, calibration curve and sensitivity. Finally, filtration-based MS Binding Assays were performed for all three monoamine transporters based on this LC-ESI-MS/MS quantification method as read out. The affinities determined in saturation experiments for (R,R)-D-84 toward hDAT, for (S,S)-reboxetine toward hNET, and for (S)-citalopram toward hSERT, respectively, were in good accordance with results from literature, clearly demonstrating that the established MS Binding Assays have the potential to be an efficient alternative to radioligand binding assays widely used for this purpose so far.

MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand

“…During method development it was further recognized that Org25543 is prone to extensive adhesion to container materials when it is dissolved in pure water, especially to polypropylene surfaces of tubes, which leads to a decrease of the free concentration of the analyte as compared to the nominal concentration. This phenomenon has already been observed during method development in other studies [35,43] . To solve this problem we used, as successfully done in former cases, N,N ‐dimethylacetamide (DMA) as organic co‐solvent.…”

“…In the last step of the binding experiment, the target‐bound‐marker remaining on the filters has to be liberated and eluted. In former MS Binding Assays the solvents acetonitrile and methanol have already been shown to be suitable for this task [30–39] . We decided to select acetonitrile as elution solvent due to the fact that it is advantageous for the chromatography, especially for peak shapes, when the composition of the sample solvent of the final sample is identical with that of the mobile phase of the employed LC‐ESI‐MS/MS method which in the present case consists of a mixture of acetonitrile and 5 mM ammonium bicarbonate buffer in a ratio of 80 : 20 ( v / v ).…”

“…As the determination of binding affinities is in general (e. g., no living cells are needed) less elaborate than the performance of functional assays and binding affinities by being direct proportional to the Gibbs free energy (▵ G 0 ) are required as basis for molecular modelling studies, we aimed at the development of a binding assay addressing GlyT2. To this end we intended to use the concept of MS Binding Assays, which was established in our group during the last years and was employed already for GlyT1 and several other target proteins [28–39] . MS Binding Assays (as described in Section 2 3…”

Benocyclidine (BTCP) as Non‐labelled Reporter Ligand for MS Binding Assays for the PCP Ion Channel Binding Site of the Desensitized Torpedo Nicotinic Acetylcholine Receptor (nAChR)

“…Thus, fluorescence based assays have been established using rac-quinacrine (Figure 1; 5) or ethidium bromide (Figure 1; 6) addressing the nAChR ion channel [12,20,21] , which however suffer from background radiation and other interfering effects. An interesting alternative to radioligand binding assays are MS Binding Assays, the concept of which has been introduced by our group [22][23][24][25] in recent years. In the present study we wanted to probe the applicability of this concept for the PCP ion channel binding site of desensitized Torpedo nAChR.…”