MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D1 and D5 dopamine receptors. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method for the selective D1 dopamine receptor antagonist SCH23390 ((5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol) was established and validated, using its 8-bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D1 and D5 receptors. Following the concept of MS Binding Assays, saturation experiments for D1 and D5 receptors were performed, as well as competition experiments for D1 receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D1 and D5 receptors are well-suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.

(+)-R,R-D-84 ((+)-R,R-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol) is a promising pharmacological tool for the dopamine transporter (DAT), due to its high affinity and selectivity for this target. In this study, an analytical method to ascertain the enantiomeric purity of this compound was established. For this purpose, a high-performance liquid chromatographic (HPLC) method, based on a cellulose derived chiral stationary phase (CSP) was developed. The method was characterized concerning its specificity, linearity, and range. It was shown that the method is suitable to determine an enantiomeric excess of up to 99.8%. With only a few adjustments, this analytical CSP-HPLC method is also well suited to separate (+)-R,R-D-84 from its enantiomer in a semipreparative scale.

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.

In this work, we present label-free, mass-spectrometry-based binding assays (MS Binding Assays), targeting the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) in simultaneous binding experiments. Using a validated LC-ESI-MS/MS method for quantification of the selective dopamine transporter inhibitor (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol ((R,R)-D-84), the selective norepinephrine transporter inhibitor (S,S)-reboxetine, and the selective serotonin reuptake inhibitor (S)-citalopram, binding affinities at the three monoamine transporters could be characterized simultaneously in a single binding experiment. The performed simultaneous saturation and competition experiments yielded results that are in good accordance with those determined in MS Binding Assays addressing the monoamine transporters individually. The results obtained from this study underscore the potential of MS Binding Assays for simultaneous affinity determination at different targets, which is difficult to accomplish with conventional radioligand binding assays.