Negative allosteric modulators interacting with the ifenprodil binding site of NMDA receptors with GluN2B subunit are of particular interest for the treatment of psychiatric and neurodegenerative disorders. In order to eliminate the radioligand 3 Hifenprodil in the standard radioactive binding assay, a novel MS binding assay was established and validated making use of the quantitative determination of non-radioactive ifenprodil by triple quadrupole MS. Kinetic, saturation and competitive experiments were carried out in a stepwise and iterative manner to optimize the final assay conditions. Under optimized assay conditions, the MS binding assay provided a K d value for the marker ifenprodil ( K d = 11 nM), which is comparable with the K d value obtained in the radioligand binding assay for 3 Hifenprodil ( K d = 7.6 nM). Although the MS binding assay led to higher K i values for known NMDA receptor antagonists, both assays showed the same trend of K i values for these inhibitors. • MS-based binding assay for the ifenprodil binding site of the NMDA receptor. • Quantification of the marker ifenprodil by triple quadrupole MS. • Careful validation of the analytical methods. • Iterative optimization of the assay conditions by kinetics, saturation and competitive experiments. • Contribution to sustainable and environmentally friendly chemical methods.
Grey et al. (Sun,) studied this question.