Development and validation of an LC-ESI-MS/MS method for the triple reuptake inhibitor indatraline enabling its...

We herein present the first LC-MS/MS quantification method for indatraline, a highly potent nonselective inhibitor of the three monoamine transporters (for dopamine, DAT; norepinephrine, NET; serotonin, SERT), and its application to MS Binding Assays. For HPLC, an R18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L(-1), pH 10.0) in a ratio of 90:10 (v/v) at a flow rate of 600 μL min(-1) was used. Recording indatraline at m/z 292.2/261.0 and ((2)H(7))-indatraline, employed as internal standard, at m/z 299.2/268.0 allowed reliable quantification from 5 pmol L(-1) (LLOQ) to 5 nmol L(-1) in biological matrices without additional sample preparation. Validation of the developed quantification method showed that selectivity, calibration standard curve, accuracy, as well as precision meet the criteria of the CDER guideline. Applying this method to mass spectrometry (MS) Binding Assays, a label-free MS-based alternative to conventional radioligand binding assays, binding of indatraline's eutomer, (1R,3S)-indatraline, towards NET could be characterized directly for the first time, revealing an equilibrium dissociation constant (K d) of 805 pmol L(-1). Additionally, it could be shown that the established MS Binding Assays enable characterization of test compounds in competition experiments. As the established setup is based on a 96-well format and an LC MS/MS method with a short chromatographic cycle time (1.5 min), the developed MS Binding Assays enable considerable throughput and are therefore well suited as substitute for corresponding radioligand binding assays.