Simultaneous quantification of EVT201, a novel partial positive allosteric GABAA receptor modulator, and its two metabolites in human plasma by UHPLC/MS/MS

A sensitive, accurate and rapid UHPLC-MS/MS method was developed and validated for the simultaneous determination of EVT201 and its two metabolites, Ro46-1927 and Ro18-5528, in human plasma. IN-EVT201 was used as the internal standard (IS). Plasma samples were extracted using ethyl acetate after being alkalized with saturated sodium carbonate solution. Chromatographic separation was carried out on an Acquity BEH C18 column (2.1 x 50 mm, 1.7 mu m) with a gradient mobile phase at a flow-rate of 0.50 mL/min. The analytical run time was 5.5 min. For mass spectrometric detection, multiple reaction monitoring (MRM) was used. The MRM ion transitions were m/z 373.2 -> 58.0 for EVT201, m/z 359.2 -> 316.1 for Ro46-1927, m/z 291.2-4 -> 274.1 for Ro18-5528 and m/z 379.3 -> 64.0 for the IS. The linear range was 0.2-200 ng/mL for each analyte, with a correlation coefficient (r) over 0.9900. The intra/inter-precision was within 7.9% and 4.5% for EVT201, 13.2% and 6.3% for Ro46-1927, 3.7% and 4.1% for Ro 18-5528. For the accuracy, the relative bias of intra-/inter-run was no more than 6.4% and 4.6% for EVT201, 9.4% and 7.9% for Ro46-1927, -6.9% and -7.5% for Ro18-5528. The validated method was successfully applied to the analysis of more than 1500 samples from a Phase I clinical trial. The incurred sample reanalysis (ISR) of 146 samples from the study was evaluated and the results met the acceptance criteria. It indicated that the method could be used for the pharmacokinetic study of EVT201. (C) 2018 Elsevier B.V. All rights reserved.