Development and validation of LC-MS/MS methods for the determination of EVT201 and its five metabolites in human urine: Application to a mass balance study.

In this study, two simple and accurate LC-MS/MS methods were firstly developed and validated to quantify EVT201, a new partial GABAA receptor agonist used for the treatment of insomnia, and its metabolites comprising M1, M2, M3, M4 and M6 in human urine. The analytes in urine samples were determined after simple dilution, and ideal chromatographic separations were obtained on C18 columns using gradient elution. The assays were performed in MRM mode on AB QTRAP 5500 tandem mass spectrometry (ESI+). The concentration ranges (ng/mL) of analytes in human urine were as follows: EVT201, 1.00 to 36.0; M1, 1.40 to 308; M2, 2.00 to 72.0; M3, 5.00 to 1100; M4, 2.00 to 300; and M6, 2.80 to 420. The methods were fully validated including selectivity, carryover, matrix effect, recovery, linearity, accuracy, precision, dilution integrity and stability, and acceptable criteria were obtained. The methods were successfully applied to a mass balance study of EVT201. The results showed that the total cumulative urinary excretion rate of EVT201 and its five metabolites was 74.25 ± 6.50%, which suggested that EVT201 had high oral bioavailability, and urinary elimination was its major excretion pathway in human.