High-performance liquid chromatographic method for determination of DN-2327, a novel non-benzodiazepine anxiolytic, and/or its active metabolite in human plasma and urine.

A sensitive and precise high-performance liquid chromatographic procedure has been developed for the determination of a new non-benzodiazepine anxiolytic agent, DN-2327 (I), and its pharmacologically active metabolite, MII (II), in human plasma and urine. Extraction of I, II, and the internal standard from plasma and urine samples were achieved using solid-phase extraction. Separation of the analytes was performed on a reversed-phase C18 column. The effluent was monitored with fluorescence detection at excitation and emission maxima of 328 and 367 nm, respectively. The work-up procedure was reproducible and recovered more than 92% of I and II from either plasma or urine. The chromatographic system for plasma and urine extracts allowed complete resolution of I and II from the internal standard with excellent selectivity. For each analyte, the lower detection limits were 0.1 and 1 ng/ml in plasma and urine, respectively. For each analyte, standard curves were linear in the ranges of 0.1-50 and 1-500 ng/ml in plasma and urine, respectively. The method was highly precise, with coefficients of variation for each analyte in quality controls that were generally below 7 and 5% for plasma and urine samples, respectively. The accuracy of the method was good with the deviations between added and calculated concentrations of each analyte being typically within +/- 10% and +/- 5.6% for plasma and urine samples, respectively. The stability of I and II in standard solutions, plasma and urine samples protected from laboratory light was excellent, with no evidence of degradation after 72 h at room temperature, five months at 4 degrees C, or three months at -20 degrees C.