A new rapid, specific and sensitive reversed-phase HPLC method has been developed for simultaneous measurement of the R- and S-enantiomers of DN-2327 (I), a novel non-benzodiazepine anxiolytic, and those of its pharmacologically active metabolite, M(II) (II), in human plasma or urine. Extraction of all the enantiomers and internal standard was achieved using solid-phase extraction on C-8 columns. Resolution was achieved using a Chiral-AGP column with mobile phase comprising 6.5% (v/v) acetonitrile in 50 mM potassium acetate buffer, pH *q3 3.10, at typical flow-rates of 0.35 ml/min for plasma and 1.0 ml/min for urine assays. Fluorescence detection was employed using excitation and emission maxima of 328 and 367 nm, respectively. Analytes were well resolved and no interfering endogenous peaks were observed either from plasma or urine. Standard curves for urine were linear for concentrations up to 500 ng/ml for R- and S-II, with correlation coefficients higher than 0.994 and limit of quantitation (LOQ) of 1 ng/ml for each enantiomer. The LOQ in plasma was 0.1 ng/ml for each of the four enantiomers. The precision and accuracy of the method for the enantiomers of both I and II were good for plasma and urine with coefficients of variations typically within 10%. The stability of the R- and S-enantiomers of II and I in plasma and those of II in urine was excellent, with no evidence of degradation or interconversion during storage and handling.
