SENSITIVE LIQUID-CHROMATOGRAPHIC MASS-SPECTROMETRIC ASSAY FOR THE SIMULTANEOUS QUANTITATION OF NEFAZODONE AND ITS METABOLITES HYDROXYNEFAZODONE M-CHLOROPHENYLPIPERAZINE AND TRIAZOLE-DIONE IN HUMAN PLASMA USINGSINGLE-ION MONITORING

A sensitive, selective, accurate, precise and reproducible high-perfor mance liquid chromatographic-mass spectrometric (LC-MS) assay was deve loped and validated for the simultaneous determination of nefazodone ( NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the i nternal standard (LS.). The method involved simultaneous protein preci pitation with acetonitrile and liquid-liquid extraction with methylene chloride, after which the organic layer was evaporated to dryness. Th e residue was reconstituted in 25% acetonitrile in 10 mM ammonium form ate (pH 4.0), and an aliquot was injected onto a BDS Hypersil C-18 col umn at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile in 55:45 (v/v) was used in a n isocratic condition. The mass spectrometer was programmed to admit t he protonated molecules at m/z 197.0 (mCPP), 372.0 (I.S.) 470.4 (NEF), 458.1 (Dione), and 486.2 (OH-NEF). Standard curves were Linear (r(2)g reater than or equal to 0.995) over the concentration range of 4-2000 ng/ml for Dione and 2-500 ng/ml for other analytes. The lowest standar d concentrations were the lower limit of quantitation for each analyte . The mean predicted quality control (QC) concentrations for all analy tes deviated less than -12.1% from the corresponding nominal values; t he intra-assay and inter-assay precision of the assay for all analytes were within 7.0% relative standard deviation. All analytes including I.S. were stable in the injection solvent at room temperature for at l east 24 h. The extraction recovery of the various analytes ranged from 67.3 to 86.5%. The validated assay was applied to a pharmacokinetic s tudy of nefazodone. (C) 1998 Published by Elsevier Science B.V. All ri ghts reserved.