Sensitive liquid chromatographic–mass spectrometric assay for the simultaneous quantitation of nefazodone and its metabolites hydroxynefazodone m-chlorophenylpiperazine and triazole-dione in human plasma using single-ion monitoring

Abstract

A sensitive, selective, accurate, precise and reproducible high-performance liquid chromatographic–mass spectrometric (LC–MS) assay was developed 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 internal standard (I.S.). The method involved simultaneous protein precipitation with acetonitrile and liquid–liquid extraction with methylene chloride, after which the organic layer was evaporated to dryness. The residue was reconstituted in 25% acetonitrile in 10 m M ammonium formate (pH 4.0), and an aliquot was injected onto a BDS Hypersil C 18 column at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 m M ammonium formate (pH 4) and acetonitrile in 55:45 (v/v) was used in an isocratic condition. The mass spectrometer was programmed to admit the 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≥0.995) over the concentration range of 4–1000 ng/ml for Dione and 2–500 ng/ml for other analytes. The lowest standard concentrations were the lower limit of quantitation for each analyte. The mean predicted quality control (QC) concentrations for all analytes deviated less than −12.1% from the corresponding nominal values; the 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 least 24 h. The extraction recovery of the various analytes ranged from 67.3 to 86.5%. The validated assay was applied to a pharmacokinetic study of nefazodone.