Simultaneous quantitation of d7-nefazodone, nefazodone, d7-hydroxynefazodone, hydroxynefazodone, m-chlorophenylpiperazine and triazole-dione in humanplasma by liquid chromatographic-mass spectrometry

A rapid and sensitive LC-MS assay was developed and validated for the simul taneous determination of d7-nefazodone (d7-NEF), nefazodone (NEF), d7-hydro xynefazodone (d7-OH-NEF), hydroxynefazodone (ON-NEF), m-chlorophenylpiperaz ine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ ) as the internal standard (IS). A 0. I mt aliquot of the plasma sample was precipitated with 0.1 mt of acetonitrile and vortexed for 2 min. After cen trifugation. 50 mu L of supernatant was mixed with 100 mu L of 10 mM ammoni um formate (pH = 4.0), and a 50 mu L aliquot was injected onto a BDS Hypers il C18 column at a flow rate of 0.3 mL/min. The mobile phase consisting of 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, was used in an isocratic system. The mass spectrometer was programmed to admit the proton ated molecules at m/z 477.2 (d7-NEF), 493.3 (d7-OH-NEF), 197.0 (mCPP), 372. 0 (IS), 470.4 (NEF), 458.0 (Dione) and 486.2 (OH-NEF). Standard curves were linear (r(2) greater than or equal to 0.994) over the concentration range of 4-1000 ng/mL for Dione and 2-500 ng/mL for all other analytes. The lowes t standard concentrations were the lower limits of quantitation for each an alyte. The mean predicted quality control concentrations for all analytes d eviated by less than 14.3% from the corresponding nominal values; the intra -assay and inter-assay precisions of the assay for all analytes were within 10.5% relative standard deviation. All analytes including the internal sta ndard were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 79.2 to 109.1%. The validated assay was applied to the analysis of clinical samples obtained from a human subject who simultaneously received d7-NEF and NEF o rally. Copyright (C) 2000 John Wiley & Sons, Ltd.