DETERMINATION OF A NON–PEPTIDE OXYTOCIN RECEPTOR ANTAGONIST IN HUMAN PLASMA BY AUTOMATED PRE–COLUMN DERIVATIZATION AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

Abstract

A sensitive and selective method for the determination of 1-(((7,7-dimethyl-2(S)-(2(S)-amino-4-(methylsulfonyl) butyramido)-bicyclo[2.2.1]-heptan-1(S)-yl) methyl) sulfonyl) - 4 - (2 - methyl-phenyl)-piperazine (L-368,899) in human plasma is described. The method is based on liquid-liquid extraction followed by automated pre-column chemical derivatization of the primary amino group of the drug and an internal standard with 2,3-naphthalene dicarboxaldehyde and N-acetylcysteamine to form fluorescent benzo[f]isoindole derivatives. The derivatives are separated from endogenous interferences using column-switching high-performance liquid chromatography (HPLC) and quantified with fluorescence detection (FD). The derivatives were initially injected onto a Zorbax SB-CN column and “heart cut” onto a Waters SymmetryTM C8 column for final separation prior to fluorescence detection. The assay in human plasma has been validated in the concentration range of 1 to 25 ng/mL. The performance of the HPLC-FD assay was evaluated by comparing the results of the analysis of selected clinical samples using an independent method based on HPLC with tandem mass spectrometric detection (LC-MS/MS). A good correlation between the data obtained using the two methods was found confirming the selectivity and reliability of the HPLC-FD method.