Determination of a non-peptide oxytocin receptor antagonist in human plasma by automated precolumn derivatization and high performance liquid chromatography with fluorescence detection

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-methylphenyl)-piperazine (L-368,894) in human pl asma 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-sw itching high-performance liquid chromatography (HPLC) and quantified with f luorescence detection (FD). The derivatives were initially injected onto a Zorbax SB-CN column and "heart cut" onto a Waters Symmetry(TM) C8 column fo r 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 compar ing the results of the analysis of selected clinical samples using an indep endent method based on HPLC with tandem mass spectrometric detection (LC-MS /MS). A good correlation between the data obtained using the two methods was foun d confirming the selectivity and reliability of the HPLC-FD method.