LC-MS/MS method for the determination of carbamathione in human plasma

Liquid chromatography-tandem mass spectrometry methodology is described for the determination of S-(N,N-diethylcarbamoyl)glutathione (carbamathione) in human plasma samples. Sample preparation consisted of a straightforward perchloric acid medicated protein precipitation, with the resulting supernatant containing the carbamathione (recovery ~98%). For optimized chromatography/mass spec detection a carbamathione analog, S-(N,N-di-i-propylcarbamoyl)glutathione, was synthesized and used as the internal standard. Carbamathione was found to be stable over the pH 1-8 region over the timeframe necessary for the various operations of the analytical method. Separation was accomplished via reversed-phase gradient elution chromatography with analyte elution and re-equilibration accomplished within 8 min. Calibration was established and validated over the concentration range of 0.5-50 nM, which is adequate to support clinical investigations. Intra- and inter-day accuracy and precision determined and found to be <4% and <10%, respectively. The methodology was utilized to demonstrate the carbamathione plasma-time profile of a human volunteer dosed with disulfiram (250 mg/d). Interestingly, an unknown but apparently related metabolite was observed with each human plasma sample analyzed.