Determination of l-lysine N-acetylcysteinate and its mono- and dimeric related compounds by liquid chromatography–mass spectrometry

Abstract

Methods for the simultaneous determination of N-acetylcysteine, l-lysine, l-cysteine, l-cystine, N, N′-diacetylcystine and N, S-diacetylcysteine in aqueous solutions were developed. The method is based on the on-line coupling of liquid chromatography (LC) to tandem mass spectrometry (MS–MS) with an atmospheric pressure chemical ionisation (APCI) interface. The optimal LC phase system consisted of a C 18 stationary phase and an acidic solution of formic acid in acetonitrile as the mobile phase, while d, l-phenylalanine was selected as an internal reference compound. The best transitions for MS–MS detection were selected after optimisation of the APCI ionisation yield in full scan. The optimisation was performed by loop injection. By coupling LC to MS–MS in the APCI+ (positive ion mode) as well as in the multiple reaction monitoring mode, satisfactory results were obtained with a mixture of the seven compounds, each at a concentration level of 1 μg/ml. For a further improvement of the detection limit of the dimeric compounds, electrospray ionisation was then investigated. By use of this interface a quantitation limit down to 10 −8 M (3 ng/ml) could be achieved for both dimers. The developed method provides improved selectivity based on the combination of chromatography and mass spectrometry thus facilitating identification.