Abstract
An approach presented recently for the high-performance liquid chromatographic determination of dl- and l-cystines and cysteines as phenylthiocarbamyl (PTC) derivatives has been completed (i) with the determination of d-cystine/cysteine, (ii) by a study relating to the spectral characteristics of the PTC cystine/cysteine derivatives performed with diode-array detection and (iii) by the fragment analysis of the PTC-cyst(e)ine derivatives, applying the extremely soft conditions of the liquid chromatography-atmosphere pressure ionization mass spectrometry (LC-API-MS). In accordance with earlier experiences it has been repeatedly demonstrated that cystines and cysteines can be determined on the basis of two or four derivatives, with the same retention times and molar responses, without any special pretreatment, furnishing a simple possibility for the determination of the total cystine/cyteine in protein hydrolysates. The UV spectra of all four PTC derivatives provided by diode-array-detected purity parameters (PuP) showed that the corresponding PTC derivatives of dl-l- and d-cystine and -cysteine are identical and homogeneous, and do not show any characteristic differences. On the basis of the fragmentation patterns of seventeen PTC-amino acids and those of six PTC-cyst(e)ines, obtained under the very soft conditions of the LC-API-MS procedure, it can be stated that two characteristic PTC derivatives are formed: (i) the main product (“a” and “b” diastereoisomers, >80% of the total), shown to be the fragment of m/ z = 255, a monomeric cysteine derivative, and (ii) the other characteristic constituent (“c” and “d” stereoisomers, <20% of the total), consisting of a fragments of m/ z = 255 and its oxidised form of m/ z = 287.
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