SEPARATION OF CYCLOSPORINES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHYAND MASS-SPECTROMETRIC STUDY OF CYCLOSPORINE METABOLITES

A semi-preparative (high-performance liquid chromatography) method for separation of cyclosporin metabolites in a fungal fermentation sample was developed. By using the optimized chromatographic separation, 20 cyclosporin metabolites in a process sample were isolated, and their m olecular masses measured by double focusing sector mass spectrometry. The structures of some of the cyclosporin congeners were investigated by tandem sector mass spectrometry using fast atom bombardment ionizat ion, The isobaric cyclosporins D ([Val(2)]CS) and G ([Nva(2)]CS) were differentiated by significantly different relative intensities of a fr agment ion at m/z 30 [CH4N](+) from a-precursor ion at m/z 72 [C4H10N] (+). Otherwise the tandem mass spectrometry (MS/MS) spectra of the fra gment ions of the two isomers are very similar. Cyclosporin A and iso- cyclosporin A were also analysed by high energy MS/MS. No significant fragment ions were found to provide distinctions between them. Relativ ely good overviews of process samples containing very similar structur es were achieved by combining LC separation, molecular ion recognition and MS/MS characterization. (C) 1998 John Wiley & Sons, Ltd.