JOURNAL OF MASS SPECTROMETRY, VOL. 30, 940-948 (1995) Distinguishing Isobaric Amino Acids in Sequence Analysis of Cyclosporins by Fast Atom Bombardment and Linked-scan Mass Spectrometry Vladimir HavliEekt Institute of Microbiology, Academy of Sciences zyxwvutsrq of the Czech Republic, Videkka 1083, 142 20 Prague 4, Czech Republic Alexandr Jegorov Galena Co., Research Unit, BraniSovska 31, 370 05 Ceske Budijovice, Czech Republic Petr Sedmera Institute of Microbiology, Academy of Sciences of the Czech Republic, Vide6ska 1083, 142 20 Prague 4, Czech Republic Winfried Wagner-Redeker Finnigan MAT GmbH, Barkhausenstrasse 2,28197 Bremen, Germany Miroslav Ryska Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic An analytical protocol suggested previously for the mass spectrometric sequence analysis of cyclosporins was extended by addition of methods for discrimination among isobaric amino acids differing either in the nature of the side-chain or N-substitution. The former goal was achieved by comparison of zyxwv B/E linked-scan mass spectra of corresponding collisionally activated imrnonium ions with those of standard amino acids and the latter by compari- son of B/E fragment ion spectra of IM zyxwvutsr + HI and IMd + *HI + ions. The use of the improved protocol is illustrated by the structure elucidation of a novel natural cyclosporin, [ Leu91CS. INTRODUCTION Cyclosporins are cyclic undecapeptides produced by various fungi imperfe~ti.'-~ The most important repre- sentative cyclosporin A (Fig. l), cyclo[-MeBmt'-Abu2- Sar3 - MeLeu4 - Val5 - MeLeu6 - Ah7- D - Alas - MeLeu9- MeLe~'~-MeVal''-], where MeBmt = (2S,3R,4R,6E)- zyxwvu 3-hydroxy-4-methyl-2-methylamino)oct-6-enoic acid, is t Author to whom correspondence should be addressed. MeLeu CK,3,CH3 MeVal a potent immunosuppressant widely used in human medicine to prevent rejection of transplanted organs such as bone marrow, heart, kidney and liver.5 Since even small changes in cyclosporin structure substan- tially influence the biological activities,6 it is necessary to control carefully the presence of cyclosporin A con- geners and degradation products in the substance used for the drug preparation. Similarly, major attention should also be devoted to cyclosporin zyx A metabolites in order to achieve the optimum immunosuppression and minimum toxicity during medical a p p l i c a t i ~ n . ~ , ~ Until recently, the contribution of mass spectrometry to the structure elucidation of novel cyclosporins was of y3 MeBmt CH II HC, Abu CHo zyxwvu StU CH3-N - CH-CO -N-CH- C-N-CH-CO-N- CH- C-N- CH2 CH3 I - CH\3,CH3 HO, FF I CH I CHZ CH3 CH CH3 CH CH3 CHZ CH3 I I 1 [ I I I s I 10 11 II 1 I 0 II I co 0 ?3 I MeLeu 0 co t r I I CH3-N I ~ II 7 I 0 11 6 I !Idi ,CH- CH2- CH o I 0 H zyxwvutsr b k 0 N-CH3 OC - CH -N - c- CH-N - C- CH - N- C-CH-N- C- CH I I I II I I I 1 cH3 0 CH2 CH3 ,cv CH2 CH3 Hi-,--- CH3CH3 LH - 1 CH / \ D-Ma CH3 CH3 Val c;;s\cH3 Meku MeLeu Figure 1. Structure of cyclosporin A. CCC 1076-5174/95/070940-09 zyxwvuts 0 1995 by John Wiley & Sons, Ltd. Received 30 December 1994 Accepted 24 February 1995