Structural elucidation studies of erythromycins by electrospray tandem mass spectrometry II

Erythromycin A (EryA), sec‐butyl erythromycin B (SEryB), oleandomycin (Olean) and a synthetic derivative, roxithromycin (Rox), were used to investigate the fragmentation of polyketide macrolide antibiotics by collision induced dissociation (CID) tandem mass spectrometry (MS/MS). Analyses were performed with two commercially available mass spectrometers: a Q‐TOF hybrid quadrupole time‐of‐flight instrument and a BioApex II (4.7 Tesla) Fourier transform ion cyclotron resonance (FTICR) instrument both equipped with electrospray ionisation (ESI) sources. One of the first fragmentation processes is the loss of an H2O molecule from the [M+H]+ ion. EryA has three hydroxyl groups on the polyketide ring and loses three H2O molecules during CID. This study indicates that these facts are not necessarily related. Deuterium exchange experiments were carried out in order to isotopically label free hydroxyl groups. 18O‐exchange experiments were also carried out in order to label the carbonyl group at the 9‐position. In EryA and its analogue the first H2O loss shifts in mass from loss of 18 Da to loss of 20 Da in deuterated solvents. For both molecules the loss also shifts in mass from loss of 18 Da to loss of 20 Da during the 18O‐exchange experiments. This suggests that the first loss of H2O is from the 9‐position carbonyl group, indicating that this, and not the nitrogen of the amino sugar, is the site of protonation of the activated MH+ ions. For Rox the initial loss of H2O is replaced by loss of the 9‐position oxime group, the rest of the fragmentation sequence being the same as for EryA. For Olean, there is no H2O loss from the parent ion. The results have allowed the proposal of a mechanism for the first loss of H2O in the EryA MS/MS fragmentation. Copyright © 1999 John Wiley & Sons, Ltd.