Ion trap MS/MS of intact testosterone and epitestosterone conjugates - Adducts, fragile ions and the advantages of derivatisation

David A. Cowan, Andrew T. Kicman, Carlos Kubli-Garfias, Helen J. Welchman

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

In ion trap mass spectrometry, fragile ions may fragment under the application of resonance ejection during precursor mass isolation, reducing MS/MS spectral intensity. In this study the steroidal epimers testosterone glucuronide (TG) and epitestosterone glucuronide (EG) have been chosen as a model for exploring whether compound structure is linked to ion trap fragility. Both compounds form multiple adducts by ESI-MS, namely protonation, ammonium and sodium, however, the mass spectrum of EG displays a more intense ammonium adduct peak than TG. [TG + NH4](+), [EG + NH4](+) and [EG + H](+) were found to be fragile ions. To explain the differences in adduct formation and fragility, molecular modelling was employed. Ammonium adduction was localised to the glucuronide ring oxygens and while EG has eight possible adduction sites, only seven were located for TG explaining the increased ammonium adduct abundance with EG. In EG the bond between the steroid and the glucuronide was slightly longer and the oxygen in this bond was more basic than TG. This shows that the EG bond is weaker which may contribute to the fact that [EG + H](+) but not [TG + H](+) is fragile. To investigate whether stability could be restored by chemical means, EG was derivatised with tris(trimethoxyphenyl)phosphonium chloride or methylated on the carboxylic acid and Girard P or methoxylamine on the 3-keto group. Derivatisation of the steroid rather than the glucuronide eliminated fragility and using a charged derivative eliminated adduct formation. This work demonstrates the importance of carefully considering the nature of the derivative and the site of derivatisation.
Original languageEnglish
Pages (from-to)621 - 628
Number of pages8
JournalSteroids
Volume73
Issue number6
DOIs
Publication statusPublished - Jul 2008

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