Tandem mass spectrometry of bilin tetrapyrroles by electrospray ionization and collision-induced dissociation

RATIONALE Bilins are metabolic products of hosts and bacteria on porphyrins, and are markers of health state and human waste contamination. Although bilin tandem mass spectrometry reports exist, their fragmentation behavior as a function of structure has not been compared, nor has fragmentation been examined as a function of collision energy. METHODS The fragmentation of bilins generated by positive ion mode electrospray ionization is examined by collision-induced dissociation (CID). CID on a quadrupole ion trap and on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer as a function of collision energy is compared. Methyl esterification was used to deduce which product ions contain the inner pyrrole rings. FT-ICR high mass accuracy measurements were used to determine the formulas of the resultant product ions. RESULTS The central carbon's bonding to the inner pyrrole rings influences fragmentation. Bilirubin is unique because fragmentation adjacent to the central methylene group between innermost rings predominates, and loss of a terminal pyrrole is observed only with helium collision gas. The other bilins lose the terminal pyrroles first; as CID energy is increased, additional fragmentation due to neutral losses of small molecules such as H₂O, CO, CO₂, and methanol occurs. CONCLUSIONS Based on these observations, fragmentation schemes for the bilins are proposed that are strongly dependent on the molecular structure and collision energy; only bilirubin fragmentation is influenced significantly by the collision gas used. This report should have value in identification of this class of molecules for biomarker detection.