Examination of the Fragmentation Behavior of Hemin and Bilin Tetrapyrroles by Electrospray Ionization and Collision-induced Dissociation

Abstract

Bilin tetrapyrroles are metabolic products of the breakdown of porphyrins within a species. In the case of mammals, these bilins are formed by the catabolism of heme and can be utilized as either biomarkers in disease or as an indicator of human waste contamination. Although a small subset of bilin tandem mass spectrometry reports exist, limited data is available in online databases for their fragmentation. The use of fragmentation data is important for metabolomics analyses to determine the identity of compounds detected within a sample. Therefore, in this study, the fragmentation of bilins generated by positive ion mode electrospray ionization is examined by collision-induced dissociation (CID) as a function of collision energy on an FT-ICR MS. The use of the FT-ICR MS allows for high mass accuracy measurements, and thus the formulas of resultant product ions can be ascertained. Based on our observations, fragmentation behavior for hemin, biliverdin and its dimethyl ester, phycocyanobilin, bilirubin, bilirubin conjugate, mesobilirubin, urobilin, and stercobilin are discussed in the context of the molecular structure and collision energy. This report provides insight into the identification of structures within this class of molecules for untargeted analyses.

Figure 1. Outline of the metabolic degradation of heme; metabolites indicated subsequent to the bilirubin conjugate are produced by microflora in the colon. Highlighted are the portions of the chemical structures which exhibited a change from the previous compound. Phycocyanobilin and biliverdin dimethyl ester were both investigated, but are not present in the human metabolic pathway, with their respective precursor being biliverdin.

Table 1. Comparison of the fragmentation pattern of all of the bilins tested at 10V. Reported are the observed exact masses of fragment ions in m/z with reported ppm error in parenthesis (nd = not detected).