Identification of a biliverdin geometric isomer by means of HPLC/ESI-MS and NMR spectroscopy. Differentiation of the isomers by using fragmentation "in-source"

Abstract

Abstract: A commercially available biliverdin sample was analyzed by means of HPLC/ESI-MS and NMR spectroscopy. It was been found that beside the main IXα 5Z,10Z,15Z isomer, the sample contains also the geometric isomer IXα 5Z,10Z,15E. It was also found the isomers behave differentially upon "in-source" fragmentation in negative ion mode (in contrast to the their behavior upon "in-source" fragmentation in positive ion mode and to their behavior upon MS/MS fragmentation in both modes): the relative abundances of deprotonated molecules and fragment ions are significantly different for both isomers, which can be used as an analytical tool to differentiate between the isomers.

Keywords: Biliverdin; Electrospray ionization; Geometric isomer; Mass spectrometry; NMR spectroscopy.

Fig. 1

Single ion chromatograms of ions [BV + H]⁺ (top) and [BV−H]⁻ (bottom)

Fig. 2

¹H NMR spectrum of biliverdin sample in CD₃CN/D₂O (298 K)

Fig. 3

¹H NMR spectrum of biliverdin sample in CD₃OD/D₂O/NaOD (298 K): full spectrum (top), selected regions (middle and bottom)

Fig. 4

ESI mass spectra of bilverdin isomers obtained upon HPLC-ESI/MS analysis (CV = 50 V)

Fig. 5

The breakdown plots of ions [2BV−H]⁻, [BV−H]⁻ and main fragment ion at m/z = 285, against cone voltage (V). The abundances of ions correspond to the respective peak areas obtained upon HPLC-ESI/MS analysis