In this study, we evaluated, by electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using a quadrupole orthogonal time-of-flight (QqToF)-MS/MS hybrid instrument, the gas-phase fragmentations of some commercially available biotinyl reagents. The biotin reagents used were: psoralen-BPE 1, p-diazobenzoyl biocytin (DBB) 2, photoreactive biotin 3, biotinyl-hexaethyleneglycol dimer 4, and the sulfo-SBED 5. The results showed that, during ESI-MS and CID-MS/MS analyses, the biotin reagents followed a similar gas-phase fragmentation pattern and the cleavages usually occurred at either end of the spacer arm of the biotin reagents. In general we have observed that the CID-MS/MS fragmentation routes of the five precursor protonated molecules obtained from the biotin linkers 1-5 afforded a series of product ions formed essentially by similar routes. The genesis and the structural identities of all the product ions obtained from the biotin linkers 1-5 have been assigned. All the exact mass assignments of the protonated molecules and the product ions were verified by conducting separate CID-MS/MS analysis of the deuterium-labelled precursor ions.

The fragmentation patterns of a series of six novel synthesized benzopyranopyrimidine derivatives 1-6, possessing the same 2-oxo-2H-benzopyrano[2,3-d]pyrimidine backbone structure, were investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) techniques using a quadrupole orthogonal time-of-flight (QqToF)-hybrid instrument. The series of six pure benzopyranopyrimidine compounds contained three constitutional isobaric isomers (compounds 4-6). A simple methodology, based on the use of ESI (positive ion mode) and increasing the declustering potential in the atmospheric pressure/vacuum interface resulting in collision-induced dissociation (CID), was used to enhance the formation of the product ions. In general, the novel synthetic benzopyranopyrimidine derivatives 1-6 afforded exact accurate masses for the protonated molecules. This led to the confirmation of both molecular masses and chemical structures of the studied compounds. The breakdown routes of the protonated molecules were rationalized by conducting low-energy CID-MS/MS analyses. It was shown that the MS/MS fragmentation routes for the protonated molecules 1 and 2 were similar, and that the MS/MS fragmentations of the constitutional isobaric protonated molecules 5 and 6 were identical. It was also shown that the gas-phase CID fragmentations of 5 and 6 were different from that of their constitutional isomer 4. Finally, the ESI-MS and CID-MS/MS analyses of the protonated molecules that were obtained from the monodeuterated benzopyranopyrimidine derivatives 1-6 confirmed the values obtained for the exact masses, the precise structural assignments of all product ions and all the pathways described in the proposed CID fragmentations.